Bio

Clinical Focus


  • Cancer > Cutaneous (Dermatologic) Oncology
  • General Dermatology
  • Dermatology

Academic Appointments


Administrative Appointments


  • Program Committee, Cancer Biology (2005 - 2012)

Honors & Awards


  • Salvador E. Luria Lecture, Massachusetts Institute of Technology (2012)
  • Montagna Lecture, Society for Investigative Dermatology (2012)
  • Alfred Marchionini Research Prize, Alfred Marchionini Foundation (2011)
  • CE.R.I.E.S. Award, Chanel Research and Technology (2010)
  • Early Career Scientist, Howard Hughes Medical Institute (2009-2015)
  • Senior Scholar Award in Aging, Ellison Medical Foundation (2009)
  • Elected Member, American Society for Clinical Investigation (2009)
  • Vilcek Prize for Creative Promise, Vilcek Foundation (2009)
  • New Faculty Award, California Institute for Regenerative Medicine (2008-2013)
  • Research Scholar Award, American Cancer Society (2007-2010)
  • Scholar Award, Damon Runyon Cancer Research Foundation (2006-2008)
  • Clinical Scientist Career Development Award (K08), NIH (2004-2009)
  • Physician-Scientist Career Development Award, Dermatology Foundation (2004)
  • Young Investigator Award, American Academy of Dermatology (2003)

Professional Education


  • Internship:Santa Clara Valley Medical Center (2001) CA
  • Fellowship:Stanford University School of Medicine (2004) CA
  • Residency:Stanford University School of Medicine (2003) CA
  • Medical Education:Harvard Medical School (2000) MA
  • Board Certification: Dermatology, American Board of Dermatology (2004)
  • Ph.D., MIT, Biology (1998)
  • A.B., Harvard, Biochemistry (1994)

Research & Scholarship

Current Research and Scholarly Interests


The same genetic blueprint gives rise to thousands of cell types that make up the human body. Intricate mechanisms govern the choice to make skin, heart, or brain cells. These different cell types must be correctly arranged in spatial patterns to make functioning tissues and organs. In many organisms with continual turnover of cells, the genome faces the additional challenge of ensuring the faithful transmission of information throughout a lifetime—over decades in the case of humans. Thus, how one genome encodes thousands of patterns in space and time is of central importance to biology and medicine. Inappropriate activation of genes can give rise to birth defects, premature aging, or cancer, among many other diseases. Restoration of proper organ function often requires restoring homeostatic gene regulation.

Long Noncoding RNAs and Positional Identity
As a practicing dermatologist, I am fascinated by what makes human skin from different parts of the body different, a fact that guides the diagnosis and treatment of many skin diseases. Why do long hairs grow on the scalp but not on our palms or soles? How do cells know where they are located in the body, and how do they remember this information?

We discovered that one class of skin cells, the fibroblasts, encode the positional identity of skin via specific markings on their chromatin, the DNA-protein complex where genes reside. Based on the chromatin configurations of specific genes, most notably the HOX genes, fibroblasts differentially activate hundreds of genes based on their the cell’s location along three anatomic axes—anterior-posterior (head to tail), proximal-distal (close or far away from the trunk), and dermal-nondermal (surface or internal organ). This in effect creates a global positioning system for all cells to navigate.

These studies also revealed a surprising abundance of long intergenic long noncoding RNAs (also known as lincRNAs, a newly recognized type of genes that do not code forencode proteins) that are involved in programming chromatin states. We are particularly fascinated by HOTAIR, the first known lincRNA that can regulate the chromatin state of genes on distantly located chromosomes. We now appreciate that the genome is pervasively transcribed to give rise to thousands of lincRNAs, which are likely to play key roles in the gene regulation of diverse biological states and disease. We are interested in understanding how lincRNAs control gene activity, and in deciphering the rules that will allow the functions of thousands of lincRNAs to be predicted and studied.

Large-Scale Gene Regulatory Programs in Cancer Metastasis and Self-Renewal
In contrast to the orderly acquisition of positional identity, cancer progression is characterized by abrogation of normal positional boundaries, especially in metastasis, which is the leading cause of cancer death. We and many others have previously identified gene expression signatures (GES ), composed of dozens to hundreds of genes, that distinguish indolent human cancers from those prone to metastasis; these signatures can provide improved prognostic prediction for cancer patients. Furthermore, we have developed methods to pinpoint master regulators of GES—singular control points that can toggle the activity of the entire genetic program. This allows complex gene programs observed in human cancers to be easily recapitulated in the laboratory as models for drug development. This has enabled the creation of faithful laboratory models of human cancer types, identified specific drugs that can target these cancers, and revealed the hierarchy of transcriptional programs involved in the generation of cancer stem cells—the cells that continually repopulate a tumor or its metastases.

Clinical Trials


  • A Pilot Study of Imatinib Mesylate in Steroid Refractory Chronic Graft Versus Host Disease Not Recruiting

    To determine if subjects with steroid refractory cGVHD can tolerate imatinib mesylate and whether their cGVHD responds to imatinib mesylate.

    Stanford is currently not accepting patients for this trial. For more information, please contact Joanne Otani, (650) 721 - 2372.

    View full details

Teaching

2013-14 Courses


Publications

Journal Articles


  • Landscape and variation of RNA secondary structure across the human transcriptome. Nature Wan, Y., Qu, K., Zhang, Q. C., Flynn, R. A., Manor, O., Ouyang, Z., Zhang, J., Spitale, R. C., Snyder, M. P., Segal, E., Chang, H. Y. 2014; 505 (7485): 706-709

    Abstract

    In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of 'riboSNitches' versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3' untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation.

    View details for DOI 10.1038/nature12946

    View details for PubMedID 24476892

  • Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position NATURE METHODS Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y., Greenleaf, W. J. 2013; 10 (12): 1213-?

    Abstract

    We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500-50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes. Using ATAC-seq maps of human CD4(+) T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individual's epigenome on a timescale compatible with clinical decision-making.

    View details for DOI 10.1038/NMETH.2688

    View details for Web of Science ID 000327698100025

    View details for PubMedID 24097267

  • Hierarchical Mechanisms for Direct Reprogramming of Fibroblasts to Neurons CELL Wapinski, O. L., Vierbuchen, T., Qu, K., Lee, Q. Y., Chanda, S., Fuentes, D. R., Giresi, P. G., Ng, Y. H., Marro, S., Neff, N. F., Drechsel, D., Martynoga, B., Castro, D. S., Webb, A. E., Suedhof, T. C., Brunet, A., Guillemot, F., Chang, H. Y., Wernig, M. 2013; 155 (3): 621-635

    Abstract

    Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.

    View details for DOI 10.1016/j.cell.2013.09.028

    View details for Web of Science ID 000326571800016

    View details for PubMedID 24243019

  • Long Noncoding RNAs: Cellular Address Codes in Development and Disease CELL Batista, P. J., Chang, H. Y. 2013; 152 (6): 1298-1307

    Abstract

    In biology as in real estate, location is a cardinal organizational principle that dictates the accessibility and flow of informational traffic. An essential question in nuclear organization is the nature of the address code--how objects are placed and later searched for and retrieved. Long noncoding RNAs (lncRNAs) have emerged as key components of the address code, allowing protein complexes, genes, and chromosomes to be trafficked to appropriate locations and subject to proper activation and deactivation. lncRNA-based mechanisms control cell fates during development, and their dysregulation underlies some human disorders caused by chromosomal deletions and translocations.

    View details for DOI 10.1016/j.cell.2013.02.012

    View details for Web of Science ID 000316192500011

    View details for PubMedID 23498938

  • Control of somatic tissue differentiation by the long non-coding RNA TINCR NATURE Kretz, M., Siprashvili, Z., Chu, C., Webster, D. E., Zehnder, A., Qu, K., Lee, C. S., Flockhart, R. J., Groff, A. F., Chow, J., Johnston, D., Kim, G. E., Spitale, R. C., Flynn, R. A., Zheng, G. X., Aiyer, S., Raj, A., Rinn, J. L., Chang, H. Y., Khavari, P. A. 2013; 493 (7431): 231-U245

    Abstract

    Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25-nucleotide 'TINCR box' motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR-STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression.

    View details for DOI 10.1038/nature11661

    View details for Web of Science ID 000313259600041

    View details for PubMedID 23201690

  • A mammalian pseudogene lncRNA at the interface of inflammation and anti-inflammatory therapeutics. eLife Rapicavoli, N. A., Qu, K., Zhang, J., Mikhail, M., Laberge, R., Chang, H. Y. 2013; 2

    Abstract

    Pseudogenes are thought to be inactive gene sequences, but recent evidence of extensive pseudogene transcription raised the question of potential function. Here we discover and characterize the sets of mouse lncRNAs induced by inflammatory signaling via TNFα. TNFα regulates hundreds of lncRNAs, including 54 pseudogene lncRNAs, several of which show exquisitely selective expression in response to specific cytokines and microbial components in a NF-κB-dependent manner. Lethe, a pseudogene lncRNA, is selectively induced by proinflammatory cytokines via NF-κB or glucocorticoid receptor agonist, and functions in negative feedback signaling to NF-κB. Lethe interacts with NF-κB subunit RelA to inhibit RelA DNA binding and target gene activation. Lethe level decreases with organismal age, a physiological state associated with increased NF-κB activity. These findings suggest that expression of pseudogenes lncRNAs are actively regulated and constitute functional regulators of inflammatory signaling. DOI:http://dx.doi.org/10.7554/eLife.00762.001.

    View details for DOI 10.7554/eLife.00762

    View details for PubMedID 23898399

  • Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature methods Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y., Greenleaf, W. J. 2013; 10 (12): 1213-8

    Abstract

    We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500-50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes. Using ATAC-seq maps of human CD4(+) T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individual's epigenome on a timescale compatible with clinical decision-making.

    View details for DOI 10.1038/nmeth.2688

    View details for PubMedID 24097267

  • Aging, Rejuvenation, and Epigenetic Reprogramming: Resetting the Aging Clock CELL Rando, T. A., Chang, H. Y. 2012; 148 (1-2): 46-57

    Abstract

    The underlying cause of aging remains one of the central mysteries of biology. Recent studies in several different systems suggest that not only may the rate of aging be modified by environmental and genetic factors, but also that the aging clock can be reversed, restoring characteristics of youthfulness to aged cells and tissues. This Review focuses on the emerging biology of rejuvenation through the lens of epigenetic reprogramming. By defining youthfulness and senescence as epigenetic states, a framework for asking new questions about the aging process emerges.

    View details for DOI 10.1016/j.cell.2012.01.003

    View details for Web of Science ID 000299540700013

    View details for PubMedID 22265401

  • A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression NATURE Wang, K. C., Yang, Y. W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B. R., Protacio, A., Flynn, R. A., Gupta, R. A., Wysocka, J., Lei, M., Dekker, J., Helms, J. A., Chang, H. Y. 2011; 472 (7341): 120-U158

    Abstract

    The genome is extensively transcribed into long intergenic noncoding RNAs (lincRNAs), many of which are implicated in gene silencing. Potential roles of lincRNAs in gene activation are much less understood. Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control. Some locus control elements and enhancers transcribe lincRNAs, hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3' to 5'of the cluster. Here we identify HOTTIP, a lincRNA transcribed from the 5' tip of the HOXA locus that coordinates the activation of several 5' HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation.

    View details for DOI 10.1038/nature09819

    View details for Web of Science ID 000289199400049

    View details for PubMedID 21423168

  • Targeted disruption of hotair leads to homeotic transformation and gene derepression. Cell reports Li, L., Liu, B., Wapinski, O. L., Tsai, M., Qu, K., Zhang, J., Carlson, J. C., Lin, M., Fang, F., Gupta, R. A., Helms, J. A., Chang, H. Y. 2013; 5 (1): 3-12

    Abstract

    Long noncoding RNAs (lncRNAs) are thought to be prevalent regulators of gene expression, but the consequences of lncRNA inactivation in vivo are mostly unknown. Here, we show that targeted deletion of mouse Hotair lncRNA leads to derepression of hundreds of genes, resulting in homeotic transformation of the spine and malformation of metacarpal-carpal bones. RNA sequencing and conditional inactivation reveal an ongoing requirement of Hotair to repress HoxD genes and several imprinted loci such as Dlk1-Meg3 and Igf2-H19 without affecting imprinting choice. Hotair binds to both Polycomb repressive complex 2, which methylates histone H3 at lysine 27 (H3K27), and Lsd1 complex, which demethylates histone H3 at lysine 4 (H3K4) in vivo. Hotair inactivation causes H3K4me3 gain and, to a lesser extent, H3K27me3 loss at target genes. These results reveal the function and mechanisms of Hotair lncRNA in enforcing a silent chromatin state at Hox and additional genes.

    View details for DOI 10.1016/j.celrep.2013.09.003

    View details for PubMedID 24075995

  • Genome-wide mapping of RNA structure using nuclease digestion and high-throughput sequencing. Nature protocols Wan, Y., Qu, K., Ouyang, Z., Chang, H. Y. 2013; 8 (5): 849-869

    Abstract

    RNA structure is important for RNA function and regulation, and there is growing interest in determining the RNA structure of many transcripts. Here we provide a detailed protocol for the parallel analysis of RNA structure (PARS) for probing RNA secondary structures genome-wide. In this method, enzymatic footprinting is coupled to high-throughput sequencing to provide secondary structure data for thousands of RNAs simultaneously. The entire experimental protocol takes ?5 d to complete, and sequencing and data analysis take an additional 6-8 d. PARS was developed using the yeast genome as proof of principle, but its approach should be applicable to probing RNA structures from different transcriptomes and structural dynamics under diverse solution conditions.

    View details for DOI 10.1038/nprot.2013.045

    View details for PubMedID 23558785

  • Cytotopic localization by long noncoding RNAs CURRENT OPINION IN CELL BIOLOGY Batista, P. J., Chang, H. Y. 2013; 25 (2): 195-199

    Abstract

    Cells are highly organized structures. In addition to membrane delimited organelles, proteins and RNAs can organize themselves into specific domains. Some examples include stress granules and subnuclear bodies. This level of organization is essential for the correct execution of multiple processes in the cell, ranging from cell signaling to assembly of structures such as the ribosomes. Here we will review evidence that noncoding RNAs play a critical role in the establishment and regulation of these domains. The unique abilities of RNA to mark the genome in a gene-specific and condition-specific manner and to serve as tethers nominate them as ideal molecular address codes.

    View details for DOI 10.1016/j.ceb.2012.12.001

    View details for Web of Science ID 000317886100008

    View details for PubMedID 23279909

  • The NeST Long ncRNA Controls Microbial Susceptibility and Epigenetic Activation of the Interferon-gamma Locus CELL Gomez, J. A., Wapinski, O. L., Yang, Y. W., Bureau, J., Gopinath, S., Monack, D. M., Chang, H. Y., Brahic, M., Kirkegaard, K. 2013; 152 (4): 743-754

    Abstract

    Long noncoding RNAs (lncRNAs) are increasingly appreciated as regulators of cell-specific gene expression. Here, an enhancer-like lncRNA termed NeST (nettoie Salmonella pas Theiler's [cleanup Salmonella not Theiler's]) is shown to be causal for all phenotypes conferred by murine viral susceptibility locus Tmevp3. This locus was defined by crosses between SJL/J and B10.S mice and contains several candidate genes, including NeST. The SJL/J-derived locus confers higher lncRNA expression, increased interferon-? (IFN-?) abundance in activated CD8(+) T cells, increased Theiler's virus persistence, and decreased Salmonella enterica pathogenesis. Transgenic expression of NeST lncRNA alone was sufficient to confer all phenotypes of the SJL/J locus. NeST RNA was found to bind WDR5, a component of the histone H3 lysine 4 methyltransferase complex, and to alter histone 3 methylation at the IFN-? locus. Thus, this lncRNA regulates epigenetic marking of IFN-?-encoding chromatin, expression of IFN-?, and susceptibility to a viral and a bacterial pathogen.

    View details for DOI 10.1016/j.cell.2013.01.015

    View details for Web of Science ID 000314945600010

    View details for PubMedID 23415224

  • Rejuvenation of Gene Expression Pattern of Aged Human Skin by Broadband Light Treatment: A Pilot Study JOURNAL OF INVESTIGATIVE DERMATOLOGY Chang, A. L., Bitter, P. H., Qu, K., Lin, M., Rapicavoli, N. A., Chang, H. Y. 2013; 133 (2): 394-402

    Abstract

    Studies in model organisms suggest that aged cells can be functionally rejuvenated, but whether this concept applies to human skin is unclear. Here we apply 3'-end sequencing for expression quantification ("3-seq") to discover the gene expression program associated with human photoaging and intrinsic skin aging (collectively termed "skin aging"), and the impact of broadband light (BBL) treatment. We find that skin aging was associated with a significantly altered expression level of 2,265 coding and noncoding RNAs, of which 1,293 became "rejuvenated" after BBL treatment; i.e., they became more similar to their expression level in youthful skin. Rejuvenated genes (RGs) included several known key regulators of organismal longevity and their proximal long noncoding RNAs. Skin aging is not associated with systematic changes in 3'-end mRNA processing. Hence, BBL treatment can restore gene expression pattern of photoaged and intrinsically aged human skin to resemble young skin. In addition, our data reveal, to our knowledge, a previously unreported set of targets that may lead to new insights into the human skin aging process.

    View details for DOI 10.1038/jid.2012.287

    View details for Web of Science ID 000313668000018

    View details for PubMedID 22931923

  • SeqFold: Genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data GENOME RESEARCH Ouyang, Z., Snyder, M. P., Chang, H. Y. 2013; 23 (2): 377-387

    Abstract

    We present an integrative approach, SeqFold, that combines high-throughput RNA structure profiling data with computational prediction for genome-scale reconstruction of RNA secondary structures. SeqFold transforms experimental RNA structure information into a structure preference profile (SPP) and uses it to select stable RNA structure candidates representing the structure ensemble. Under a high-dimensional classification framework, SeqFold efficiently matches a given SPP to the most likely cluster of structures sampled from the Boltzmann-weighted ensemble. SeqFold is able to incorporate diverse types of RNA structure profiling data, including parallel analysis of RNA structure (PARS), selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), fragmentation sequencing (FragSeq) data generated by deep sequencing, and conventional SHAPE data. Using the known structures of a wide range of mRNAs and noncoding RNAs as benchmarks, we demonstrate that SeqFold outperforms or matches existing approaches in accuracy and is more robust to noise in experimental data. Application of SeqFold to reconstruct the secondary structures of the yeast transcriptome reveals the diverse impact of RNA secondary structure on gene regulation, including translation efficiency, transcription initiation, and protein-RNA interactions. SeqFold can be easily adapted to incorporate any new types of high-throughput RNA structure profiling data and is widely applicable to analyze RNA structures in any transcriptome.

    View details for DOI 10.1101/gr.138545.112

    View details for Web of Science ID 000314323100016

    View details for PubMedID 23064747

  • RNA SHAPE analysis in living cells NATURE CHEMICAL BIOLOGY Spitale, R. C., Crisalli, P., Flynn, R. A., Torre, E. A., Kool, E. T., Chang, H. Y. 2013; 9 (1): 18-?

    Abstract

    RNA structure has important roles in practically every facet of gene regulation, but the paucity of in vivo structural probes limits current understanding. Here we design, synthesize and demonstrate two new chemical probes that enable selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) in living cells. RNA structures in human, mouse, fly, yeast and bacterial cells are read out at single-nucleotide resolution, revealing tertiary contacts and RNA-protein interactions.

    View details for DOI 10.1038/NCHEMBIO.1131

    View details for Web of Science ID 000312484200007

    View details for PubMedID 23178934

  • Systematic reconstruction of RNA functional motifs with high-throughput microfluidics NATURE METHODS Martin, L., Meier, M., Lyons, S. M., Sit, R. V., Marzluff, W. F., Quake, S. R., Chang, H. Y. 2012; 9 (12): 1192-U85

    Abstract

    We present RNA-mechanically induced trapping of molecular interactions (RNA-MITOMI), a microfluidic platform that allows integrated synthesis and functional assays for programmable RNA libraries. The interaction of a comprehensive library of RNA mutants with stem-loop-binding protein precisely defined the RNA structural and sequence features that govern affinity. The functional motif reconstructed in a single experiment on our platform uncovers new binding specificities and enriches interpretation of phylogenetic data.

    View details for DOI 10.1038/NMETH.2225

    View details for Web of Science ID 000312093500022

    View details for PubMedID 23142872

  • Identification of proteins binding coding and non-coding human RNAs using protein microarrays BMC GENOMICS Siprashvili, Z., Webster, D. E., Kretz, M., Johnston, D., Rinn, J. L., Chang, H. Y., Khavari, P. A. 2012; 13

    Abstract

    The regulation and function of mammalian RNAs has been increasingly appreciated to operate via RNA-protein interactions. With the recent discovery of thousands of novel human RNA molecules by high-throughput RNA sequencing, efficient methods to uncover RNA-protein interactions are urgently required. Existing methods to study proteins associated with a given RNA are laborious and require substantial amounts of cell-derived starting material. To overcome these limitations, we have developed a rapid and large-scale approach to characterize binding of in vitro transcribed labeled RNA to ~9,400 human recombinant proteins spotted on protein microarrays.We have optimized methodology to probe human protein microarrays with full-length RNA molecules and have identified 137 RNA-protein interactions specific for 10 coding and non-coding RNAs. Those proteins showed strong enrichment for common human RNA binding domains such as RRM, RBD, as well as K homology and CCCH type zinc finger motifs. Previously unknown RNA-protein interactions were discovered using this technique, and these interactions were biochemically verified between TP53 mRNA and Staufen1 protein as well as between HRAS mRNA and CNBP protein. Functional characterization of the interaction between Staufen 1 protein and TP53 mRNA revealed a novel role for Staufen 1 in preserving TP53 RNA stability.Our approach demonstrates a scalable methodology, allowing rapid and efficient identification of novel human RNA-protein interactions using RNA hybridization to human protein microarrays. Biochemical validation of newly identified interactions between TP53-Stau1 and HRAS-CNBP using reciprocal pull-down experiments, both in vitro and in vivo, demonstrates the utility of this approach to study uncharacterized RNA-protein interactions.

    View details for DOI 10.1186/1471-2164-13-633

    View details for Web of Science ID 000314649200001

    View details for PubMedID 23157412

  • Genome-wide Measurement of RNA Folding Energies MOLECULAR CELL Wan, Y., Qu, K., Ouyang, Z., Kertesz, M., Li, J., Tibshirani, R., Makino, D. L., Nutter, R. C., Segal, E., Chang, H. Y. 2012; 48 (2): 169-181

    Abstract

    RNA structural transitions are important in the function and regulation of RNAs. Here, we reveal a layer of transcriptome organization in the form of RNA folding energies. By probing yeast RNA structures at different temperatures, we obtained relative melting temperatures (Tm) for RNA structures in over 4000 transcripts. Specific signatures of RNA Tm demarcated the polarity of mRNA open reading frames and highlighted numerous candidate regulatory RNA motifs in 3' untranslated regions. RNA Tm distinguished noncoding versus coding RNAs and identified mRNAs with distinct cellular functions. We identified thousands of putative RNA thermometers, and their presence is predictive of the pattern of RNA decay in vivo during heat shock. The exosome complex recognizes unpaired bases during heat shock to degrade these RNAs, coupling intrinsic structural stabilities to gene regulation. Thus, genome-wide structural dynamics of RNA can parse functional elements of the transcriptome and reveal diverse biological insights.

    View details for DOI 10.1016/j.molcel.2012.08.008

    View details for Web of Science ID 000310423800004

    View details for PubMedID 22981864

  • Detection of Long Non-Coding RNA in Archival Tissue: Correlation with Polycomb Protein Expression in Primary and Metastatic Breast Carcinoma PLOS ONE Chisholm, K. M., Wan, Y., Li, R., Montgomery, K. D., Chang, H. Y., West, R. B. 2012; 7 (10)

    Abstract

    A major function of long non-coding RNAs (lncRNAs) is regulating gene expression through changes in chromatin state. Experimental evidence suggests that in cancer, they can influence Polycomb Repressive Complexes (PRC) to retarget to an occupancy pattern resembling that of the embryonic state. We have previously demonstrated that the expression level of lncRNA in the HOX locus, including HOTAIR, is a predictor of breast cancer metastasis. In this current project, RNA in situ hybridization of probes to three different lncRNAs (HOTAIR, ncHoxA1, and ncHoxD4), as well a immunohistochemical staining of EZH2, is undertaken in formalin-fixed paraffin-embedded breast cancer tissues in a high throughput tissue microarray format to correlate expression with clinicopathologic features. Though overall EZH2 and HOTAIR expression levels were highly correlated, the subset of cases with strong HOTAIR expression correlated with ER and PR positivity, while the subset of cases with strong EZH2 expression correlated with an increased proliferation rate, ER and PR negativity, HER2 underexpression, and triple negativity. Co-expression of HOTAIR and EZH2 trended with a worse outcome. In matched primary and metastatic cancers, both HOTAIR and EZH2 had increased expression in the metastatic carcinomas. This is the first study to show that RNA in situ hybridization of formalin fixed paraffin-embedded clinical material can be used to measure levels of long non-coding RNAs. This approach offers a method to make observations on lncRNAs that may influence the cancer epigenome in a tissue-based technique.

    View details for DOI 10.1371/journal.pone.0047998

    View details for Web of Science ID 000310261800028

    View details for PubMedID 23133536

  • Transcriptome sequencing in Sezary syndrome identifies Sezary cell and mycosis fungoides-associated lncRNAs and novel transcripts BLOOD Lee, C. S., Ungewickell, A., Bhaduri, A., Qu, K., Webster, D. E., Armstrong, R., Weng, W., Aros, C. J., Mah, A., Chen, R. O., Lin, M., Sundram, U., Chang, H. Y., Kretz, M., Kim, Y. H., Khavari, P. A. 2012; 120 (16): 3288-3297

    Abstract

    Sézary syndrome (SS) is an aggressive cutaneous T-cell lymphoma (CTCL) of unknown etiology in which malignant cells circulate in the peripheral blood. To identify viral elements, gene fusions, and gene expression patterns associated with this lymphoma, flow cytometry was used to obtain matched pure populations of malignant Sézary cells (SCs) versus nonmalignant CD4(+) T cells from 3 patients for whole transcriptome, paired-end sequencing with an average depth of 112 million reads per sample. Pathway analysis of differentially expressed genes identified mis-regulation of PI3K/Akt, TGF?, and NF-?B pathways as well as T-cell receptor signaling. Bioinformatic analysis did not detect either nonhuman transcripts to support a viral etiology of SS or recurrently expressed gene fusions, but it did identify 21 SC-associated annotated long noncoding RNAs (lncRNAs). Transcriptome assembly by multiple algorithms identified 13 differentially expressed unannotated transcripts termed Sézary cell-associated transcripts (SeCATs) that include 12 predicted lncRNAs and a novel transcript with coding potential. High-throughput sequencing targeting the 3' end of polyadenylated transcripts in archived tumors from 24 additional patients with tumor-stage CTCL confirmed the differential expression of SC-associated lncRNAs and SeCATs in CTCL. Our findings characterize the SS transcriptome and support recent reports that implicate lncRNA dysregulation in human malignancies.

    View details for DOI 10.1182/blood-2012-04-423061

    View details for Web of Science ID 000311619200020

    View details for PubMedID 22936659

  • A Molecular Signature for Purified Definitive Endoderm Guides Differentiation and Isolation of Endoderm from Mouse and Human Embryonic Stem Cells STEM CELLS AND DEVELOPMENT Wang, P., McKnight, K. D., Wong, D. J., Rodriguez, R. T., Sugiyama, T., Gu, X., Ghodasara, A., Qu, K., Chang, H. Y., Kim, S. K. 2012; 21 (12): 2273-2287

    Abstract

    Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as liver, pancreas, and intestine. However, purification of DE in mammals has not been achieved, limiting the molecular "definition" of endoderm, and hindering our understanding of DE development and attempts to produce endoderm from sources such as embryonic stem (ES) cells. Here, we describe purification of mouse DE using fluorescence-activated cell sorting (FACS) and mice harboring a transgene encoding enhanced green fluorescent protein (eGFP) inserted into the Sox17 locus, which is expressed in the embryonic endoderm. Comparison of patterns of signaling pathway activation in native mouse DE and endoderm-like cells generated from ES cells produced novel culture modifications that generated Sox17-eGFP? progeny whose gene expression resembled DE more closely than achieved with standard methods. These studies also produced new FACS methods for purifying DE from nontransgenic mice and mouse ES cell cultures. Parallel studies of a new human SOX17-eGFP ES cell line allowed analysis of endoderm differentiation in vitro, leading to culture modifications that enhanced expression of an endoderm-like signature. This work should accelerate our understanding of mechanisms regulating DE development in mice and humans, and guide further use of ES cells for tissue replacement.

    View details for DOI 10.1089/scd.2011.0416

    View details for Web of Science ID 000307295500018

    View details for PubMedID 22236333

  • The Transcription Factor ZNF217 Is a Prognostic Biomarker and Therapeutic Target during Breast Cancer Progression CANCER DISCOVERY Littlepage, L. E., Adler, A. S., Kouros-Mehr, H., Huang, G., Chou, J., Krig, S. R., Griffith, O. L., Korkola, J. E., Qu, K., Lawson, D. A., Xue, Q., Sternlicht, M. D., Dijkgraaf, G. J., Yaswen, P., Rugo, H. S., Sweeney, C. A., Collins, C. C., Gray, J. W., Chang, H. Y., Werb, Z. 2012; 2 (7): 638-651

    Abstract

    The transcription factor ZNF217 is a candidate oncogene in the amplicon on chromosome 20q13 that occurs in 20% to 30% of primary human breast cancers and that correlates with poor prognosis. We show that Znf217 overexpression drives aberrant differentiation and signaling events, promotes increased self-renewal capacity, mesenchymal marker expression, motility, and metastasis, and represses an adult tissue stem cell gene signature downregulated in cancers. By in silico screening, we identified candidate therapeutics that at low concentrations inhibit growth of cancer cells expressing high ZNF217. We show that the nucleoside analogue triciribine inhibits ZNF217-induced tumor growth and chemotherapy resistance and inhibits signaling events [e.g., phospho-AKT, phospho-mitogen-activated protein kinase (MAPK)] in vivo. Our data suggest that ZNF217 is a biomarker of poor prognosis and a therapeutic target in patients with breast cancer and that triciribine may be part of a personalized treatment strategy in patients overexpressing ZNF217. Because ZNF217 is amplified in numerous cancers, these results have implications for other cancers.This study finds that ZNF217 is a poor prognostic indicator and therapeutic target in patients with breast cancer and may be a strong biomarker of triciribine treatment efficacy in patients. Because previous clinical trials for triciribine did not include biomarkers of treatment efficacy, this study provides a rationale for revisiting triciribine in the clinical setting as a therapy for patients with breast cancer who overexpress ZNF217.

    View details for DOI 10.1158/2159-8290.CD-12-0093

    View details for Web of Science ID 000306365800042

    View details for PubMedID 22728437

  • Active chromatin and noncoding RNAs: an intimate relationship CURRENT OPINION IN GENETICS & DEVELOPMENT Flynn, R. A., Chang, H. Y. 2012; 22 (2): 172-178

    Abstract

    Eukaryotic genomes are packaged into chromatin, where diverse histone modifications can demarcate chromatin domains that facilitate or block gene expression. While silent chromatin has been associated with long noncoding RNAs (lncRNAs) for some time, new studies suggest that noncoding RNAs also modulate the active chromatin state. Divergent, antisense, and enhancer-like intergenic noncoding RNAs can either activate or repress gene expression by altering histone H3 lysine 4 methylation. An emerging class of enhancer-like lncRNAs may link chromosome structure to chromatin state and establish active chromatin domains. The confluence of several new technologies promises to rapidly expand this fascinating topic of investigation.

    View details for DOI 10.1016/j.gde.2011.11.002

    View details for Web of Science ID 000304338000016

    View details for PubMedID 22154525

  • Suppression of progenitor differentiation requires the long noncoding RNA ANCR GENES & DEVELOPMENT Kretz, M., Webster, D. E., Flockhart, R. J., Lee, C. S., Zehnder, A., Lopez-Pajares, V., Qu, K., Zheng, G. X., Chow, J., Kim, G. E., Rinn, J. L., Chang, H. Y., Siprashvili, Z., Khavari, P. A. 2012; 26 (4): 338-343

    Abstract

    Long noncoding RNAs (lncRNAs) regulate diverse processes, yet a potential role for lncRNAs in maintaining the undifferentiated state in somatic tissue progenitor cells remains uncharacterized. We used transcriptome sequencing and tiling arrays to compare lncRNA expression in epidermal progenitor populations versus differentiating cells. We identified ANCR (anti-differentiation ncRNA) as an 855-base-pair lncRNA down-regulated during differentiation. Depleting ANCR in progenitor-containing populations, without any other stimuli, led to rapid differentiation gene induction. In epidermis, ANCR loss abolished the normal exclusion of differentiation from the progenitor-containing compartment. The ANCR lncRNA is thus required to enforce the undifferentiated cell state within epidermis.

    View details for DOI 10.1101/gad.182121.111

    View details for Web of Science ID 000300626800004

    View details for PubMedID 22302877

  • Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers GENOME BIOLOGY Brunner, A. L., Beck, A. H., Edris, B., Sweeney, R. T., Zhu, S. X., Li, R., Montgomery, K., Varma, S., Gilks, T., Guo, X., Foley, J. W., Witten, D. M., Giacomini, C. P., Flynn, R. A., Pollack, J. R., Tibshirani, R., Chang, H. Y., van de Rijn, M., West, R. B. 2012; 13 (8)
  • Genome Regulation by Long Noncoding RNAs ANNUAL REVIEW OF BIOCHEMISTRY, VOL 81 Rinn, J. L., Chang, H. Y. 2012; 81: 145-166

    Abstract

    The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. The discovery of extensive transcription of large RNA transcripts that do not code for proteins, termed long noncoding RNAs (lncRNAs), provides an important new perspective on the centrality of RNA in gene regulation. Here, we discuss genome-scale strategies to discover and characterize lncRNAs. An emerging theme from multiple model systems is that lncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators and then target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, lncRNAs can function as modular scaffolds to specify higher-order organization in RNP complexes and in chromatin states. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs for proper gene control across all kingdoms of life.

    View details for DOI 10.1146/annurev-biochem-051410-092902

    View details for Web of Science ID 000305765500008

    View details for PubMedID 22663078

  • Chromatin isolation by RNA purification (ChIRP). Journal of visualized experiments : JoVE Chu, C., Quinn, J., Chang, H. Y. 2012

    Abstract

    Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression (1,2,3,4,5,6,7). The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion (8,9). While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation (10,11). However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide( 3,12,13), but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex (14); HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex (13). Prior studies mapping RNA occupancy at chromatin have revealed substantial insights (15,16), but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution (17). This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

    View details for DOI 10.3791/3912

    View details for PubMedID 22472705

  • High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation LAB ON A CHIP Wu, A. R., Kawahara, T. L., Rapicavoli, N. A., van Riggelen, J., Shroff, E. H., Xu, L., Felsher, D. W., Chang, H. Y., Quake, S. R. 2012; 12 (12): 2190-2198

    Abstract

    Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein-DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery.

    View details for DOI 10.1039/c2lc21290k

    View details for Web of Science ID 000304448700012

    View details for PubMedID 22566096

  • Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions MOLECULAR CELL Chu, C., Qu, K., Zhong, F. L., Artandi, S. E., Chang, H. Y. 2011; 44 (4): 667-678

    Abstract

    Long noncoding RNAs (lncRNAs) are key regulators of chromatin state, yet the nature and sites of RNA-chromatin interaction are mostly unknown. Here we introduce Chromatin Isolation by RNA Purification (ChIRP), where tiling oligonucleotides retrieve specific lncRNAs with bound protein and DNA sequences, which are enumerated by deep sequencing. ChIRP-seq of three lncRNAs reveal that RNA occupancy sites in the genome are focal, sequence-specific, and numerous. Drosophila roX2 RNA occupies male X-linked gene bodies with increasing tendency toward the 3' end, peaking at CES sites. Human telomerase RNA TERC occupies telomeres and Wnt pathway genes. HOTAIR lncRNA preferentially occupies a GA-rich DNA motif to nucleate broad domains of Polycomb occupancy and histone H3 lysine 27 trimethylation. HOTAIR occupancy occurs independently of EZH2, suggesting the order of RNA guidance of Polycomb occupancy. ChIRP-seq is generally applicable to illuminate the intersection of RNA and chromatin with newfound precision genome wide.

    View details for DOI 10.1016/j.molcel.2011.08.027

    View details for Web of Science ID 000297387800017

    View details for PubMedID 21963238

  • Direct Lineage Conversion of Terminally Differentiated Hepatocytes to Functional Neurons CELL STEM CELL Marro, S., Pang, Z. P., Yang, N., Tsai, M., Qu, K., Chang, H. Y., Suedhof, T. C., Wernig, M. 2011; 9 (4): 374-382

    Abstract

    Several recent studies have showed that mouse and human fibroblasts can be directly reprogrammed into induced neuronal (iN) cells, bypassing a pluripotent intermediate state. However, fibroblasts represent heterogeneous mesenchymal progenitor cells that potentially contain neural crest lineages, and the cell of origin remained undefined. This raises the fundamental question of whether lineage reprogramming is possible between cell types derived from different germ layers. Here, we demonstrate that terminally differentiated hepatocytes can be directly converted into functional iN cells. Importantly, single-cell and genome-wide expression analyses showed that fibroblast- and hepatocyte-derived iN cells not only induced a neuronal transcriptional program, but also silenced their donor transcriptome. The remaining donor signature decreased over time and could not support functional hepatocyte properties. Thus, the reprogramming factors lead to a binary lineage switch decision rather than an induction of hybrid phenotypes, but iN cells retain a small but detectable epigenetic memory of their donor cells.

    View details for DOI 10.1016/j.stem.2011.09.002

    View details for Web of Science ID 000296041200015

    View details for PubMedID 21962918

  • Molecular Mechanisms of Long Noncoding RNAs MOLECULAR CELL Wang, K. C., Chang, H. Y. 2011; 43 (6): 904-914

    Abstract

    Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. Here we discuss the emerging archetypes of molecular functions that lncRNAs execute-as signals, decoys, guides, and scaffolds. For each archetype, examples from several disparate biological contexts illustrate the commonality of the molecular mechanisms, and these mechanistic views provide useful explanations and predictions of biological outcomes. These archetypes of lncRNA function may be a useful framework to consider how lncRNAs acquire properties as biological signal transducers and hint at their possible origins in evolution. As new lncRNAs are being discovered at a rapid pace, the molecular mechanisms of lncRNAs are likely to be enriched and diversified.

    View details for DOI 10.1016/j.molcel.2011.08.018

    View details for Web of Science ID 000295309800006

    View details for PubMedID 21925379

  • Disruption of PPAR gamma/beta-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival JOURNAL OF CLINICAL INVESTIGATION Alastalo, T., Li, M., Perez, V. D., Pham, D., Sawada, H., Wang, J. K., Koskenvuo, M., Wang, L., Freeman, B. A., Chang, H. Y., Rabinovitch, M. 2011; 121 (9): 3735-3746

    Abstract

    Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with pulmonary arterial hypertension (PAH) can impair pulmonary arterial EC (PAEC) function. This can adversely affect EC survival and promote SMC proliferation. We hypothesized that interventions to normalize expression of genes that are targets of BMPR2 signaling could restore PAEC function and prevent or reverse PAH. Here we have characterized, in human PAECs, a BMPR2-mediated transcriptional complex between PPAR? and ?-catenin and shown that disruption of this complex impaired BMP-mediated PAEC survival. Using whole genome-wide ChIP-Chip promoter analysis and gene expression microarrays, we delineated PPAR?/?-catenin-dependent transcription of target genes including APLN, which encodes apelin. We documented reduced PAEC expression of apelin in PAH patients versus controls. In cell culture experiments, we showed that apelin-deficient PAECs were prone to apoptosis and promoted pulmonary arterial SMC (PASMC) proliferation. Conversely, we established that apelin, like BMPR2 ligands, suppressed proliferation and induced apoptosis of PASMCs. Consistent with these functions, administration of apelin reversed PAH in mice with reduced production of apelin resulting from deletion of PPAR? in ECs. Taken together, our findings suggest that apelin could be effective in treating PAH by rescuing BMPR2 and PAEC dysfunction.

    View details for DOI 10.1172/JCI43382

    View details for Web of Science ID 000294753700038

    View details for PubMedID 21821917

  • Understanding the transcriptome through RNA structure NATURE REVIEWS GENETICS Wan, Y., Kertesz, M., Spitale, R. C., Segal, E., Chang, H. Y. 2011; 12 (9): 641-655

    Abstract

    RNA structure is crucial for gene regulation and function. In the past, transcriptomes have largely been parsed by primary sequences and expression levels, but it is now becoming feasible to annotate and compare transcriptomes based on RNA structure. In addition to computational prediction methods, the recent advent of experimental techniques to probe RNA structure by high-throughput sequencing has enabled genome-wide measurements of RNA structure and has provided the first picture of the structural organization of a eukaryotic transcriptome - the 'RNA structurome'. With additional advances in method refinement and interpretation, structural views of the transcriptome should help to identify and validate regulatory RNA motifs that are involved in diverse cellular processes and thereby increase understanding of RNA function.

    View details for DOI 10.1038/nrg3049

    View details for Web of Science ID 000294004100010

    View details for PubMedID 21850044

  • Crystal structure of the N-terminal region of human Ash2L shows a winged-helix motif involved in DNA binding EMBO REPORTS Chen, Y., Wan, B., Wang, K. C., Cao, F., Yang, Y., Protacio, A., Dou, Y., Chang, H. Y., Lei, M. 2011; 12 (8): 797-803

    Abstract

    Ash2L is a core component of the MLL family histone methyltransferases and has an important role in regulating the methylation of histone H3 on lysine 4. Here, we report the crystal structure of the N-terminal domain of Ash2L and reveal a new function of Ash2L. The structure shows that Ash2L contains an atypical PHD finger that does not have histone tail-binding activity. Unexpectedly, the structure shows a previously unrecognized winged-helix motif that directly binds to DNA. The DNA-binding-deficient mutants of Ash2L reduced Ash2L localization to the HOX locus. Strikingly, a single mutation in Ash2L(WH) (K131A) breaks the chromatin domain boundary, suggesting that Ash2L also has a role in chromosome demarcation.

    View details for DOI 10.1038/embor.2011.101

    View details for Web of Science ID 000293326500014

    View details for PubMedID 21660059

  • Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters NATURE GENETICS Hung, T., Wang, Y., Lin, M. F., Koegel, A. K., Kotake, Y., Grant, G. D., Horlings, H. M., Shah, N., Umbricht, C., Wang, P., Wang, Y., Kong, B., Langerod, A., Borresen-Dale, A., Kim, S. K., van de Vijver, M., Sukumar, S., Whitfield, M. L., Kellis, M., Xiong, Y., Wong, D. J., Chang, H. Y. 2011; 43 (7): 621-U196

    Abstract

    Transcription of long noncoding RNAs (lncRNAs) within gene regulatory elements can modulate gene activity in response to external stimuli, but the scope and functions of such activity are not known. Here we use an ultrahigh-density array that tiles the promoters of 56 cell-cycle genes to interrogate 108 samples representing diverse perturbations. We identify 216 transcribed regions that encode putative lncRNAs, many with RT-PCR-validated periodic expression during the cell cycle, show altered expression in human cancers and are regulated in expression by specific oncogenic stimuli, stem cell differentiation or DNA damage. DNA damage induces five lncRNAs from the CDKN1A promoter, and one such lncRNA, named PANDA, is induced in a p53-dependent manner. PANDA interacts with the transcription factor NF-YA to limit expression of pro-apoptotic genes; PANDA depletion markedly sensitized human fibroblasts to apoptosis by doxorubicin. These findings suggest potentially widespread roles for promoter lncRNAs in cell-growth control.

    View details for DOI 10.1038/ng.848

    View details for Web of Science ID 000292184600005

    View details for PubMedID 21642992

  • Long noncoding RNAs and human disease TRENDS IN CELL BIOLOGY Wapinski, O., Chang, H. Y. 2011; 21 (6): 354-361

    Abstract

    A new class of transcripts, long noncoding RNAs (lncRNAs), has been recently found to be pervasively transcribed in the genome. Multiple lines of evidence increasingly link mutations and dysregulations of lncRNAs to diverse human diseases. Alterations in the primary structure, secondary structure, and expression levels of lncRNAs as well as their cognate RNA-binding proteins underlie diseases ranging from neurodegeneration to cancer. Recent progress suggests that the involvement of lncRNAs in human diseases could be far more prevalent than previously appreciated. We review the evidence linking lncRNAs to diverse human diseases and highlight fundamental concepts in lncRNA biology that still need to be clarified to provide a robust framework for lncRNA genetics.

    View details for DOI 10.1016/j.tcb.2011.04.001

    View details for Web of Science ID 000292238200005

    View details for PubMedID 21550244

  • Dynamic Chromatin Localization of Sirt6 Shapes Stress- and Aging-Related Transcriptional Networks PLOS GENETICS Kawahara, T. L., Rapicavoli, N. A., Wu, A. R., Qu, K., Quake, S. R., Chang, H. Y. 2011; 7 (6)

    Abstract

    The sirtuin Sirt6 is a NAD-dependent histone deacetylase that is implicated in gene regulation and lifespan control. Sirt6 can interact with the stress-responsive transcription factor NF-?B and regulate some NF-?B target genes, but the full scope of Sirt6 target genes as well as dynamics of Sirt6 occupancy on chromatin are not known. Here we map Sirt6 occupancy on mouse promoters genome-wide and show that Sirt6 occupancy is highly dynamic in response to TNF-?. More than half of Sirt6 target genes are only revealed upon stress-signaling. The majority of genes bound by NF-?B subunit RelA recruit Sirt6, and dynamic Sirt6 relocalization is largely driven in a RelA-dependent manner. Integrative analysis with global gene expression patterns in wild-type, Sirt6-/-, and double Sirt6-/- RelA-/- cells reveals the epistatic relationships between Sirt6 and RelA in shaping diverse temporal patterns of gene expression. Genes under the direct joint control of Sirt6 and RelA include several with prominent roles in cell senescence and organismal aging. These data suggest dynamic chromatin relocalization of Sirt6 as a key output of NF-?B signaling in stress response and aging.

    View details for DOI 10.1371/journal.pgen.1002153

    View details for Web of Science ID 000292386300062

    View details for PubMedID 21738489

  • RNA templating the epigenome Long noncoding RNAs as molecular scaffolds EPIGENETICS Spitale, R. C., Tsai, M., Chang, H. Y. 2011; 6 (5): 539-543

    Abstract

    Cellular pathways must be synergized, controlled and organized to manage homeostasis. To achieve high selectivity within the crowded cellular milieu the cell utilizes scaffolding complexes whose role is to bring molecules in proximity thereby controlling and enhancing intermolecular interactions and signaling events. To date, scaffolds have been shown to be composed of proteinaceous units; however, recent evidence has supported the idea that non-coding RNAs may also play a similar role. In this point of view article we discuss recent data on ncRNA scaffolds, with particular focus on ncRNA HOTAIR. Using our current knowledge of signaling networks we discuss the role that RNA may play in writing and regulating histone modifications and the information needed for correct gene expression. Further, we speculate on additional, yet undiscovered roles that ncRNAs may be playing as molecular scaffolds.

    View details for DOI 10.4161/epi.6.5.15221

    View details for Web of Science ID 000290203600001

    View details for PubMedID 21393997

  • Long Intergenic Noncoding RNAs: New Links in Cancer Progression CANCER RESEARCH Tsai, M., Spitale, R. C., Chang, H. Y. 2011; 71 (1): 3-7

    Abstract

    The process of cancer metastasis involves a series of sequential and complex steps. Here we give a perspective on recent results regarding noncoding transcription in cancer progression, focusing on the emerging role of long intergenic noncoding RNAs (lincRNAs). LincRNAs target chromatin modification complexes or RNA-binding proteins to alter gene expression programs. Similarly to miRNAs, lincRNAs exhibit distinct gene expression patterns in primary tumors and metastases. We discuss how lincRNAs can be used for cancer diagnosis and prognosis and serve as potential therapeutic targets.

    View details for DOI 10.1158/0008-5472.CAN-10-2483

    View details for Web of Science ID 000285826800001

    View details for PubMedID 21199792

  • Noncoding RNA Landmarks of Pluripotency and Reprogramming CELL STEM CELL Ouyang, Z., Zheng, G. X., Chang, H. Y. 2010; 7 (6): 649-650

    View details for DOI 10.1016/j.stem.2010.11.018

    View details for Web of Science ID 000285537700006

    View details for PubMedID 21112559

  • G1 arrest and differentiation can occur independently of Rb family function JOURNAL OF CELL BIOLOGY Wirt, S. E., Adler, A. S., Gebala, V., Weimann, J. M., Schaffer, B. E., Saddic, L. A., Viatour, P., Vogel, H., Chang, H. Y., Meissner, A., Sage, J. 2010; 191 (4): 809-825

    Abstract

    The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9-11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.

    View details for DOI 10.1083/jcb.201003048

    View details for Web of Science ID 000284737200014

    View details for PubMedID 21059851

  • Genome-wide measurement of RNA secondary structure in yeast NATURE Kertesz, M., Wan, Y., Mazor, E., Rinn, J. L., Nutter, R. C., Chang, H. Y., Segal, E. 2010; 467 (7311): 103-107

    Abstract

    The structures of RNA molecules are often important for their function and regulation, yet there are no experimental techniques for genome-scale measurement of RNA structure. Here we describe a novel strategy termed parallel analysis of RNA structure (PARS), which is based on deep sequencing fragments of RNAs that were treated with structure-specific enzymes, thus providing simultaneous in vitro profiling of the secondary structure of thousands of RNA species at single nucleotide resolution. We apply PARS to profile the secondary structure of the messenger RNAs (mRNAs) of the budding yeast Saccharomyces cerevisiae and obtain structural profiles for over 3,000 distinct transcripts. Analysis of these profiles reveals several RNA structural properties of yeast transcripts, including the existence of more secondary structure over coding regions compared with untranslated regions, a three-nucleotide periodicity of secondary structure across coding regions and an anti-correlation between the efficiency with which an mRNA is translated and the structure over its translation start site. PARS is readily applicable to other organisms and to profiling RNA structure in diverse conditions, thus enabling studies of the dynamics of secondary structure at a genomic scale.

    View details for DOI 10.1038/nature09322

    View details for Web of Science ID 000281461200044

    View details for PubMedID 20811459

  • Long noncoding RNA in genome regulation Prospects and mechanisms RNA BIOLOGY Hung, T., Chang, H. Y. 2010; 7 (5): 582-585

    Abstract

    Long noncoding RNAs (lncRNAs) are pervasively transcribed and critical regulators of the epigenome[1, 2]. These long, polyadenylated RNAs do not code for proteins, but function directly as RNAs, recruiting chromatin modifiers to mediate transcriptional changes in processes ranging from X-inactivation (XIST) to imprinting (H19)[3]. The recent discovery that lncRNA HOTAIR can link chromatin changes to cancer metastasis[4] furthers the relevance of lncRNAs to human disease. Here, we discuss lncRNAs as regulatory modules and explore the implications for disease pathogenesis. Although large-scale analyses of mammalian transcriptomes have revealed that more than 50% of transcripts have no protein coding potential[2, 5, 6], the functions of these putative transcripts are largely unknown. A subset of these noncoding transcripts are termed long noncoding RNAs (lncRNAs), based on an arbitrary minimum length of 200 nucleotides. LncRNAs are roughly classified based on their position relative to protein-coding genes: intergenic (between genes), intragenic/intronic (within genes), and antisense[2]. Initial efforts to characterize these molecules demonstrated that they function in cis, regulating their immediate genomic neighbors. Examples include AIR, XIST, and Kcnq1ot (reviewed in [1, 7, 8]), which recruit chromatin modifying complexes to silence adjacent sites. The scope of lncRNAs in gene regulation was advanced with the finding that lncRNA HOTAIR exhibited trans regulatory capacities. HOTAIR is transcribed at the intersection of opposing chromatin domains in the HOXC locus, but targets Polycomb Repressive Complex 2 (PRC2) to silence 40 kilobases of HOXD[9], a locus involved in developmental patterning. A subsequent study revealed that HOTAIR is overexpressed in approximately one quarter of human breast cancers, directing PRC2 to approximately 800 ectopic sites in the genome, which leads to histone H3 lysine 27 trimethylation and changes in gene expression[4]. The impacts of lncRNA-mediated chromatin changes are noteworthy: not only did HOTAIR drive metastasis in a mouse model, but HOTAIR expression in human breast cancer was found to be an independent prognostic marker for death and metastasis[4]. The fact that HOTAIR drives chromatin reprogramming genome-wide suggests that long-range regulation by lncRNAs may be a widespread mechanism. This is supported by a study showing that > 20% of tested lncRNAs are bound by PRC2 and other chromatin modifiers[10]. Furthermore, this is an underestimate of the total RNAs involved in chromatin modification, as PRC2 target genes also transcribe smaller 50-200 nt RNAs that interact with SUZ12 to mediate gene repression[11]. These findings provoke questions regarding the initial triggers for HOTAIR overexpression and whether understanding of lncRNA mechanics may have clinical relevance.

    View details for Web of Science ID 000288456500014

    View details for PubMedID 20930520

  • HOTAIR Flight of noncoding RNAs in cancer metastasis CELL CYCLE Wan, Y., Chang, H. Y. 2010; 9 (17): 3391-3392

    View details for DOI 10.4161/cc.9.17.13122

    View details for Web of Science ID 000281621700004

    View details for PubMedID 20864820

  • Long Noncoding RNA as Modular Scaffold of Histone Modification Complexes SCIENCE Tsai, M., Manor, O., Wan, Y., Mosammaparast, N., Wang, J. K., Lan, F., Shi, Y., Segal, E., Chang, H. Y. 2010; 329 (5992): 689-693

    Abstract

    Long intergenic noncoding RNAs (lincRNAs) regulate chromatin states and epigenetic inheritance. Here, we show that the lincRNA HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5' domain of HOTAIR binds polycomb repressive complex 2 (PRC2), whereas a 3' domain of HOTAIR binds the LSD1/CoREST/REST complex. The ability to tether two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1 and coordinates targeting of PRC2 and LSD1 to chromatin for coupled histone H3 lysine 27 methylation and lysine 4 demethylation. Our results suggest that lincRNAs may serve as scaffolds by providing binding surfaces to assemble select histone modification enzymes, thereby specifying the pattern of histone modifications on target genes.

    View details for DOI 10.1126/science.1192002

    View details for Web of Science ID 000280602700041

    View details for PubMedID 20616235

  • Tumor suppression by the histone demethylase UTX CELL CYCLE Tsai, M., Wang, J. K., Chang, H. Y. 2010; 9 (11): 2043-2044

    View details for Web of Science ID 000279148400001

    View details for PubMedID 20505331

  • Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis NATURE Gupta, R. A., Shah, N., Wang, K. C., Kim, J., Horlings, H. M., Wong, D. J., Tsai, M., Hung, T., Argani, P., Rinn, J. L., Wang, Y., Brzoska, P., Kong, B., Li, R., West, R. B., van de Vijver, M. J., Sukumar, S., Chang, H. Y. 2010; 464 (7291): 1071-U148

    Abstract

    Large intervening non-coding RNAs (lincRNAs) are pervasively transcribed in the genome yet their potential involvement in human disease is not well understood. Recent studies of dosage compensation, imprinting, and homeotic gene expression suggest that individual lincRNAs can function as the interface between DNA and specific chromatin remodelling activities. Here we show that lincRNAs in the HOX loci become systematically dysregulated during breast cancer progression. The lincRNA termed HOTAIR is increased in expression in primary breast tumours and metastases, and HOTAIR expression level in primary tumours is a powerful predictor of eventual metastasis and death. Enforced expression of HOTAIR in epithelial cancer cells induced genome-wide re-targeting of Polycomb repressive complex 2 (PRC2) to an occupancy pattern more resembling embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness and metastasis in a manner dependent on PRC2. Conversely, loss of HOTAIR can inhibit cancer invasiveness, particularly in cells that possess excessive PRC2 activity. These findings indicate that lincRNAs have active roles in modulating the cancer epigenome and may be important targets for cancer diagnosis and therapy.

    View details for DOI 10.1038/nature08975

    View details for Web of Science ID 000276635000045

    View details for PubMedID 20393566

  • A TGF beta-Responsive Gene Signature Is Associated with a Subset of Diffuse Scleroderma with Increased Disease Severity JOURNAL OF INVESTIGATIVE DERMATOLOGY Sargent, J. L., Milano, A., Bhattacharyya, S., Varga, J., Connolly, M. K., Chang, H. Y., Whitfield, M. L. 2010; 130 (3): 694-705

    Abstract

    Systemic sclerosis is a complex disease with widespread skin fibrosis and variable visceral organ involvement. Since transforming growth factor-beta (TGFbeta) has been implicated in driving fibrosis in systemic sclerosis, a mechanism-derived gene expression signature was used to assay TGFbeta-responsive gene expression in the skin of patients with systemic sclerosis (SSc). Primary dermal fibroblasts from patients with diffuse SSc (dSSc) and healthy controls were treated with TGFbeta, and the genome-wide gene expression was measured on DNA microarrays over a time course of 24 hours. Eight hundred and ninety-four probes representing 674 uniquely annotated genes were identified as TGFbeta responsive. Expression of the TGFbeta-responsive signature was examined in skin biopsies from 17 dSSc, seven limited SSc (lSSc), three morphea patients, and six healthy controls. The TGFbeta-responsive signature was expressed in 10 out of 17 dSSc skin biopsies, but was not found in lSSc, morphea, or healthy control biopsies. Expression of dSSC the TGFbeta-responsive signature stratifies patients into two major groups, one of which corresponds to the "diffuse-proliferation" intrinsic subset that showed higher modified Rodnan skin score and a higher likelihood of scleroderma lung disease. The TGFbeta-responsive signature is found in only a subset of dSSc patients who could be targeted by specific therapies.

    View details for DOI 10.1038/jid.2009.318

    View details for Web of Science ID 000275017600013

    View details for PubMedID 19812599

  • The histone demethylase UTX enables RB-dependent cell fate control GENES & DEVELOPMENT Wang, J. K., Tsai, M., Poulin, G., Adler, A. S., Chen, S., Liu, H., Shi, Y., Chang, H. Y. 2010; 24 (4): 327-332

    Abstract

    Trimethylation of histone H3 on Lys 27 (H3K27me3) is key for cell fate regulation. The H3K27me3 demethylase UTX functions in development and tumor suppression with undefined mechanisms. Here, genome-wide chromatin occupancy analysis of UTX and associated histone modifications reveals distinct classes of UTX target genes, including genes encoding Retinoblastoma (RB)-binding proteins. UTX removes H3K27me3 and maintains expression of several RB-binding proteins, enabling cell cycle arrest. Genetic interactions in mammalian cells and Caenorhabditis elegans show that UTX regulates cell fates via RB-dependent pathways. Thus, UTX defines an evolutionarily conserved mechanism to enable coordinate transcription of a RB network in cell fate control.

    View details for DOI 10.1101/gad.1882610

    View details for Web of Science ID 000274577100001

    View details for PubMedID 20123895

  • Anatomic Demarcation of Cells: Genes to Patterns SCIENCE Chang, H. Y. 2009; 326 (5957): 1206-1207

    Abstract

    An organizing principle of the diverse cell types in multicellular organisms is their anatomic location. In turn, anatomic location is patterned by the positional identities of cells along developmental axes. Recent progress in functional genomics and chromatin biology illustrates how cells use specific gene expression programs to encode location. Dynamic chromatin states of key genes, notably the Hox loci, serve as the internal representation in cells of their positional identity within the animal.

    View details for DOI 10.1126/science.1175686

    View details for Web of Science ID 000272117900032

    View details for PubMedID 19965461

  • Identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, K. S., Espinosa, I., Chao, M., Wong, D., Ailles, L., Diehn, M., Gill, H., Presti, J., Chang, H. Y., van de Rijn, M., Shortliffe, L., Weissman, I. L. 2009; 106 (33): 14016-14021

    Abstract

    Major clinical issues in bladder cancer include the identification of prediction markers and novel therapeutic targets for invasive bladder cancer. In the current study, we describe the isolation and characterization of a tumor-initiating cell (T-IC) subpopulation in primary human bladder cancer, based on the expression of markers similar to that of normal bladder basal cells (Lineage-CD44(+)CK5(+)CK20(-)). The bladder T-IC subpopulation was defined functionally by its enriched ability to induce xenograft tumors in vivo that recapitulated the heterogeneity of the original tumor. Further, molecular analysis of more than 300 bladder cancer specimens revealed heterogeneity among activated oncogenic pathways in T-IC (e.g., 80% Gli1, 45% Stat3, 10% Bmi-1, and 5% beta-catenin). Despite this molecular heterogeneity, we identified a unique bladder T-IC gene signature by gene chip analysis. This T-IC gene signature, which effectively distinguishes muscle-invasive bladder cancer with worse clinical prognosis from non-muscle-invasive (superficial) cancer, has significant clinical value. It also can predict the progression of a subset of recurring non-muscle-invasive cancers. Finally, we found that CD47, a protein that provides an inhibitory signal for macrophage phagocytosis, is highly expressed in bladder T-ICs compared with the rest of the tumor. Blockade of CD47 by a mAb resulted in macrophage engulfment of bladder cancer cells in vitro. In summary, we have identified a T-IC subpopulation with potential prognostic and therapeutic value for invasive bladder cancer.

    View details for DOI 10.1073/pnas.0906549106

    View details for Web of Science ID 000269078700071

    View details for PubMedID 19666525

  • Gene dates, parties and galas Symposium on Chromatin Dynamics and Higher Order Organization EMBO REPORTS Chang, H. Y., Cuvier, O., Dekker, J. 2009; 10 (7): 689-693

    View details for DOI 10.1038/embor.2009.136

    View details for Web of Science ID 000267599700009

    View details for PubMedID 19525922

  • Tumor Vasculature Is Regulated by PHD2-Mediated Angiogenesis and Bone Marrow-Derived Cell Recruitment CANCER CELL Chan, D. A., Kawahara, T. L., Sutphin, P. D., Chang, H. Y., Chi, J., Giaccia, A. J. 2009; 15 (6): 527-538

    Abstract

    Sustained angiogenesis, through either local sprouting (angiogenesis) or the recruitment of bone marrow-derived cells (BMDCs) (vasculogenesis), is essential to the development of a tumor. How BMDCs are recruited to the tumor and their contribution to the tumor vasculature is poorly understood. Here, we demonstrate that both IL-8 and angiogenin contribute to the complementary pathways of angiogenesis and BMDC mobilization to increase tumor growth. These two factors are regulated by PHD2 in a HIF-independent but NF-kappaB-dependent manner. PHD2 levels are decreased in human cancers, compared with corresponding normal tissue, and correlate with an increase in mature blood vessels. Thus, PHD2 plays a critical role in regulating tumor angiogenesis.

    View details for DOI 10.1016/j.ccr.2009.04.010

    View details for Web of Science ID 000266686500010

    View details for PubMedID 19477431

  • Modeling Inducible Human Tissue Neoplasia Identifies an Extracellular Matrix Interaction Network Involved in Cancer Progression CANCER CELL Reuter, J. A., Ortiz-Urda, S., Kretz, M., Garcia, J., Scholl, F. A., Pasmooij, A. M., Cassarino, D., Chang, H. Y., Khavari, P. A. 2009; 15 (6): 477-488

    Abstract

    To elucidate mechanisms of cancer progression, we generated inducible human neoplasia in three-dimensionally intact epithelial tissue. Gene expression profiling of both epithelia and stroma at specific time points during tumor progression revealed sequential enrichment of genes mediating discrete biologic functions in each tissue compartment. A core cancer progression signature was distilled using the increased signaling specificity of downstream oncogene effectors and subjected to network modeling. Network topology predicted that tumor development depends on specific extracellular matrix-interacting network hubs. Blockade of one such hub, the beta1 integrin subunit, disrupted network gene expression and attenuated tumorigenesis in vivo. Thus, integrating network modeling and temporal gene expression analysis of inducible human neoplasia provides an approach to prioritize and characterize genes functioning in cancer progression.

    View details for DOI 10.1016/j.ccr.2009.04.002

    View details for Web of Science ID 000266686500006

    View details for PubMedID 19477427

  • Regeneration, repair and remembering identity: the three Rs of Hox gene expression TRENDS IN CELL BIOLOGY Wang, K. C., Helms, J. A., Chang, H. Y. 2009; 19 (6): 268-275

    Abstract

    Hox genes encode transcription factors that specify embryonic positional identity in cells and guide tissue differentiation. Recent advances have greatly increased our understanding of the epigenetic mechanisms that ensure the faithful expression of Hox genes in adult cells and which involve the interplay of histone methylation, demethylation and intergenic transcription of long non-coding RNAs. The transcriptional memory of Hox genes poses both an opportunity and a challenge for regenerative medicine. Matching the positional identity of transplanted stem cells with that of the host environment, as reflected by their respective Hox profiles, is likely to be required to achieve regenerative healing. Strategies to manipulate the plasticity of Hox gene expression will probably become a major focus in regenerative medicine.

    View details for DOI 10.1016/j.tcb.2009.03.007

    View details for Web of Science ID 000267449500004

    View details for PubMedID 19428253

  • Hierarchical Maintenance of MLL Myeloid Leukemia Stem Cells Employs a Transcriptional Program Shared with Embryonic Rather Than Adult Stem Cells CELL STEM CELL Somervaille, T. C., Matheny, C. J., Spencer, G. J., Iwasaki, M., Rinn, J. L., Witten, D. M., Chang, H. Y., Shurtleff, S. A., Downing, J. R., Cleary, M. L. 2009; 4 (2): 129-140

    Abstract

    The genetic programs that promote retention of self-renewing leukemia stem cells (LSCs) at the apex of cellular hierarchies in acute myeloid leukemia (AML) are not known. In a mouse model of human AML, LSCs exhibit variable frequencies that correlate with the initiating MLL oncogene and are maintained in a self-renewing state by a transcriptional subprogram more akin to that of embryonic stem cells (ESCs) than to that of adult stem cells. The transcription/chromatin regulatory factors Myb, Hmgb3, and Cbx5 are critical components of the program and suffice for Hoxa/Meis-independent immortalization of myeloid progenitors when coexpressed, establishing the cooperative and essential role of an ESC-like LSC maintenance program ancillary to the leukemia-initiating MLL/Hox/Meis program. Enriched expression of LSC maintenance and ESC-like program genes in normal myeloid progenitors and poor-prognosis human malignancies links the frequency of aberrantly self-renewing progenitor-like cancer stem cells (CSCs) to prognosis in human cancer.

    View details for DOI 10.1016/j.stem.2008.11.015

    View details for Web of Science ID 000263213400010

    View details for PubMedID 19200802

  • Molecular Framework for Response to Imatinib Mesylate in Systemic Sclerosis ARTHRITIS AND RHEUMATISM Chung, L., Fiorentino, D. F., Benbarak, M. J., Adler, A. S., Mariano, M. M., Paniagua, R. T., Milano, A., Connolly, M. K., Ratiner, B. D., Wiskocil, R. L., Whitfield, M. L., Chang, H. Y., Robinson, W. H. 2009; 60 (2): 584-591

    Abstract

    Systemic sclerosis (SSc) is an autoimmune disease in which the tyrosine kinases platelet-derived growth factor receptor (PDGFR) and Abl are hypothesized to contribute to the fibrosis and vasculopathy of the skin and internal organs. Herein we describe 2 patients with early diffuse cutaneous SSc (dcSSc) who experienced reductions in cutaneous sclerosis in response to therapy with the tyrosine kinase inhibitor imatinib mesylate. Immunohistochemical analyses of skin biopsy specimens demonstrated reductions of phosphorylated PDGFRbeta and Abl with imatinib therapy. By gene expression profiling, an imatinib-responsive signature specific to dcSSc was identified (P < 10(-8)). The response of these patients and the findings of the analyses suggest that PDGFRbeta and Abl play critical, synergistic roles in the pathogenesis of SSc, and that imatinib targets a gene expression program that is frequently dysregulated in dcSSc.

    View details for DOI 10.1002/art.24221

    View details for Web of Science ID 000263276400032

    View details for PubMedID 19180499

  • ING4 Mediates Crosstalk between Histone H3 K4 Trimethylation and H3 Acetylation to Attenuate Cellular Transformation MOLECULAR CELL Hung, T., Binda, O., Champagne, K. S., Kuo, A. J., Johnson, K., Chang, H. Y., Simon, M. D., Kutateladze, T. G., Gozani, O. 2009; 33 (2): 248-256

    Abstract

    Aberrations in chromatin dynamics play a fundamental role in tumorigenesis, yet relatively little is known of the molecular mechanisms linking histone lysine methylation to neoplastic disease. ING4 (Inhibitor of Growth 4) is a native subunit of an HBO1 histone acetyltransferase (HAT) complex and a tumor suppressor protein. Here we show a critical role for specific recognition of histone H3 trimethylated at lysine 4 (H3K4me3) by the ING4 PHD finger in mediating ING4 gene expression and tumor suppressor functions. The interaction between ING4 and H3K4me3 augments HBO1 acetylation activity on H3 tails and drives H3 acetylation at ING4 target promoters. Further, ING4 facilitates apoptosis in response to genotoxic stress and inhibits anchorage-independent cell growth, and these functions depend on ING4 interactions with H3K4me3. Together, our results demonstrate a mechanism for brokering crosstalk between H3K4 methylation and H3 acetylation and reveal a molecular link between chromatin modulation and tumor suppressor mechanisms.

    View details for DOI 10.1016/j.molcel.2008.12.016

    View details for Web of Science ID 000263204500015

    View details for PubMedID 19187765

  • SIRT6 Links Histone H3 Lysine 9 Deacetylation to NF-kappa B-Dependent Gene Expression and Organismal Life Span CELL Kawahara, T. L., Michishita, E., Adler, A. S., Damian, M., Berber, E., Lin, M., McCord, R. A., Ongaigui, K. C., Boxer, L. D., Chang, H. Y., Chua, K. F. 2009; 136 (1): 62-74

    Abstract

    Members of the sirtuin (SIRT) family of NAD-dependent deacetylases promote longevity in multiple organisms. Deficiency of mammalian SIRT6 leads to shortened life span and an aging-like phenotype in mice, but the underlying molecular mechanisms are unclear. Here we show that SIRT6 functions at chromatin to attenuate NF-kappaB signaling. SIRT6 interacts with the NF-kappaB RELA subunit and deacetylates histone H3 lysine 9 (H3K9) at NF-kappaB target gene promoters. In SIRT6-deficient cells, hyperacetylation of H3K9 at these target promoters is associated with increased RELA promoter occupancy and enhanced NF-kappaB-dependent modulation of gene expression, apoptosis, and cellular senescence. Computational genomics analyses revealed increased activity of NF-kappaB-driven gene expression programs in multiple Sirt6-deficient tissues in vivo. Moreover, haploinsufficiency of RelA rescues the early lethality and degenerative syndrome of Sirt6-deficient mice. We propose that SIRT6 attenuates NF-kappaB signaling via H3K9 deacetylation at chromatin, and hyperactive NF-kappaB signaling may contribute to premature and normal aging.

    View details for DOI 10.1016/j.cell.2008.10.052

    View details for Web of Science ID 000262318400015

    View details for PubMedID 19135889

  • Genome-Wide Views of Chromatin Structure ANNUAL REVIEW OF BIOCHEMISTRY Rando, O. J., Chang, H. Y. 2009; 78: 245-271

    Abstract

    Eukaryotic genomes are packaged into a nucleoprotein complex known as chromatin, which affects most processes that occur on DNA. Along with genetic and biochemical studies of resident chromatin proteins and their modifying enzymes, mapping of chromatin structure in vivo is one of the main pillars in our understanding of how chromatin relates to cellular processes. In this review, we discuss the use of genomic technologies to characterize chromatin structure in vivo, with a focus on data from budding yeast and humans. The picture emerging from these studies is the detailed chromatin structure of a typical gene, where the typical behavior gives insight into the mechanisms and deep rules that establish chromatin structure. Important deviation from the archetype is also observed, usually as a consequence of unique regulatory mechanisms at special genomic loci. Chromatin structure shows substantial conservation from yeast to humans, but mammalian chromatin has additional layers of complexity that likely relate to the requirements of multicellularity such as the need to establish faithful gene regulatory mechanisms for cell differentiation.

    View details for DOI 10.1146/annurev.biochem.78.071107.134639

    View details for Web of Science ID 000268069200011

    View details for PubMedID 19317649

  • Global Expression Profiling in Atopic Eczema Reveals Reciprocal Expression of Inflammatory and Lipid Genes PLOS ONE Saaf, A. M., Tengvall-Linder, M., Chang, H. Y., Adler, A. S., Wahlgren, C., Scheynius, A., Nordenskjold, M., Bradley, M. 2008; 3 (12)

    Abstract

    Atopic eczema (AE) is a common chronic inflammatory skin disorder. In order to dissect the genetic background several linkage and genetic association studies have been performed. Yet very little is known about specific genes involved in this complex skin disease, and the underlying molecular mechanisms are not fully understood.We used human DNA microarrays to identify a molecular picture of the programmed responses of the human genome to AE. The transcriptional program was analyzed in skin biopsy samples from lesional and patch-tested skin from AE patients sensitized to Malassezia sympodialis (M. sympodialis), and corresponding biopsies from healthy individuals. The most notable feature of the global gene-expression pattern observed in AE skin was a reciprocal expression of induced inflammatory genes and repressed lipid metabolism genes. The overall transcriptional response in M. sympodialis patch-tested AE skin was similar to the gene-expression signature identified in lesional AE skin. In the constellation of genes differentially expressed in AE skin compared to healthy control skin, we have identified several potential susceptibility genes that may play a critical role in the pathological condition of AE. Many of these genes, including genes with a role in immune responses, lipid homeostasis, and epidermal differentiation, are localized on chromosomal regions previously linked to AE.Through genome-wide expression profiling, we were able to discover a distinct reciprocal expression pattern of induced inflammatory genes and repressed lipid metabolism genes in skin from AE patients. We found a significant enrichment of differentially expressed genes in AE with cytobands associated to the disease, and furthermore new chromosomal regions were found that could potentially guide future region-specific linkage mapping in AE. The full data set is available at http://microarray-pubs.stanford.edu/eczema.

    View details for DOI 10.1371/journal.pone.0004017

    View details for Web of Science ID 000265463000012

    View details for PubMedID 19107207

  • Stemness, cancer and cancer stem cells CELL CYCLE Wong, D. J., Segal, E., Chang, H. Y. 2008; 7 (23): 3622-3624

    Abstract

    The ability of cancers to grow indefinitely has fueled the idea that cancer and stem cells may have common underlying mechanisms. Detailed gene expression maps have now shown the diversity and distinctiveness in gene expression programs associated with stemness in embryonic and adult stem cells. These maps have further revealed a shared transcriptional program in embryonic stem cells (ESC) and cancer stem cells. Surprisingly, forced activation of an ESC-like gene expression program in adult epithelial cells can reprogram them into human cancer stem cells and achieve pathologic self-renewal. The ability to create induced cancer stem cells (iCSC) may provide opportunities to better define the biology of cancer stem cells in order to trace or eliminate them in human patients.

    View details for Web of Science ID 000261260100003

    View details for PubMedID 19029796

  • Combining biological gene expression signatures in predicting outcome in breast cancer: An alternative to supervised classification EUROPEAN JOURNAL OF CANCER Nuyten, D. S., Hastie, T., Chi, J. A., Chang, H. Y., van de Vijver, M. J. 2008; 44 (15): 2319-2329

    Abstract

    Gene expression profiling has been extensively used to predict outcome in breast cancer patients. We have previously reported on biological hypothesis-driven analysis of gene expression profiling data and we wished to extend this approach through the combinations of various gene signatures to improve the prediction of outcome in breast cancer.We have used gene expression data (25.000 gene probes) from a previously published study of tumours from 295 early stage breast cancer patients from the Netherlands Cancer Institute using updated follow-up. Tumours were assigned to three prognostic groups using the previously reported Wound-response and hypoxia-response signatures, and the outcome in each of these subgroups was evaluated.We have assigned invasive breast carcinomas from 295 stages I and II breast cancer patients to three groups based on gene expression profiles subdivided by the wound-response signature (WS) and hypoxia-response signature (HS). These three groups are (1) quiescent WS/non-hypoxic HS; (2) activated WS/non-hypoxic HS or quiescent WS/hypoxic tumours and (3) activated WS/hypoxic HS. The overall survival at 15 years for patients with tumours in groups 1, 2 and 3 are 79%, 59% and 27%, respectively. In multivariate analysis, this signature is not only independent of clinical and pathological risk factors; it is also the strongest predictor of outcome. Compared to a previously identified 70-gene prognosis profile, obtained with supervised classification, the combination of signatures performs roughly equally well and might have additional value in the ER-negative subgroup. In the subgroup of lymph node positive patients, the combination signature outperforms the 70-gene signature in multivariate analysis. In addition, in multivariate analysis, the WS/HS combination is a stronger predictor of outcome compared to the recently reported invasiveness gene signature combined with the WS.A combination of biological gene expression signatures can be used to identify a powerful and independent predictor for outcome in breast cancer patients.

    View details for DOI 10.1016/j.ejca.2008.07.015

    View details for Web of Science ID 000261020800031

    View details for PubMedID 18715778

  • Deletional tolerance mediated by extrathymic Aire-expressing cells SCIENCE Gardner, J. M., DeVoss, J. J., Friedman, R. S., Wong, D. J., Tan, Y. X., Zhou, X., Johannes, K. P., Su, M. A., Chang, H. Y., Krummel, M. F., Anderson, M. S. 2008; 321 (5890): 843-847

    Abstract

    The prevention of autoimmunity requires the elimination of self-reactive T cells during their development and maturation. The expression of diverse self-antigens by stromal cells in the thymus is essential to this process and depends, in part, on the activity of the autoimmune regulator (Aire) gene. Here we report the identification of extrathymic Aire-expressing cells (eTACs) resident within the secondary lymphoid organs. These stromally derived eTACs express a diverse array of distinct self-antigens and are capable of interacting with and deleting naïve autoreactive T cells. Using two-photon microscopy, we observed stable antigen-specific interactions between eTACs and autoreactive T cells. We propose that such a secondary network of self-antigen-expressing stromal cells may help reinforce immune tolerance by preventing the maturation of autoreactive T cells that escape thymic negative selection.

    View details for DOI 10.1126/science.1159407

    View details for Web of Science ID 000258261000048

    View details for PubMedID 18687966

  • Control of differentiation in a self-renewing mammalian tissue by the histone demethylase JMJD3 GENES & DEVELOPMENT Sen, G. L., Webster, D. E., Barragan, D. I., Chang, H. Y., Khavari, P. A. 2008; 22 (14): 1865-1870

    Abstract

    The recent discovery of H3K27me3 demethylases suggests that H3K27me3 may dynamically regulate gene expression, but this potential role in mammalian tissue homeostasis remains uncharacterized. In the epidermis, a tissue that balances stem cell self-renewal with differentiation, H3K27me3, occupies the promoters of many differentiation genes. During calcium-induced differentiation, H3K27me3 was erased at these promoters in concert with loss of PcG protein occupancy and increased binding by the H3K27me3 demethylase, JMJD3. Within epidermal tissue, JMJD3 depletion blocked differentiation, while active JMJD3 dominantly induced it. These results indicate that epigenetic derepression by JMJD3 controls mammalian epidermal differentiation.

    View details for DOI 10.1101/gad.1673508

    View details for Web of Science ID 000257643400003

    View details for PubMedID 18628393

  • Mechanisms of an autoimmunity syndrome in mice caused by a dominant mutation in Aire JOURNAL OF CLINICAL INVESTIGATION Su, M. A., Giang, K., Zumer, K., Jiang, H., Oven, I., Rinn, J. L., DeVoss, J. J., Johannes, K. P., Lu, W., Gardner, J., Chang, A., Bubulya, P., Chang, H. Y., Peterlin, B. M., Anderson, M. S. 2008; 118 (5): 1712-1726

    Abstract

    Homozygous loss-of-function mutations in AIRE cause autoimmune polyglandular syndrome type 1 (APS 1), which manifests in a classic triad of hypoparathyroidism, adrenal insufficiency, and candidiasis. Interestingly, a kindred with a specific G228W AIRE variant presented with an autosomal dominant autoimmune phenotype distinct from APS 1. We utilized a novel G228W-knockin mouse model to show that this variant acted in a dominant-negative manner to cause a unique autoimmunity syndrome. In addition, the expression of a large number of Aire-regulated thymic antigens was partially inhibited in these animals, demonstrating the importance of quantitative changes in thymic antigen expression in determining organ-specific autoimmunity. Furthermore, the dominant-negative effect of the G228W variant was exerted through recruitment of WT Aire away from active sites of transcription in the nucleus of medullary thymic epithelial cells in vivo. Together, these results may demonstrate a mechanism by which autoimmune predisposition to phenotypes distinct from APS 1 can be mediated in a dominant-negative fashion by Aire.

    View details for DOI 10.1172/JCI34523

    View details for Web of Science ID 000255490100016

    View details for PubMedID 18414681

  • Module map of stem cell genes guides creation of epithelial cancer stem cells CELL STEM CELL Wong, D. J., Liu, H., Ridky, T. W., Cassarino, D., Segal, E., Chang, H. Y. 2008; 2 (4): 333-344

    Abstract

    Self-renewal is a hallmark of stem cells and cancer, but existence of a shared stemness program remains controversial. Here, we construct a gene module map to systematically relate transcriptional programs in embryonic stem cells (ESCs), adult tissue stem cells, and human cancers. This map reveals two predominant gene modules that distinguish ESCs and adult tissue stem cells. The ESC-like transcriptional program is activated in diverse human epithelial cancers and strongly predicts metastasis and death. c-Myc, but not other oncogenes, is sufficient to reactivate the ESC-like program in normal and cancer cells. In primary human keratinocytes transformed by Ras and I kappa B alpha, c-Myc increases the fraction of tumor-initiating cells by 150-fold, enabling tumor formation and serial propagation with as few as 500 cells. c-Myc-enhanced tumor initiation is cell-autonomous and independent of genomic instability. Thus, activation of an ESC-like transcriptional program in differentiated adult cells may induce pathologic self-renewal characteristic of cancer stem cells.

    View details for DOI 10.1016/j.stem.2008.02.009

    View details for Web of Science ID 000255327000010

    View details for PubMedID 18397753

  • A systems biology approach to anatomic diversity of skin JOURNAL OF INVESTIGATIVE DERMATOLOGY Rinn, J. L., Wang, J. K., Liu, H., Montgomery, K., van de Rijn, M., Chang, H. Y. 2008; 128 (4): 776-782

    Abstract

    Human skin exhibits exquisite site-specific morphologies and functions. How are these site-specific differences specified during development, maintained in adult homeostasis, and potentially perturbed by disease processes? Here, we review progress in understanding the anatomic patterning of fibroblasts, a major constituent cell type of the dermis and key participant in epithelial-mesenchymal interactions. The gene expression programs of human fibroblasts largely reflect the superimposition of three gene expression profiles that demarcate the fibroblast's position relative to three developmental axes. The HOX family of homeodomain transcription factors is implicated in specifying site-specific transcriptional programs. The use of gene, tiling, and tissue microarrays together gives a comprehensive view of the gene regulation involved in patterning the skin.

    View details for DOI 10.1038/sj.jid.5700986

    View details for Web of Science ID 000254330300005

    View details for PubMedID 18337710

  • SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin NATURE Michishita, E., McCord, R. A., Berber, E., Kioi, M., Padilla-Nash, H., Damian, M., Cheung, P., Kusumoto, R., Kawahara, T. L., Barrett, J. C., Chang, H. Y., Bohr, V. A., Ried, T., Gozani, O., Chua, K. F. 2008; 452 (7186): 492-U16

    Abstract

    The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.

    View details for DOI 10.1038/nature06736

    View details for Web of Science ID 000254341300036

    View details for PubMedID 18337721

  • Reversal of aging by NF kappa B blockade CELL CYCLE Adler, A. S., Kawahara, T. L., Segal, E., Chang, H. Y. 2008; 7 (5): 556-559

    Abstract

    Genetic studies in model organisms such as yeast, worms, flies, and mice leading to lifespan extension suggest that longevity is subject to regulation. In addition, various system-wide interventions in old animals can reverse features of aging. To better understand these processes, much effort has been put into the study of aging on a molecular level. In particular, genome-wide microarray analysis of differently aged individual organisms or tissues has been used to track the global expression changes that occur during normal aging. Although these studies consistently implicate specific pathways in aging processes, there is little conservation between the individual genes that change. To circumvent this problem, we have recently developed a novel computational approach to discover transcription factors that may be responsible for driving global expression changes with age. We identified the transcription factor NFkappaB as a candidate activator of aging-related transcriptional changes in multiple human and mouse tissues. Genetic blockade of NFkappaB in the skin of chronologically aged mice reversed the global gene expression program and tissue characteristics to those of young mice, demonstrating for the first time that disruption of a single gene is sufficient to reverse features of aging, at least for the short-term.

    View details for Web of Science ID 000254365700002

    View details for PubMedID 18256548

  • Systematic functional characterization of cis-regulatory motifs in human core promoters GENOME RESEARCH Sinha, S., Adler, A. S., Field, Y., Chang, H. Y., Segal, E. 2008; 18 (3): 477-488

    Abstract

    A large number of cis-regulatory motifs involved in transcriptional control have been identified, but the regulatory context and biological processes in which many of them function are unknown. Here, we computationally identify the sets of human core promoters targeted by motifs, and systematically characterize their function by using a robust gene-set-based approach and diverse sources of biological data. We find that the target sets of most motifs contain both genes with similar function and genes that are coregulated in vivo, thereby suggesting both the biological process regulated by the motifs and the conditions in which this regulation may occur. Our analysis also identifies many motifs whose target sets are predicted to be regulated by a common microRNA, suggesting a connection between transcriptional and post-transcriptional control processes. Finally, we predict novel roles for uncharacterized motifs in the regulation of specific biological processes and certain types of human cancer, and experimentally validate four such predictions, suggesting regulatory roles for four uncharacterized motifs in cell cycle progression. Our analysis thus provides a concrete framework for uncovering the biological function of cis-regulatory motifs genome wide.

    View details for DOI 10.1101/gr.6828808

    View details for Web of Science ID 000253766700014

    View details for PubMedID 18256240

  • A dermal HOX transcriptional program regulates site-specific epidermal fate GENES & DEVELOPMENT Rinn, J. L., Wang, J. K., Allen, N., Brugmann, S. A., Mikels, A. J., Liu, H., Ridky, T. W., Stadler, H. S., Nusse, R., Helms, J. A., Chang, H. Y. 2008; 22 (3): 303-307

    Abstract

    Reciprocal epithelial-mesenchymal interactions shape site-specific development of skin. Here we show that site-specific HOX expression in fibroblasts is cell-autonomous and epigenetically maintained. The distal-specific gene HOXA13 is continually required to maintain the distal-specific transcriptional program in adult fibroblasts, including expression of WNT5A, a morphogen required for distal development. The ability of distal fibroblasts to induce epidermal keratin 9, a distal-specific gene, is abrogated by depletion of HOXA13, but rescued by addition of WNT5A. Thus, maintenance of appropriate HOX transcriptional program in adult fibroblasts may serve as a source of positional memory to differentially pattern the epithelia during homeostasis and regeneration.

    View details for DOI 10.1101/gad.1610508

    View details for Web of Science ID 000253170400005

    View details for PubMedID 18245445

  • CSN5 isopeptidase activity links COP9 signalosome activation to breast cancer progression CANCER RESEARCH Adler, A. S., Littlepage, L. E., Lin, M., Kawahara, T. L., Wong, D. J., Werb, Z., Chang, H. Y. 2008; 68 (2): 506-515

    Abstract

    CSN5 has been implicated as a candidate oncogene in human breast cancers by genetic linkage with activation of the poor-prognosis, wound response gene expression signature. CSN5 is a subunit of the eight-protein COP9 signalosome, a signaling complex with multiple biochemical activities; the mechanism of CSN5 action in cancer development remains poorly understood. Here, we show that CSN5 isopeptidase activity is essential for breast epithelial transformation and progression. Amplification of CSN5 is required for transformation of primary human breast epithelial cells by defined oncogenes. The transforming effects of CSN5 require CSN subunits for assembly of the full COP9 signalosome and the isopeptidase activity of CSN5, which potentiates the transcriptional activity of MYC. Transgenic inhibition of CSN5 isopeptidase activity blocks breast cancer progression evoked by MYC and RAS in vivo. These results highlight CSN5 isopeptidase activity in breast cancer progression, suggesting it as a therapeutic target in aggressive human breast cancers.

    View details for DOI 10.1158/0008-5472.CAN-07-3060

    View details for Web of Science ID 000252503800023

    View details for PubMedID 18199546

  • Revealing targeted therapy for human cancer by gene module maps CANCER RESEARCH Wong, D. J., Nuyten, D. S., Regev, A., Lin, M., Adler, A. S., Segal, E., van de Vijver, M. J., Chang, H. Y. 2008; 68 (2): 369-378

    Abstract

    A major goal of cancer research is to match specific therapies to molecular targets in cancer. Genome-scale expression profiling has identified new subtypes of cancer based on consistent patterns of variation in gene expression, leading to improved prognostic predictions. However, how these new genetic subtypes of cancers should be treated is unknown. Here, we show that a gene module map can guide the prospective identification of targeted therapies for genetic subtypes of cancer. By visualizing genome-scale gene expression in cancer as combinations of activated and deactivated functional modules, gene module maps can reveal specific functional pathways associated with each subtype that might be susceptible to targeted therapies. We show that in human breast cancers, activation of a poor-prognosis "wound signature" is strongly associated with induction of both a mitochondria gene module and a proteasome gene module. We found that 3-bromopyruvic acid, which inhibits glycolysis, selectively killed breast cells expressing the mitochondria and wound signatures. In addition, inhibition of proteasome activity by bortezomib, a drug approved for human use in multiple myeloma, abrogated wound signature expression and selectively killed breast cells expressing the wound signature. Thus, gene module maps may enable rapid translation of complex genomic signatures in human disease to targeted therapeutic strategies.

    View details for DOI 10.1158/0008-5472.CAN-07-0382

    View details for Web of Science ID 000252503800007

    View details for PubMedID 18199530

  • Motif module map reveals enforcement of aging by continual NF-kappa B activity GENES & DEVELOPMENT Adler, A. S., Sinha, S., Kawahara, T. L., Zhang, J. Y., Segal, E., Chang, H. Y. 2007; 21 (24): 3244-3257

    Abstract

    Aging is characterized by specific alterations in gene expression, but their underlying mechanisms and functional consequences are not well understood. Here we develop a systematic approach to identify combinatorial cis-regulatory motifs that drive age-dependent gene expression across different tissues and organisms. Integrated analysis of 365 microarrays spanning nine tissue types predicted fourteen motifs as major regulators of age-dependent gene expression in human and mouse. The motif most strongly associated with aging was that of the transcription factor NF-kappaB. Inducible genetic blockade of NF-kappaB for 2 wk in the epidermis of chronologically aged mice reverted the tissue characteristics and global gene expression programs to those of young mice. Age-specific NF-kappaB blockade and orthogonal cell cycle interventions revealed that NF-kappaB controls cell cycle exit and gene expression signature of aging in parallel but not sequential pathways. These results identify a conserved network of regulatory pathways underlying mammalian aging and show that NF-kappaB is continually required to enforce many features of aging in a tissue-specific manner.

    View details for DOI 10.1101/gad.1588507

    View details for Web of Science ID 000251627200005

    View details for PubMedID 18055696

  • A histone H3 lysine 27 demethylase regulates animal posterior development NATURE Lan, F., Bayliss, P. E., Rinn, J. L., Whetstine, J. R., Wang, J. K., Chen, S., Iwase, S., Alpatov, R., Issaeva, I., Canaani, E., Roberts, T. M., Chang, H. Y., Shi, Y. 2007; 449 (7163): 689-U3

    Abstract

    The recent discovery of a large number of histone demethylases suggests a central role for these enzymes in regulating histone methylation dynamics. Histone H3K27 trimethylation (H3K27me3) has been linked to polycomb-group-protein-mediated suppression of Hox genes and animal body patterning, X-chromosome inactivation and possibly maintenance of embryonic stem cell (ESC) identity. An imbalance of H3K27 methylation owing to overexpression of the methylase EZH2 has been implicated in metastatic prostate and aggressive breast cancers. Here we show that the JmjC-domain-containing related proteins UTX and JMJD3 catalyse demethylation of H3K27me3/2. UTX is enriched around the transcription start sites of many HOX genes in primary human fibroblasts, in which HOX genes are differentially expressed, but is selectively excluded from the HOX loci in ESCs, in which HOX genes are largely silent. Consistently, RNA interference inhibition of UTX led to increased H3K27me3 levels at some HOX gene promoters. Importantly, morpholino oligonucleotide inhibition of a zebrafish UTX homologue resulted in mis-regulation of hox genes and a striking posterior developmental defect, which was partially rescued by wild-type, but not by catalytically inactive, human UTX. Taken together, these findings identify a small family of H3K27 demethylases with important, evolutionarily conserved roles in H3K27 methylation regulation and in animal anterior-posterior development.

    View details for DOI 10.1038/nature06192

    View details for Web of Science ID 000250045000036

    View details for PubMedID 17851529

  • Turning skin into embryonic stem cells NATURE MEDICINE Chang, H. Y., Cotsarelis, G. 2007; 13 (7): 783-784

    View details for DOI 10.1038/nm0707-783

    View details for Web of Science ID 000247902800016

    View details for PubMedID 17618266

  • Functional demarcation of active and silent chromatin domains in human HOX loci by Noncoding RNAs CELL Rinn, J. L., Kertesz, M., Wang, J. K., Squazzo, S. L., Xu, X., Brugmann, S. A., Goodnough, L. H., Helms, J. A., Farnham, P. J., Segal, E., Chang, H. Y. 2007; 129 (7): 1311-1323

    Abstract

    Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. Here we characterize the transcriptional landscape of the four human HOX loci at five base pair resolution in 11 anatomic sites and identify 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes and possess unique sequence motifs, and their expression demarcates broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. We identified a 2.2 kilobase ncRNA residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states.

    View details for DOI 10.1016/j.cell.2007.05.022

    View details for Web of Science ID 000247911400017

    View details for PubMedID 17604720

  • Decoding global gene expression programs in liver cancer by noninvasive imaging NATURE BIOTECHNOLOGY Segal, E., Sirlin, C. B., Ooi, C., Adler, A. S., Gollub, J., Chen, X., Chan, B. K., Matcuk, G. R., Barry, C. T., Chang, H. Y., Kuo, M. D. 2007; 25 (6): 675-680

    Abstract

    Paralleling the diversity of genetic and protein activities, pathologic human tissues also exhibit diverse radiographic features. Here we show that dynamic imaging traits in non-invasive computed tomography (CT) systematically correlate with the global gene expression programs of primary human liver cancer. Combinations of twenty-eight imaging traits can reconstruct 78% of the global gene expression profiles, revealing cell proliferation, liver synthetic function, and patient prognosis. Thus, genomic activity of human liver cancers can be decoded by noninvasive imaging, thereby enabling noninvasive, serial and frequent molecular profiling for personalized medicine.

    View details for DOI 10.1038/nbt1306

    View details for Web of Science ID 000247077500025

    View details for PubMedID 17515910

  • A transcriptional program mediating entry into cellular quiescence PLOS GENETICS Liu, H., Adler, A. S., Segal, E., Chang, H. Y. 2007; 3 (6): 996-1008

    Abstract

    The balance of quiescence and cell division is critical for tissue homeostasis and organismal health. Serum stimulation of fibroblasts is well studied as a classic model of entry into the cell division cycle, but the induction of cellular quiescence, such as by serum deprivation (SD), is much less understood. Here we show that SS and SD activate distinct early transcriptional responses genome-wide that converge on a late symmetric transcriptional program. Several serum deprivation early response genes (SDERGs), including the putative tumor suppressor genes SALL2 and MXI1, are required for cessation of DNA synthesis in response to SD and induction of additional SD genes. SDERGs are coordinately repressed in many types of human cancers compared to their normal counterparts, and repression of SDERGs predicts increased risk of cancer progression and death in human breast cancers. These results identify a gene expression program uniquely responsive to loss of growth factor signaling; members of SDERGs may constitute novel growth inhibitors that prevent cancer.

    View details for DOI 10.1371/journal.pgen.0030091

    View details for Web of Science ID 000248349300013

    View details for PubMedID 17559306

  • Genome-wide analysis of KAP1 binding suggests autoregulation of KRAB-ZNFs PLOS GENETICS O'Geen, H., Squazzo, S. L., Iyengar, S., Blahnik, K., Rinn, J. L., Chang, H. Y., Green, R., Farnham, P. J. 2007; 3 (6): 916-926

    Abstract

    We performed a genome-scale chromatin immunoprecipitation (ChIP)-chip comparison of two modifications (trimethylation of lysine 9 [H3me3K9] and trimethylation of lysine 27 [H3me3K27]) of histone H3 in Ntera2 testicular carcinoma cells and in three different anatomical sources of primary human fibroblasts. We found that in each of the cell types the two modifications were differentially enriched at the promoters of the two largest classes of transcription factors. Specifically, zinc finger (ZNF) genes were bound by H3me3K9 and homeobox genes were bound by H3me3K27. We have previously shown that the Polycomb repressive complex 2 is responsible for mediating trimethylation of lysine 27 of histone H3 in human cancer cells. In contrast, there is little overlap between H3me3K9 targets and components of the Polycomb repressive complex 2, suggesting that a different histone methyltransferase is responsible for the H3me3K9 modification. Previous studies have shown that SETDB1 can trimethylate H3 on lysine 9, using in vitro or artificial tethering assays. SETDB1 is thought to be recruited to chromatin by complexes containing the KAP1 corepressor. To determine if a KAP1-containing complex mediates trimethylation of the identified H3me3K9 targets, we performed ChIP-chip assays and identified KAP1 target genes using human 5-kb promoter arrays. We found that a large number of genes of ZNF transcription factors were bound by both KAP1 and H3me3K9 in normal and cancer cells. To expand our studies of KAP1, we next performed a complete genomic analysis of KAP1 binding using a 38-array tiling set, identifying ~7,000 KAP1 binding sites. The identified KAP1 targets were highly enriched for C2H2 ZNFs, especially those containing Krüppel-associated box (KRAB) domains. Interestingly, although most KAP1 binding sites were within core promoter regions, the binding sites near ZNF genes were greatly enriched within transcribed regions of the target genes. Because KAP1 is recruited to the DNA via interaction with KRAB-ZNF proteins, we suggest that expression of KRAB-ZNF genes may be controlled via an auto-regulatory mechanism involving KAP1.

    View details for DOI 10.1371/journal.pgen.0030089

    View details for Web of Science ID 000248349300006

    View details for PubMedID 17542650

  • Patterning skin pigmentation via dickkopf JOURNAL OF INVESTIGATIVE DERMATOLOGY Chang, H. Y. 2007; 127 (5): 994-995

    Abstract

    One of the striking regional variations in skin is its pigmentation. Yamaguchi et al. further dissect the mechanism of regional pigmentation by assessing the effects of dickkopf 1 (DKK1), an antagonist of the Wnt pathway produced in lightly pigmented skin, on melanocyte gene expression. The results provide a plethora of candidate genes that may mediate DKK1's inhibitory effects on melanocyte function.

    View details for DOI 10.1038/sj.jid.5700636

    View details for Web of Science ID 000245889900004

    View details for PubMedID 17435783

  • GSK3 beta mediates suppression of cyclin D2 expression by tumor suppressor PTEN ONCOGENE Huang, W., Chang, H. Y., Fei, T., Wu, H., Chen, Y. 2007; 26 (17): 2471-2482

    Abstract

    PTEN, encoding a lipid phosphatase, is a tumor suppressor gene and is mutated in various types of cancers. It is reported to regulate G1 to S phase transition of the cell cycle by influencing the expression, protein stability and subcellular location of cyclin D1. Here, we provide evidence that PTEN modulates the transcription and protein stability of cyclin D2. Targeted deletion of Pten in mouse embryonic fibroblasts (MEFs) endowed cells with greater potential to overcome G1 arrest than wild-type MEFs and led to the elevated expression of cyclin D2, which was suppressed by the introduction of PTEN. We further defined a pathway involving GSK3beta and beta-catenin/TCF in PTEN-mediated suppression of cyclin D2 transcription. LiCl, an inhibitor of GSK3beta, abolished inhibitory effect of PTEN on cyclin D2 expression, and TCF members could directly bind to the promoter of cyclin D2 and regulate its transcription in a CREB-dependent manner. Our results indicate that the downregulation of cyclin D2 expression by PTEN is mediated by the GSK3beta/beta-catenin/TCF pathway in cooperation with CREB, and suggest a convergence from the PI-3 kinase/PTEN pathway and the Wnt pathway in modulation of cyclin D2 expression.

    View details for DOI 10.1038/sj.onc.1210033

    View details for Web of Science ID 000245831000007

    View details for PubMedID 17043650

  • Spontaneous autoimmunity prevented by thymic expression of a single self-antigen JOURNAL OF EXPERIMENTAL MEDICINE DeVoss, J., Hou, Y., Johannes, K., Lu, W., Liou, G. I., Rinn, J., Chang, H., Caspi, R., Fong, L., Anderson, M. S. 2006; 203 (12): 2727-2735

    Abstract

    The expression of self-antigen in the thymus is believed to be responsible for the deletion of autoreactive T lymphocytes, a critical process in the maintenance of unresponsiveness to self. The Autoimmune regulator (Aire) gene, which is defective in the disorder autoimmune polyglandular syndrome type 1, has been shown to promote the thymic expression of self-antigens. A clear link, however, between specific thymic self-antigens and a single autoimmune phenotype in this model has been lacking. We show that autoimmune eye disease in aire-deficient mice develops as a result of loss of thymic expression of a single eye antigen, interphotoreceptor retinoid-binding protein (IRBP). In addition, lack of IRBP expression solely in the thymus, even in the presence of aire expression, is sufficient to trigger spontaneous eye-specific autoimmunity. These results suggest that failure of thymic expression of selective single self-antigens can be sufficient to cause organ-specific autoimmune disease, even in otherwise self-tolerant individuals.

    View details for DOI 10.1084/jem.20061864

    View details for Web of Science ID 000242339700015

    View details for PubMedID 17116738

  • Bone morphogenetic protein antagonist gremlin 1 is widely expressed by cancer-associated stromal cells and can promote tumor cell proliferation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sneddon, J. B., Zhen, H. H., Montgomery, K., van de Rijn, M., Tward, A. D., West, R., Gladstone, H., Chang, H. Y., Morganroth, G. S., Oro, A. E., Brown, P. O. 2006; 103 (40): 14842-14847

    Abstract

    Although tissue microenvironments play critical roles in epithelial development and tumorigenesis, the factors mediating these effects are poorly understood. In this work, we used a genomic approach to identify factors produced by cells in the microenvironment of basal cell carcinoma (BCC) of the skin, one of the most common human cancers. The global gene expression programs of stromal cell cultures derived from human BCCs showed consistent, systematic differences from those derived from nontumor skin. The gene most consistently expressed at a higher level in BCC tumor stromal cells compared with those from nontumor skin was GREMLIN 1, which encodes a secreted antagonist of the bone morphogenetic protein (BMP) pathway. BMPs and their antagonists are known to play a crucial role in stem and progenitor cell biology as regulators of the balance between expansion and differentiation. Consistent with the hypothesis that BMP antagonists might have a similar role in cancer, we found GREMLIN 1 expression in the stroma of human BCC tumors but not in normal skin in vivo. Furthermore, BMP 2 and 4 are expressed by BCC cells. Ex vivo, BMP inhibits, and Gremlin 1 promotes, proliferation of cultured BCC cells. We further found that GREMLIN 1 is expressed by stromal cells in many carcinomas but not in the corresponding normal tissue counterparts that we examined. Our data suggest that BMP antagonists may be important constituents of tumor stroma, providing a favorable microenvironment for cancer cell survival and expansion in many cancers.

    View details for DOI 10.1073/pnas.0606857103

    View details for Web of Science ID 000241069300037

    View details for PubMedID 17003113

  • Anatomic demarcation by positional variation in fibroblast gene expression programs PLOS GENETICS Rinn, J. L., Bondre, C., Gladstone, H. B., Brown, P. O., Chang, H. Y. 2006; 2 (7): 1084-1096

    Abstract

    Fibroblasts are ubiquitous mesenchymal cells with many vital functions during development, tissue repair, and disease. Fibroblasts from different anatomic sites have distinct and characteristic gene expression patterns, but the principles that govern their molecular specialization are poorly understood. Spatial organization of cellular differentiation may be achieved by unique specification of each cell type; alternatively, organization may arise by cells interpreting their position along a coordinate system. Here we test these models by analyzing the genome-wide gene expression profiles of primary fibroblast populations from 43 unique anatomical sites spanning the human body. Large-scale differences in the gene expression programs were related to three anatomic divisions: anterior-posterior (rostral-caudal), proximal-distal, and dermal versus nondermal. A set of 337 genes that varied according to these positional divisions was able to group all 47 samples by their anatomic sites of origin. Genes involved in pattern formation, cell-cell signaling, and matrix remodeling were enriched among this minimal set of positional identifier genes. Many important features of the embryonic pattern of HOX gene expression were retained in fibroblasts and were confirmed both in vitro and in vivo. Together, these findings suggest that site-specific variations in fibroblast gene expression programs are not idiosyncratic but rather are systematically related to their positional identities relative to major anatomic axes.

    View details for DOI 10.1371/journal.pgen.0020119

    View details for Web of Science ID 000239494800018

    View details for PubMedID 16895450

  • MYC can induce DNA breaks in vivo and in vitro independent of reactive oxygen species CANCER RESEARCH Ray, S., Atkuri, K. R., Deb-Basu, D., Adler, A. S., Chang, H. Y., Herzenberg, L. A., Felsher, D. W. 2006; 66 (13): 6598-6605

    Abstract

    MYC overexpression is thought to initiate tumorigenesis by inducing cellular proliferation and growth and to be restrained from causing tumorigenesis by inducing cell cycle arrest, cellular senescence, and/or apoptosis. Here we show that MYC can induce DNA breaks both in vitro and in vivo independent of increased production of reactive oxygen species (ROS). We provide an insight into the specific circumstances under which MYC generates ROS in vitro and propose a possible mechanism. We found that MYC induces DNA double-strand breaks (DSBs) independent of ROS production in murine lymphocytes in vivo as well as in normal human foreskin fibroblasts (NHFs) in vitro in normal (10%) serum, as measured by gammaH2AX staining. However, NHFs cultured in vitro in low serum (0.05%) and/or ambient oxygen saturation resulted in ROS-associated oxidative damage and DNA single-strand breaks (SSBs), as measured by Ape-1 staining. In NHFs cultured in low versus normal serum, MYC induced increased expression of CYP2C9, a gene product well known to be associated with ROS production. Specific inhibition of CYP2C9 by small interfering RNA was shown to partially inhibit MYC-induced ROS production. Hence, MYC overexpression can induce ROS and SSBs under some conditions, but generally induces widespread DSBs in vivo and in vitro independent of ROS production.

    View details for DOI 10.1158/0008-5472.CAN-05-3115

    View details for Web of Science ID 000238825800021

    View details for PubMedID 16818632

  • From description to causality - Mechanisms of gene expression signatures in cancer CELL CYCLE Adler, A. S., Chang, H. Y. 2006; 5 (11): 1148-1151

    Abstract

    Global gene expression profiles of thousands of cancer samples have been completed, giving rise to hundreds of gene expression signatures (GES). Although many expression signatures show promise in predicting patient prognosis or response to therapies, the usefulness of the signatures in understanding the underlying mechanisms of cancer has not been fully exploited. While "reverse genomic" methods can test specific hypotheses of gene regulation, they fare less well in deciphering novel or combinatorial mechanisms of gene regulation. Recently we described SLAMS (stepwise linkage analysis of microarray signatures), a novel method that can prospectively identify genetic regulators of gene expression signatures in cancer. Applying SLAMS on a poor-prognosis wound signature in human breast cancer, we identified CSN5-mediated ubiquitination of MYC as a novel mechanism to activate a biological program favoring metastasis.

    View details for Web of Science ID 000238581100006

    View details for PubMedID 16721055

  • Genetic regulators of large-scale transcriptional signatures in cancer NATURE GENETICS Adler, A. S., Lin, M. H., Horlings, H., Nuyten, D. S., Van de Vijver, M. J., Chang, H. Y. 2006; 38 (4): 421-430

    Abstract

    Gene expression signatures encompassing dozens to hundreds of genes have been associated with many important parameters of cancer, but mechanisms of their control are largely unknown. Here we present a method based on genetic linkage that can prospectively identify functional regulators driving large-scale transcriptional signatures in cancer. Using this method we show that the wound response signature, a poor-prognosis expression pattern of 512 genes in breast cancer, is induced by coordinate amplifications of MYC and CSN5 (also known as JAB1 or COPS5). This information enabled experimental recapitulation, functional assessment and mechanistic elucidation of the wound signature in breast epithelial cells.

    View details for DOI 10.1038/ng1752

    View details for Web of Science ID 000236340500014

    View details for PubMedID 16518402

  • Microarray analysis of stem cells and differentiation STEM CELL TOOLS AND OTHER EXPERIMENTAL PROTOCOLS Chang, H. Y., Thomson, J. A., Chen, X. 2006; 420: 225-254

    Abstract

    Microarrays have revolutionized molecular biology and enabled biologists to perform global analysis on the expression of tens of thousands of genes simultaneously. They have been widely used in gene discovery, biomarker determination, disease classification, and studies of gene regulation. Microarrays have been applied in stem cell research to identify major features or expression signatures that define stem cells and characterize their differentiation programs toward specific lineages. Here we provide a review of the microarray technology, including the introduction of array platforms, experimental designs, RNA isolation and amplification, array hybridization, and data analysis. We also detail examples that apply microarray technology to address several of the main questions in stem cell biology.

    View details for DOI 10.1016/S0076-6879(06)20010-7

    View details for Web of Science ID 000243420300010

    View details for PubMedID 17161699

  • Predicting a local recurrence after breast-conserving therapy by gene expression profiling BREAST CANCER RESEARCH Nuyten, D. S., Kreike, B., Hart, A. A., Chi, J. A., Sneddon, J. B., Wessels, L. F., Peterse, H. J., Bartelink, H., Brown, P. O., Chang, H. Y., van de Vijver, M. J. 2006; 8 (5)

    Abstract

    To tailor local treatment in breast cancer patients there is a need for predicting ipsilateral recurrences after breast-conserving therapy. After adequate treatment (excision with free margins and radiotherapy), young age and incompletely excised extensive intraductal component are predictors for local recurrence, but many local recurrences can still not be predicted. Here we have used gene expression profiling by microarray analysis to identify gene expression profiles that can help to predict local recurrence in individual patients.By using previously established gene expression profiles with proven value in predicting metastasis-free and overall survival (wound-response signature, 70-gene prognosis profile and hypoxia-induced profile) and training towards an optimal prediction of local recurrences in a training series, we establish a classifier for local recurrence after breast-conserving therapy.Validation of the different gene lists shows that the wound-response signature is able to separate patients with a high (29%) or low (5%) risk of a local recurrence at 10 years (sensitivity 87.5%, specificity 75%). In multivariable analysis the classifier is an independent predictor for local recurrence.Our findings indicate that gene expression profiling can identify subgroups of patients at increased risk of developing a local recurrence after breast-conserving therapy.

    View details for DOI 10.1186/bcr1614

    View details for Web of Science ID 000243169100015

    View details for PubMedID 17069664

  • Learning more from microarrays: Insights from modules and networks JOURNAL OF INVESTIGATIVE DERMATOLOGY Wong, D. J., Chang, H. Y. 2005; 125 (2): 175-182

    Abstract

    Global gene expression patterns can provide comprehensive molecular portraits of biologic diversity and complex disease states, but understanding the physiologic meaning and genetic basis of the myriad gene expression changes have been a challenge. Several new analytic strategies have now been developed to improve the interpretation of microarray data. Because genes work together in groups to carry out specific functions, defining the unit of analysis by coherent changes in biologically meaningful sets of genes, termed modules, improves our understanding of the biological processes underlying the gene expression changes. The gene module approach has been used in exploratory discovery of defective oxidative phosphorylation in diabetes mellitus and also has allowed definitive hypothesis testing on a genomic scale for the relationship between wound healing and cancer and for the oncogenic mechanism of cyclin D. To understand the genetic basis of global gene expression patterns, computational modeling of regulatory networks can highlight key regulators of the gene expression changes, and many of these predictions can now be experimentally validated using global chromatin-immunoprecipitation analysis.

    View details for DOI 10.1111/j.0022-202X.2005.23827.x

    View details for Web of Science ID 000230493200002

    View details for PubMedID 16098025

  • Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, H. Y., Nuyten, D. S., Sneddon, J. B., Hastie, T., Tibshirani, R., Sorlie, T., Dai, H. Y., He, Y. D., Van't Veer, L. J., Bartelink, H., van de Rijn, M., Brown, P. O., van de Vijver, M. J. 2005; 102 (10): 3738-3743

    Abstract

    Based on the hypothesis that features of the molecular program of normal wound healing might play an important role in cancer metastasis, we previously identified consistent features in the transcriptional response of normal fibroblasts to serum, and used this "wound-response signature" to reveal links between wound healing and cancer progression in a variety of common epithelial tumors. Here, in a consecutive series of 295 early breast cancer patients, we show that both overall survival and distant metastasis-free survival are markedly diminished in patients whose tumors expressed this wound-response signature compared to tumors that did not express this signature. A gene expression centroid of the wound-response signature provides a basis for prospectively assigning a prognostic score that can be scaled to suit different clinical purposes. The wound-response signature improves risk stratification independently of known clinico-pathologic risk factors and previously established prognostic signatures based on unsupervised hierarchical clustering ("molecular subtypes") or supervised predictors of metastasis ("70-gene prognosis signature").

    View details for DOI 10.1073/pnas.0409462102

    View details for Web of Science ID 000227533100040

    View details for PubMedID 15701700

  • Gene expression signature of fibroblast serum response predicts human cancer progression: Similarities between tumors and wounds PLOS BIOLOGY Chang, H. Y., Sneddon, J. B., Alizadeh, A. A., Sood, R., West, R. B., Montgomery, K., Chi, J. T., van de Rijn, M., Botstein, D., Brown, P. O. 2004; 2 (2): 206-214
  • Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds. PLoS biology Chang, H. Y., Sneddon, J. B., Alizadeh, A. A., Sood, R., West, R. B., Montgomery, K., Chi, J., van de Rijn, M., Botstein, D., Brown, P. O. 2004; 2 (2): E7-?

    Abstract

    Cancer invasion and metastasis have been likened to wound healing gone awry. Despite parallels in cellular behavior between cancer progression and wound healing, the molecular relationships between these two processes and their prognostic implications are unclear. In this study, based on gene expression profiles of fibroblasts from ten anatomic sites, we identify a stereotyped gene expression program in response to serum exposure that appears to reflect the multifaceted role of fibroblasts in wound healing. The genes comprising this fibroblast common serum response are coordinately regulated in many human tumors, allowing us to identify tumors with gene expression signatures suggestive of active wounds. Genes induced in the fibroblast serum-response program are expressed in tumors by the tumor cells themselves, by tumor-associated fibroblasts, or both. The molecular features that define this wound-like phenotype are evident at an early clinical stage, persist during treatment, and predict increased risk of metastasis and death in breast, lung, and gastric carcinomas. Thus, the transcriptional signature of the response of fibroblasts to serum provides a possible link between cancer progression and wound healing, as well as a powerful predictor of the clinical course in several common carcinomas.

    View details for PubMedID 14737219

  • Kinetics and specificity of Fas ligand induction in toxic epidermal necrolysis ARCHIVES OF DERMATOLOGY Chang, H. Y., Cooper, Z. A., Swetter, S. A., Marinkovich, M. P. 2004; 140 (2): 242-244

    View details for Web of Science ID 000188877100024

    View details for PubMedID 14967808

  • Endothelial cell diversity revealed by global expression profiling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chi, J. T., Chang, H. Y., Haraldsen, G., Jahnsen, F. L., Troyanskaya, O. G., Chang, D. S., Wang, Z., Rockson, S. G., van de Rijn, M., Botstein, D., Brown, P. O. 2003; 100 (19): 10623-10628

    Abstract

    The vascular system is locally specialized to accommodate widely varying blood flow and pressure and the distinct needs of individual tissues. The endothelial cells (ECs) that line the lumens of blood and lymphatic vessels play an integral role in the regional specialization of vascular structure and physiology. However, our understanding of EC diversity is limited. To explore EC specialization on a global scale, we used DNA microarrays to determine the expression profile of 53 cultured ECs. We found that ECs from different blood vessels and microvascular ECs from different tissues have distinct and characteristic gene expression profiles. Pervasive differences in gene expression patterns distinguish the ECs of large vessels from microvascular ECs. We identified groups of genes characteristic of arterial and venous endothelium. Hey2, the human homologue of the zebrafish gene gridlock, was selectively expressed in arterial ECs and induced the expression of several arterial-specific genes. Several genes critical in the establishment of left/right asymmetry were expressed preferentially in venous ECs, suggesting coordination between vascular differentiation and body plan development. Tissue-specific expression patterns in different tissue microvascular ECs suggest they are distinct differentiated cell types that play roles in the local physiology of their respective organs and tissues.

    View details for DOI 10.1073/pnas.1434429100

    View details for Web of Science ID 000185415300012

    View details for PubMedID 12963823

  • Eruptive xanthomas associated with olanzapine use ARCHIVES OF DERMATOLOGY Chang, H. Y., Ridky, T. W., Kimball, A. B., Hughes, E., Oro, A. E. 2003; 139 (8): 1045-1048

    Abstract

    Since their introduction to the US market, atypical antipsychotic drugs, such as olanzapine, have been widely prescribed for the management of psychosis and have increasingly been used in dermatologic settings for the treatment of psychogenic dermatoses. Mild hyperglycemia and hypertriglyceridemia have been documented from the use of these medications, but the range of effects on metabolism and the effects on skin are poorly characterized. OBSERVETION: We describe 3 patients who developed eruptive xanthomas, 1 of whom had relative insulin insufficiency, after starting olanzapine therapy. These cases further support the association of severe dyslipidemia with olanzapine use in selected patients.With the increasing use of atypical antipsychotic agents in the dermatologic setting, the dyslipidemia that develops in association with olanzapine use emphasizes the need for periodic metabolic studies in high-risk patients.

    View details for Web of Science ID 000184692700012

    View details for PubMedID 12925394

  • Myelogenous leukemia cutis resembling stasis dermatitis JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY Chang, H. Y., Wong, K. M., Bosenberg, M., McKee, P. H., Haynes, H. A. 2003; 49 (1): 128-129

    Abstract

    Leukemia cutis may clinically mimic many inflammatory dermatoses. A patient with myelodysplastic syndrome presented with an acute eruption of bilateral, lower-extremity, tender, indurated, brown plaques that clinically resembled chronic stasis dermatitis. Histologic study revealed a dermal myeloblastic leukemic infiltrate.

    View details for DOI 10.1067/mjd.2003.233

    View details for Web of Science ID 000184135000021

    View details for PubMedID 12833025

  • Genomewide view of gene silencing by small interfering RNAs PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chi, J. T., Chang, H. Y., Wang, N. N., Chang, D. S., Dunphy, N., Brown, P. O. 2003; 100 (11): 6343-6346

    Abstract

    RNA interference (RNAi) is an evolutionarily conserved mechanism in plant and animal cells that directs the degradation of messenger RNAs homologous to short double-stranded RNAs termed small interfering RNA (siRNA). The ability of siRNA to direct gene silencing in mammalian cells has raised the possibility that siRNA might be used to investigate gene function in a high throughput fashion or to modulate gene expression in human diseases. The specificity of siRNA-mediated silencing, a critical consideration in these applications, has not been addressed on a genomewide scale. Here we show that siRNA-induced gene silencing of transient or stably expressed mRNA is highly gene-specific and does not produce secondary effects detectable by genomewide expression profiling. A test for transitive RNAi, extension of the RNAi effect to sequences 5' of the target region that has been observed in Caenorhabditis elegans, was unable to detect this phenomenon in human cells.

    View details for DOI 10.1073/pnas.1037853100

    View details for Web of Science ID 000183190700012

    View details for PubMedID 12730368

  • Diversity, topographic differentiation, and positional memory in human fibroblasts PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, H. Y., Chi, J. T., Dudoit, S., Bondre, C., van de Rijn, M., Botstein, D., Brown, P. O. 2002; 99 (20): 12877-12882

    Abstract

    A fundamental feature of the architecture and functional design of vertebrate animals is a stroma, composed of extracellular matrix and mesenchymal cells, which provides a structural scaffold and conduit for blood and lymphatic vessels, nerves, and leukocytes. Reciprocal interactions between mesenchymal and epithelial cells are known to play a critical role in orchestrating the development and morphogenesis of tissues and organs, but the roles played by specific stromal cells in controlling the design and function of tissues remain poorly understood. The principal cells of stromal tissue are called fibroblasts, a catch-all designation that belies their diversity. We characterized genome-wide patterns of gene expression in cultured fetal and adult human fibroblasts derived from skin at different anatomical sites. Fibroblasts from each site displayed distinct and characteristic transcriptional patterns, suggesting that fibroblasts at different locations in the body should be considered distinct differentiated cell types. Notable groups of differentially expressed genes included some implicated in extracellular matrix synthesis, lipid metabolism, and cell signaling pathways that control proliferation, cell migration, and fate determination. Several genes implicated in genetic diseases were found to be expressed in fibroblasts in an anatomic pattern that paralleled the phenotypic defects. Finally, adult fibroblasts maintained key features of HOX gene expression patterns established during embryogenesis, suggesting that HOX genes may direct topographic differentiation and underlie the detailed positional memory in fibroblasts.

    View details for DOI 10.1073/pnas.162488599

    View details for Web of Science ID 000178391700069

    View details for PubMedID 12297622

  • Proteases for cell suicide: Functions and regulation of caspases MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS Chang, H. Y., Yang, X. L. 2000; 64 (4): 821-?

    Abstract

    Caspases are a large family of evolutionarily conserved proteases found from Caenorhabditis elegans to humans. Although the first caspase was identified as a processing enzyme for interleukin-1beta, genetic and biochemical data have converged to reveal that many caspases are key mediators of apoptosis, the intrinsic cell suicide program essential for development and tissue homeostasis. Each caspase is a cysteine aspartase; it employs a nucleophilic cysteine in its active site to cleave aspartic acid peptide bonds within proteins. Caspases are synthesized as inactive precursors termed procaspases; proteolytic processing of procaspase generates the tetrameric active caspase enzyme, composed of two repeating heterotypic subunits. Based on kinetic data, substrate specificity, and procaspase structure, caspases have been conceptually divided into initiators and effectors. Initiator caspases activate effector caspases in response to specific cell death signals, and effector caspases cleave various cellular proteins to trigger apoptosis. Adapter protein-mediated oligomerization of procaspases is now recognized as a universal mechanism of initiator caspase activation and underlies the control of both cell surface death receptor and mitochondrial cytochrome c-Apaf-1 apoptosis pathways. Caspase substrates have bene identified that induce each of the classic features of apoptosis, including membrane blebbing, cell body shrinkage, and DNA fragmentation. Mice deficient for caspase genes have highlighted tissue- and signal-specific pathways for apoptosis and demonstrated an independent function for caspase-1 and -11 in cytokine processing. Dysregulation of caspases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders, and increasing evidence shows that altering caspase activity can confer therapeutic benefits.

    View details for Web of Science ID 000167056400008

    View details for PubMedID 11104820

  • Dissecting Fas signaling with an altered-specificity death-domain mutant: Requirement of FADD binding for apoptosis but not Jun N-terminal kinase activation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, H. Y., Yang, X. L., Baltimore, D. 1999; 96 (4): 1252-1256

    Abstract

    Fas is a cell surface death receptor that regulates peripheral tolerance and lymphoid homeostasis. In many pathologic conditions, ectopic Fas activation mediates tissue destruction. Several proteins that can bind to the cytoplasmic death domain of Fas have been implicated in Fas signal transduction. Here we show that FADD, which couples Fas to pro-caspase-8, and, Daxx, which couples Fas to the Jun N-terminal kinase pathway, bind independently to the Fas death domain. We have isolated a death domain mutant, termed FasDelta, that selectively binds Daxx but not FADD. In tranfected tissue culture cells, FasDelta activated Jun N-terminal kinase normally but was impaired in cell death induction. These results suggest that FADD and Daxx activate two independent pathways downstream of Fas and confirm the essential role of FADD binding in apoptosis induction.

    View details for Web of Science ID 000078698400018

    View details for PubMedID 9990010

  • Activation of apoptosis signal regulating kinase 1 (ASK1) by the adapter protein Daxx SCIENCE Chang, H. Y., Nishitoh, H., Yang, X. L., ICHIJO, H., Baltimore, D. 1998; 281 (5384): 1860-1863

    Abstract

    The Fas death receptor can activate the Jun NH2-terminal kinase (JNK) pathway through the receptor-associated protein Daxx. Daxx was found to activate the JNK kinase kinase ASK1, and overexpression of a kinase-deficient ASK1 mutant inhibited Fas- and Daxx-induced apoptosis and JNK activation. Fas activation induced Daxx to interact with ASK1, which consequently relieved an inhibitory intramolecular interaction between the amino- and carboxyl-termini of ASK1, activating its kinase activity. The Daxx-ASK1 connection completes a signaling pathway from a cell surface death receptor to kinase cascades that modulate nuclear transcription factors.

    View details for Web of Science ID 000076007100056

    View details for PubMedID 9743501

  • Essential role of CED-4 oligomerization in CED-3 activation and apoptosis SCIENCE Yang, X. L., Chang, H. Y., Baltimore, D. 1998; 281 (5381): 1355-1357

    Abstract

    Control of the activation of apoptosis is important both in development and in protection against cancer. In the classic genetic model Caenorhabditis elegans, the pro-apoptotic protein CED-4 activates the CED-3 caspase and is inhibited by the Bcl-2-like protein CED-9. Both processes are mediated by protein-protein interaction. Facilitating the proximity of CED-3 zymogen molecules was found to induce caspase activation and cell death. CED-4 protein oligomerized in cells and in vitro. This oligomerization induced CED-3 proximity and competed with CED-4:CED-9 interaction. Mutations that abolished CED-4 oligomerization inactivated its ability to activate CED-3. Thus, the mechanism of control is that CED-3 in CED-3:CED-4 complexes is activated by CED-4 oligomerization, which is inhibited by binding of CED-9 to CED-4.

    View details for Web of Science ID 000075666800051

    View details for PubMedID 9721101

  • Autoproteolytic activation of pro-caspases by oligomerization MOLECULAR CELL Yang, X. L., Chang, H. Y., Baltimore, D. 1998; 1 (2): 319-325

    Abstract

    Initiation of apopotosis requires the conversion of procaspases to mature caspases. Here we show that oligomerization of pro-caspases is sufficient to induce proteolytic generation of mature caspase subunits and activation of their cell death activity. Deletion of the protein interaction motif DED from pro-caspase-8 greatly suppresses its apoptotic activity. Cell death activity can be restored by oligomerization of pro-caspase-8 protease domains by two heterologous inducible oligomerization systems. Induced oligomerization also activates the apoptotic activity of pro-caspase-1 but not pro-caspase-3. In vitro, oligomerization leads to pro-caspase processing to from the mature caspase subunits; this processing requires the intrinsic caspase activity of zymogens and proceeds via a novel order of cleavage events.

    View details for Web of Science ID 000072970500016

    View details for PubMedID 9659928

  • Daxx, a novel Fas-binding protein that activates JNK and apoptosis CELL Yang, X. L., KHOSRAVIFAR, R., Chang, H. Y., Baltimore, D. 1997; 89 (7): 1067-1076

    Abstract

    The Fas cell surface receptor induces apoptosis upon receptor oligomerization. We have identified a novel signaling protein, termed Daxx, that binds specifically to the Fas death domain. Overexpression of Daxx enhances Fas-mediated apoptosis and activates the Jun N-terminal kinase (JNK) pathway. A C-terminal portion of Daxx interacts with the Fas death domain, while a different region activates both JNK and apoptosis. The Fas-binding domain of Daxx is a dominant-negative inhibitor of both Fas-induced apoptosis and JNK activation, while the FADD death domain partially inhibits death but not JNK activation. The Daxx apoptotic pathway is sensitive to both Bcl-2 and dominant-negative JNK pathway components and acts cooperatively with the FADD pathway. Thus, Daxx and FADD define two distinct apoptotic pathways downstream of Fas.

    View details for Web of Science ID A1997XG83000011

    View details for PubMedID 9215629

  • ASYMMETRIC RETRACTION OF GROWTH CONE FILOPODIA FOLLOWING FOCAL INACTIVATION OF CALCINEURIN NATURE Chang, H. Y., Takei, K., SYDOR, A. M., Born, T., Rusnak, F., Jay, D. G. 1995; 376 (6542): 686-690

    Abstract

    The neuronal growth cone is thought to be the site of decision making in nerve growth and guidance. One likely mechanism of how the growth cone translates various extracellular cues into directed motility involves rises in intracellular calcium. A variety of physiological cues, such as adhesion molecules and neurotransmitters, increases intracellular calcium, and artificial manipulations of growth cone calcium levels affect growth cone morphology and neurite outgrowth. The molecular events downstream of calcium fluxes are incompletely understood. Here we show that calcineurin, a protein phosphatase enriched in growth cones that is dependent on calcium ions and calmodulin, functions in neurite outgrowth and directed filopodial motility in cultured chick dorsal root ganglia neurons. Cyclosporin A and FK506, inhibitors of calcineurin, delayed neuritogenesis and inhibited neurite extension. Chromophore-assisted laser inactivation of calcineurin in regions of growth cones causes localized filopodial and lamellipodial retraction and influences the direction of subsequent outgrowth. We suggest that a spatial distribution of calcineurin activity within the growth cone can regulate motility and direct outgrowth.

    View details for Web of Science ID A1995RQ67200060

    View details for PubMedID 7544441

  • ACTIVATION OF CLN1 AND CLN2 G(1) CYCLIN GENE-EXPRESSION BY BCK2 MOLECULAR AND CELLULAR BIOLOGY DICOMO, C. J., Chang, H., Arndt, K. T. 1995; 15 (4): 1835-1846

    Abstract

    The Saccharomyces cerevisiae CLN3 protein, a G1 cyclin, positively regulates the expression of CLN1 and CLN2, two additional G1 cyclins whose expression during late G1 is activated, in part, by the transcription factors SWI4 and SWI6. We isolated 12 complementation groups of mutants that require CLN3. The members of one of these complementation groups have mutations in the BCK2 gene. In a wild-type CLN3 genetic background, bck2 mutants have a normal growth rate but have a larger cell size, are more sensitive to alpha-factor, and have a modest defect in the accumulation of CLN1 and CLN2 RNA. In the absence of CLN3, bck2 mutations cause an extremely slow growth rate: the cells accumulate in late G1 with very low levels of CLN1 and CLN2 RNA. The slow growth rate and long G1 delay of bck2 cln3 mutants are cured by heterologous expression of CLN2. Moreover, overexpression of BCK2 induces very high levels of CLN1, CLN2, and HCS26 RNAs. The results suggest that BCK2 and CLN3 provide parallel activation pathways for the expression of CLN1 and CLN2 during late G1.

    View details for Web of Science ID A1995QM49200001

    View details for PubMedID 7891677

  • ACTIVE-SITE MUTANTS OF HUMAN CYCLOPHILIN-A SEPARATE PEPTIDYL-PROLYL ISOMERASE ACTIVITY FROM CYCLOSPORINE-A BINDING AND CALCINEURIN INHIBITION PROTEIN SCIENCE ZYDOWSKY, L. D., Etzkorn, F. A., Chang, H. Y., Ferguson, S. B., STOLZ, L. A., Ho, S. I., Walsh, C. T. 1992; 1 (9): 1092-1099

    Abstract

    Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin.

    View details for Web of Science ID A1992JR69300003

    View details for PubMedID 1338979

  • CYCLOSPORINE-MEDIATED INHIBITION OF BOVINE CALCINEURIN BY CYCLOPHILIN-A AND CYCLOPHILIN-B PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA SWANSON, S. K., Born, T., ZYDOWSKY, L. D., Cho, H. J., Chang, H. Y., Walsh, C. T., Rusnak, F. 1992; 89 (9): 3741-3745

    Abstract

    The Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin is inhibited by the immunosuppressant drug cyclosporin A in the presence of cyclophilin A or B. Of the two isoforms, cyclophilin B is more potent by a factor of 2-5 when either the phosphoprotein [32P]casein or the [32P]phosphoserine [Ser(32P)] form of the 19-residue bovine cardiac cAMP-dependent protein kinase regulatory subunit peptide RII, [Ser(32P)15]RII, is used as substrate. With [Ser(32P15]RII as substrate, the concentrations of the cyclosporin A.cyclophilin A and cyclosporin A.cyclophilin B complexes, which cause 50% inhibition of calcineurin activity, are 120 and 50 nM, respectively. Lowering the concentration of calcineurin 80% with [32P]casein as substrate lowered the apparent inhibition constant for each complex even further; 50% inhibition of calcineurin was observed at 40 nM for cyclosporin A.cyclophilin A, whereas it was less than 10 nM for cyclosporin A.cyclophilin B. In all inhibition assays with [32P]casein or [Ser(32P)15]RII, the concentration of calcineurin required for measurable phosphatase activity is such that these complexes behave as tight-binding inhibitors of calcineurin, and steady-state kinetics cannot be used to assess inhibition patterns or Ki values. Limited trypsinization of calcineurin produces a fragment that is still inhibited, indicating that the interaction of cyclosporin.cyclophilin with calcineurin does not require either calmodulin or Ca2+.

    View details for Web of Science ID A1992HR85300015

    View details for PubMedID 1315036

Stanford Medicine Resources: