Bio

Academic Appointments


Honors & Awards


  • Ellison Senior Scholar in Aging, Ellison Medical Foundation (2009-2013)
  • Searle Scholar, Searle Scholars Program (2007-2010)
  • Terman Fellowship, Fredrick E. Terman Foundation (2006-2008)
  • Kimmel Scholar Award, Sidney Kimmel Foundation for Cancer Research (2006-2008)
  • Career Award in the Biomedical Sciences, Burroughs Wellcome Fund (2003 - 2008)
  • Mentored Clinical Scientist Development Award (KO8), NIA (2002 - 2007)
  • Advanced Fellowship in Aging Research, Harvard/Hartford Foundation (2001-2002)

Professional Education


  • M.D., Harvard Medical School (1999)
  • Ph.D., Harvard Medical School (1999)

Research & Scholarship

Current Research and Scholarly Interests


We study the molecular mechanisms by which chromatin-signaling networks effect nuclear and epigenetic programs, and how dysregulation of these pathways leads to disease. Our work centers on the biology of lysine methylation, a principal chromatin-regulatory mechanism that directs epigenetic processes. We study how lysine methylation events are generated, sensed, and transduced, and how these chemical marks integrate with other nuclear signaling systems to govern diverse cellular functions.

Teaching

2013-14 Courses


Graduate and Fellowship Programs


Publications

Journal Articles


  • A general molecular affinity strategy for global detection and proteomic analysis of lysine methylation. Molecular cell Moore, K. E., Carlson, S. M., Camp, N. D., Cheung, P., James, R. G., Chua, K. F., Wolf-Yadlin, A., Gozani, O. 2013; 50 (3): 444-456

    Abstract

    Lysine methylation of histone proteins regulates chromatin dynamics and plays important roles in diverse physiological and pathological processes. However, beyond histone proteins, the proteome-wide extent of lysine methylation remains largely unknown. We have engineered the naturally occurring MBT domain repeats of L3MBTL1 to serve as a universal affinity reagent for detecting, enriching, and identifying proteins carrying a mono- or dimethylated lysine. The domain is broadly specific for methylated lysine ("pan-specific") and can be applied to any biological system. We have used our approach to demonstrate that SIRT1 is a substrate of the methyltransferase G9a both in vitro and in cells, to perform proteome-wide detection and enrichment of methylated proteins, and to identify candidate in-cell substrates of G9a and the related methyltransferase GLP. Together, our results demonstrate a powerful new approach for global and quantitative analysis of methylated lysine, and they represent the first systems biology understanding of lysine methylation.

    View details for DOI 10.1016/j.molcel.2013.03.005

    View details for PubMedID 23583077

  • The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome NATURE Kuo, A. J., Song, J., Cheung, P., Ishibe-Murakami, S., Yamazoe, S., Chen, J. K., Patel, D. J., Gozani, O. 2012; 484 (7392): 115-?

    Abstract

    The recognition of distinctly modified histones by specialized 'effector' proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes. Effector proteins influence DNA-templated processes, including transcription, DNA recombination and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulate DNA replication. Here we show that ORC1--a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing--contains a bromo adjacent homology (BAH) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyl-lysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins, and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at replication origins, ORC chromatin loading and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the aetiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism, and ORC1 depletion in zebrafish results in an MGS-like phenotype. We find that wild-type human ORC1, but not ORC1-H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyl-lysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal aetiological role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.

    View details for DOI 10.1038/nature10956

    View details for Web of Science ID 000302343400045

    View details for PubMedID 22398447

  • Methylation of H4 lysines 5, 8 and 12 by yeast Set5 calibrates chromatin stress responses NATURE STRUCTURAL & MOLECULAR BIOLOGY Green, E. M., Mas, G., Young, N. L., Garcia, B. A., Gozani, O. 2012; 19 (3): 361-363

    Abstract

    Methylation of histones is central to chromatin regulation, and thus previously unknown mechanisms regulating genome function can be revealed through the discovery of new histone methyl marks. Here we identify Set5 as the first histone H4 methyltransferase, which monomethylates the critical H4 lysine residues 5, 8 and 12 in budding yeast. Set5's enzymatic activity functions together with the global chromatin-modifying complexes COMPASS and NuA4 to regulate cell growth and stress responses.

    View details for DOI 10.1038/nsmb.2252

    View details for Web of Science ID 000301181900015

    View details for PubMedID 22343720

  • NSD2 Links Dimethylation of Histone H3 at Lysine 36 to Oncogenic Programming MOLECULAR CELL Kuo, A. J., Cheung, P., Chen, K., Zee, B. M., Kioi, M., Lauring, J., Xi, Y., Park, B. H., Shi, X., Garcia, B. A., Li, W., Gozani, O. 2011; 44 (4): 609-620

    Abstract

    The histone lysine methyltransferase NSD2 (MMSET/WHSC1) is implicated in diverse diseases and commonly overexpressed in multiple myeloma due to a recurrent t(4;14) chromosomal translocation. However, the precise catalytic activity of NSD2 is obscure, preventing progress in understanding how this enzyme influences chromatin biology and myeloma pathogenesis. Here, we show that dimethylation of histone H3 at lysine 36 (H3K36me2) is the principal chromatin-regulatory activity of NSD2. Catalysis of H3K36me2 by NSD2 is sufficient for gene activation. In t(4;14)-positive myeloma cells, the normal genome-wide and gene-specific distribution of H3K36me2 is obliterated, creating a chromatin landscape that selects for a transcription profile favorable for myelomagenesis. Catalytically active NSD2 confers xenograft tumor formation upon t(4;14)-negative cells and promotes oncogenic transformation of primary cells in an H3K36me2-dependent manner. Together, our findings establish H3K36me2 as the primary product generated by NSD2 and demonstrate that genomic disorganization of this canonical chromatin mark by NSD2 initiates oncogenic programming.

    View details for DOI 10.1016/j.molcel.2011.08.042

    View details for Web of Science ID 000297387800012

    View details for PubMedID 22099308

  • Lysine methylation of the NF-kappa B subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-kappa B signaling NATURE IMMUNOLOGY Levy, D., Kuo, A. J., Chang, Y., Schaefer, U., Kitson, C., Cheung, P., Espejo, A., Zee, B. M., Liu, C. L., Tangsombatvisit, S., Tennen, R. I., Kuo, A. Y., Tanjing, S., Cheung, R., Chua, K. F., Utz, P. J., Shi, X., Prinjha, R. K., Lee, K., Garcia, B. A., Bedford, M. T., Tarakhovsky, A., Cheng, X., Gozani, O. 2011; 12 (1): 29-U47

    Abstract

    Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-?B subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-?B-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-? (PKC-?) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.

    View details for DOI 10.1038/ni.1968

    View details for Web of Science ID 000285465100010

    View details for PubMedID 21131967

  • ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression NATURE Shi, X., Hong, T., Walter, K. L., Ewalt, M., Michishita, E., Hung, T., Carney, D., Pena, P., Lan, F., Kaadige, M. R., Lacoste, N., Cayrou, C., Davrazou, F., Saha, A., Cairns, B. R., Ayer, D. E., Kutateladze, T. G., Shi, Y., Cote, J., Chua, K. F., Gozani, O. 2006; 442 (7098): 96-99

    Abstract

    Dynamic regulation of diverse nuclear processes is intimately linked to covalent modifications of chromatin. Much attention has focused on methylation at lysine 4 of histone H3 (H3K4), owing to its association with euchromatic genomic regions. H3K4 can be mono-, di- or tri-methylated. Trimethylated H3K4 (H3K4me3) is preferentially detected at active genes, and is proposed to promote gene expression through recognition by transcription-activating effector molecules. Here we identify a novel class of methylated H3K4 effector domains--the PHD domains of the ING (for inhibitor of growth) family of tumour suppressor proteins. The ING PHD domains are specific and highly robust binding modules for H3K4me3 and H3K4me2. ING2, a native subunit of a repressive mSin3a-HDAC1 histone deacetylase complex, binds with high affinity to the trimethylated species. In response to DNA damage, recognition of H3K4me3 by the ING2 PHD domain stabilizes the mSin3a-HDAC1 complex at the promoters of proliferation genes. This pathway constitutes a new mechanism by which H3K4me3 functions in active gene repression. Furthermore, ING2 modulates cellular responses to genotoxic insults, and these functions are critically dependent on ING2 interaction with H3K4me3. Together, our findings establish a pivotal role for trimethylation of H3K4 in gene repression and, potentially, tumour suppressor mechanisms.

    View details for DOI 10.1038/nature04835

    View details for Web of Science ID 000238724500044

    View details for PubMedID 16728974

  • Proteome-wide enrichment of proteins modified by lysine methylation. Nature protocols Carlson, S. M., Moore, K. E., Green, E. M., Martín, G. M., Gozani, O. 2014; 9 (1): 37-50

    Abstract

    We present a protocol for using the triple malignant brain tumor domains of L3MBTL1 (3xMBT), which bind to mono- and di-methylated lysine with minimal sequence specificity, in order to enrich for such methylated lysine from cell lysates. Cells in culture are grown with amino acids containing light or heavy stable isotopic labels. Methylated proteins are enriched by incubating cell lysates with 3xMBT, or with the binding-null D355N mutant as a negative control. Quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) are then used to identify proteins that are specifically enriched by 3xMBT pull-down. The addition of a third isotopic label allows the comparison of protein lysine methylation between different biological conditions. Unlike most approaches, our strategy does not require a prior hypothesis of candidate methylated proteins, and it recognizes a wider range of methylated proteins than any available method using antibodies. Cells are prepared by growing in isotopic labeling medium for about 7 d; the process of enriching methylated proteins takes 3 d and analysis by LC-MS/MS takes another 1-2 d.

    View details for DOI 10.1038/nprot.2013.164

    View details for PubMedID 24309976

  • Nuclear phosphatidylinositol-5-phosphate regulates ING2 stability at discrete chromatin targets in response to DNA damage SCIENTIFIC REPORTS Bua, D. J., Martin, G. M., Binda, O., Gozani, O. 2013; 3

    Abstract

    ING2 (inhibitor of growth family member 2) is a component of a chromatin-regulatory complex that represses gene expression and is implicated in cellular processes that promote tumor suppression. However, few direct genomic targets of ING2 have been identified and the mechanism(s) by which ING2 selectively regulates genes remains unknown. Here we provide evidence that direct association of ING2 with the nuclear phosphoinositide phosphatidylinositol-5-phosphate (PtdIns(5)P) regulates a subset of ING2 targets in response to DNA damage. At these target genes, the binding event between ING2 and PtdIns(5)P is required for ING2 promoter occupancy and ING2-associated gene repression. Moreover, depletion of PtdIns(5)P attenuates ING2-mediated regulation of these targets in the presence of DNA damage. Taken together, these findings support a model in which PtdIns(5)P functions as a sub-nuclear trafficking factor that stabilizes ING2 at discrete genomic sites.

    View details for DOI 10.1038/srep02137

    View details for Web of Science ID 000321287400008

    View details for PubMedID 23823870

  • Chd5 requires PHD-mediated histone 3 binding for tumor suppression. Cell reports Paul, S., Kuo, A., Schalch, T., Vogel, H., Joshua-Tor, L., McCombie, W. R., Gozani, O., Hammell, M., Mills, A. A. 2013; 3 (1): 92-102

    Abstract

    Chromodomain Helicase DNA binding protein 5 (CHD5) is a tumor suppressor mapping to 1p36, a genomic region that is frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin-remodeling proteins, whether its tumor-suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N terminus of H3 through its tandem plant homeodomains (PHDs). Genome-wide chromatin immunoprecipitation studies reveal preferential binding of Chd5 to loci lacking the active mark H3K4me3 and also identify Chd5 targets implicated in cancer. Chd5 mutations that abrogate H3 binding are unable to inhibit proliferation or transcriptionally modulate target genes, which leads to tumorigenesis in vivo. Unlike wild-type Chd5, Chd5-PHD mutants are unable to induce differentiation or efficiently suppress the growth of human neuroblastoma in vivo. Our work defines Chd5 as an N-terminally unmodified H3-binding protein and provides functional evidence that this interaction orchestrates chromatin-mediated transcriptional programs critical for tumor suppression.

    View details for DOI 10.1016/j.celrep.2012.12.009

    View details for PubMedID 23318260

  • Phf19 links methylated Lys36 of histone H3 to regulation of Polycomb activity NATURE STRUCTURAL & MOLECULAR BIOLOGY Ballare, C., Lange, M., Lapinaite, A., Martin, G. M., Morey, L., Pascual, G., Liefke, R., Simon, B., Shi, Y., Gozani, O., Carlomagno, T., Aznar Benitah, S., Di Croce, L. 2012; 19 (12): 1257-?

    Abstract

    Polycomb-group proteins are transcriptional repressors with essential roles in embryonic development. Polycomb repressive complex 2 (PRC2) contains the methyltransferase activity for Lys27. However, the role of other histone modifications in regulating PRC2 activity is just beginning to be understood. Here we show that direct recognition of methylated histone H3 Lys36 (H3K36me), a mark associated with activation, by the PRC2 subunit Phf19 is required for the full enzymatic activity of the PRC2 complex. Using NMR spectroscopy, we provide structural evidence for this interaction. Furthermore, we show that Phf19 binds to a subset of PRC2 targets in mouse embryonic stem cells and that this is required for their repression and for H3K27me3 deposition. These findings show that the interaction of Phf19 with H3K36me2 and H3K36me3 is essential for PRC2 complex activity and for proper regulation of gene repression in embryonic stem cells.

    View details for DOI 10.1038/nsmb.2434

    View details for Web of Science ID 000311991700012

    View details for PubMedID 23104054

  • On silico peptide microarrays for high-resolution mapping of antibody epitopes and diverse protein-protein interactions NATURE MEDICINE Price, J. V., Tangsombatvisit, S., Xu, G., Yu, J., Levy, D., Baechler, E. C., Gozani, O., Varma, M., Utz, P. J., Liu, C. L. 2012; 18 (9): 1434-?

    Abstract

    We developed a new, silicon-based peptide array for a broad range of biological applications, including potential development as a real-time point-of-care platform. We used photolithography on silicon wafers to synthesize microarrays (Intel arrays) that contained every possible overlapping peptide within a linear protein sequence covering the N-terminal tail of human histone H2B. These arrays also included peptides with acetylated and methylated lysine residues, reflecting post-translational modifications of H2B. We defined minimum binding epitopes for commercial antibodies recognizing the modified and unmodified H2B peptides. We further found that this platform is suitable for the highly sensitive characterization of methyltransferases and kinase substrates. The Intel arrays also revealed specific H2B epitopes that are recognized by autoantibodies in individuals with systemic lupus erythematosus who have elevated disease severity. By combining emerging nonfluorescence-based detection methods with an underlying integrated circuit, we are now poised to create a truly transformative proteomics platform with applications in bioscience, drug development and clinical diagnostics.

    View details for DOI 10.1038/nm.2913

    View details for Web of Science ID 000308472300042

    View details for PubMedID 22902875

  • SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation NATURE Barber, M. F., Michishita-Kioi, E., Xi, Y., Tasselli, L., Kioi, M., Moqtaderi, Z., Tennen, R. I., Paredes, S., Young, N. L., Chen, K., Struhl, K., Garcia, B. A., Gozani, O., Li, W., Chua, K. F. 2012; 487 (7405): 114-?

    Abstract

    Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism and ageing. SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets and physiological functions have been unclear. Here we show that SIRT7 is an NAD(+)-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional repression. The spectrum of SIRT7 target genes is defined in part by its interaction with the cancer-associated E26 transformed specific (ETS) transcription factor ELK4, and comprises numerous genes with links to tumour suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to oncogenic transformation, and in patients is associated with aggressive tumour phenotypes and poor prognosis. We find that deacetylation of H3K18Ac by SIRT7 is necessary for maintaining essential features of human cancer cells, including anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7 is necessary for a global hypoacetylation of H3K18Ac associated with cellular transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly reduces the tumorigenicity of human cancer cell xenografts in mice. Together, our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular transformation programs and tumour formation in vivo.

    View details for DOI 10.1038/nature11043

    View details for Web of Science ID 000305982900061

    View details for PubMedID 22722849

  • New marks on the block Set5 methylates H4 lysines 5, 8 and 12 NUCLEUS-AUSTIN Green, E. M., Morrison, A. J., Gozani, O. 2012; 3 (4): 335-339

    Abstract

    The methylation of lysine residues in the N-terminal tails of histones is a highly conserved mechanism that regulates critical functions of chromatin, such as the control of gene expression. Using a biochemical approach, we recently identified new methylation marks on the histone H4 tail in budding yeast at lysines 5, 8 and 12, catalyzed by the previously-uncharacterized enzyme Set5. Genetic studies revealed that Set5 functions in cellular processes that also rely on the global chromatin modifying complexes COMPASS and NuA4, which methylate H3 lysine 4 and acetylate H4 lysines 5, 8 and 12, respectively. The identification of new methylation events on the H4 tail raises many intriguing questions regarding their function and their interaction with known histone modifications. Here, we analyze the insights gained about the new enzyme Set5 and the implications for new functionality added to the H4 tail.

    View details for DOI 10.4161/nucl.20695

    View details for Web of Science ID 000315929000009

    View details for PubMedID 22688645

  • Methylation by Set9 modulates FoxO3 stability and transcriptional activity AGING-US Calnan, D. R., Webb, A. E., White, J. L., Stowe, T. R., Goswami, T., Shi, X., Espejo, A., Bedford, M. T., Gozani, O., Gygi, S. P., Brunet, A. 2012; 4 (7): 462-479

    Abstract

    The FoxO family of transcription factors plays an important role in longevity and tumor suppression by regulating the expression of a wide range of target genes. FoxO3 has recently been found to be associated with extreme longevity in humans and to regulate the homeostasis of adult stem cell pools in mammals, which may contribute to longevity. The activity of FoxO3 is controlled by a variety of post-translational modifications that have been proposed to form a 'code' affecting FoxO3 subcellular localization, DNA binding ability, protein-protein interactions and protein stability. Lysine methylation is a crucial post-translational modification on histones that regulates chromatin accessibility and is a key part of the 'histone code'. However, whether lysine methylation plays a role in modulating FoxO3 activity has never been examined. Here we show that the methyltransferase Set9 directly methylates FoxO3 in vitro and in cells. Using a combination of tandem mass spectrometry and methyl-specific antibodies, we find that Set9 methylates FoxO3 at a single residue, lysine 271, a site previously known to be deacetylated by Sirt1. Methylation of FoxO3 by Set9 decreases FoxO3 protein stability, while moderately increasing FoxO3 transcriptional activity. The modulation of FoxO3 stability and activity by methylation may be critical for fine-tuning cellular responses to stress stimuli, which may in turn affect FoxO3's ability to promote tumor suppression and longevity.

    View details for Web of Science ID 000307474000004

    View details for PubMedID 22820736

  • Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation EPIGENETICS Van Aller, G. S., Reynoird, N., Barbash, O., Huddleston, M., Liu, S., Zmoos, A., McDevitt, P., Sinnamon, R., Le, B., Mas, G., Annan, R., Sage, J., Garcia, B. A., Tummino, P. J., Gozani, O., Kruger, R. G. 2012; 7 (4): 340-343

    Abstract

    Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.

    View details for Web of Science ID 000302493300004

    View details for PubMedID 22419068

  • Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies ARTHRITIS RESEARCH & THERAPY Liu, C. L., Tangsombatvisit, S., Rosenberg, J. M., Mandelbaum, G., Gillespie, E. C., Gozani, O. P., Alizadeh, A. A., Utz, P. J. 2012; 14 (1)

    Abstract

    Autoreactivity to histones is a pervasive feature of several human autoimmune disorders, including systemic lupus erythematosus (SLE). Specific post-translational modifications (PTMs) of histones within neutrophil extracellular traps (NETs) may potentially drive the process by which tolerance to these chromatin-associated proteins is broken. We hypothesized that NETs and their unique histone PTMs might be capable of inducing autoantibodies that target histones.We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen.We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the immunoglobulin G (IgG) and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE.Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified.

    View details for DOI 10.1186/ar3707

    View details for Web of Science ID 000304698800039

    View details for PubMedID 22300536

  • Structure-activity relationships of methyl-lysine reader antagonists MEDCHEMCOMM Herold, J. M., James, L. I., Korboukh, V. K., Gao, C., Coil, K. E., Bua, D. J., Norris, J. L., Kireev, D. B., Brown, P. J., Jin, J., Janzen, W. P., Gozani, O., Frye, S. V. 2012; 3 (1): 45-51

    View details for DOI 10.1039/c1md00195g

    View details for Web of Science ID 000299975300007

  • Everybody's welcome: The big tent approach to epigenetic drug discovery. Drug discovery today. Therapeutic strategies Green, E. M., Gozani, O. 2012; 9 (2-3): e75-e81

    Abstract

    The rapid expansion of epigenetics research is fueled by the increasing understanding that epigenetic processes are critical to regulating cellular development and dysfunction of epigenetic programs is responsible for a diverse set of human pathologies, including cancer, autoimmune and neurodegenerative diseases. The expansive set of components contributing to epigenetic disease mechanisms and the often reversible nature of epigenetic lesions provide prime opportunities for the development of novel therapeutic strategies. Here, we provide an overview of epigenetics and its relationship to disease, discuss current epigenetics-based therapies and suggest new avenues for the identification of therapies targeting deregulated epigenetic programs in disease.

    View details for PubMedID 23505394

  • Correction: Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies. Arthritis research & therapy Liu, C. L., Tangsombatvisit, S., Rosenberg, J. M., Mandelbaum, G., Gillespie, E. C., Gozani, O. P., Alizadeh, A. A., Utz, P. J. 2012; 14 (4): 403

    View details for PubMedID 22894771

  • A proteomic approach for the identification of novel lysine methyltransferase substrates EPIGENETICS & CHROMATIN Levy, D., Liu, C. L., Yang, Z., Newman, A. M., Alizadeh, A. A., Utz, P. J., Gozani, O. 2011; 4

    Abstract

    Signaling via protein lysine methylation has been proposed to play a central role in the regulation of many physiologic and pathologic programs. In contrast to other post-translational modifications such as phosphorylation, proteome-wide approaches to investigate lysine methylation networks do not exist.In the current study, we used the ProtoArray® platform, containing over 9,500 human proteins, and developed and optimized a system for proteome-wide identification of novel methylation events catalyzed by the protein lysine methyltransferase (PKMT) SETD6. This enzyme had previously been shown to methylate the transcription factor RelA, but it was not known whether SETD6 had other substrates. By using two independent detection approaches, we identified novel candidate substrates for SETD6, and verified that all targets tested in vitro and in cells were genuine substrates.We describe a novel proteome-wide methodology for the identification of new PKMT substrates. This technological advance may lead to a better understanding of the enzymatic activity and substrate specificity of the large number (more than 50) PKMTs present in the human proteome, most of which are uncharacterized.

    View details for DOI 10.1186/1756-8935-4-19

    View details for Web of Science ID 000296832600001

    View details for PubMedID 22024134

  • Hypoxia-induced methylation of a pontin chromatin remodeling factor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lee, J. S., Kim, Y., Bhin, J., Shin, H. R., Nam, H. J., Lee, S. H., Yoon, J., Binda, O., Gozani, O., Hwang, D., Baek, S. H. 2011; 108 (33): 13510-13515

    Abstract

    Pontin is a chromatin remodeling factor that possesses both ATPase and DNA helicase activities. Although Pontin is frequently overexpressed in human cancers of various types and implicated in oncogenic functions, the upstream signaling network leading to the regulation of Pontin that in turn affects transcription of downstream target genes has not been extensively studied. Here, we identify Pontin is methylated by G9a/GLP methyltransferases in hypoxic condition and potentiates HIF-1?-mediated activation by increasing the recruitment of p300 coactivator to a subset of HIF-1? target promoters. Intriguingly, Pontin methylation results in the increased invasive and migratory properties by activating downstream target gene, Ets1. In contrast, inhibition of Pontin methylation results in the suppression of tumorigenic and metastatic properties. Together, our data provide new approaches by targeting Pontin methylation and its downstream targets for the development of therapeutic agents for human cancers.

    View details for DOI 10.1073/pnas.1106106108

    View details for Web of Science ID 000293895100036

    View details for PubMedID 21825155

  • CUL4B: Trash Talking at Chromatin MOLECULAR CELL Green, E. M., Gozani, O. 2011; 43 (3): 321-323

    Abstract

    In this issue, Nakagawa and Xiong (2011) reveal a mechanism targeting WDR5 for proteolysis dependent on the X-linked mental retardation gene, CUL4B. This provides a link between the stability of a chromatin factor and gene expression implicated in neurological pathogenesis.

    View details for DOI 10.1016/j.molce1.2011.07.015

    View details for Web of Science ID 000293680900002

    View details for PubMedID 21816341

  • Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling NUCLEIC ACIDS RESEARCH Chang, Y., Levy, D., Horton, J. R., Peng, J., Zhang, X., Gozani, O., Cheng, X. 2011; 39 (15): 6380-6389

    Abstract

    SET domain containing 6 (SETD6) monomethylates the RelA subunit of nuclear factor kappa B (NF-?B). The ankyrin repeats of G9a-like protein (GLP) recognizes RelA monomethylated at Lys310. Adjacent to Lys310 is Ser311, a known phosphorylation site of RelA. Ser311 phosphorylation inhibits Lys310 methylation by SETD6 as well as binding of Lys310me1 by GLP. The structure of SETD6 in complex with RelA peptide containing the methylation site, in the presence of S-adenosyl-L-methionine, reveals a V-like protein structure and suggests a model for NF-?B binding to SETD6. In addition, structural modeling of the GLP ankyrin repeats bound to Lys310me1 peptide provides insight into the molecular basis for inhibition of Lys310me1 binding by Ser311 phosphorylation. Together, these findings provide a structural explanation for a key cellular signaling pathway centered on RelA Lys310 methylation, which is generated by SETD6 and recognized by GLP, and incorporate a methylation-phosphorylation switch of adjacent lysine and serine residues. Finally, SETD6 is structurally similar to the Rubisco large subunit methyltransferase. Given the restriction of Rubisco to plant species, this particular appearance of the protein lysine methyltransferase has been evolutionarily well conserved.

    View details for DOI 10.1093/nar/gkr256

    View details for Web of Science ID 000294555800013

    View details for PubMedID 21515635

  • Regulation of p53 function by lysine methylation EPIGENOMICS West, L. E., Gozani, O. 2011; 3 (3): 361-369

    Abstract

    The reversible and dynamic methylation of proteins on lysine residues can greatly increase the signaling potential of the modified factor. In addition to histones, several other nuclear factors such as the tumor suppressor and transcription factor p53 undergo lysine methylation, suggesting that this modification may be a common mechanism for modulating protein–protein interactions and key cellular signaling pathways. This article focuses on how lysine methylation events on the C-terminal tail of p53 are generated, sensed and transduced to modulate p53 functions.

    View details for DOI 10.2217/EPI.11.21

    View details for Web of Science ID 000293246400014

    View details for PubMedID 21826189

  • A Chemical Method for Labeling Lysine Methyltransferase Substrates CHEMBIOCHEM Binda, O., Boyce, M., Rush, J. S., Palaniappan, K. K., Bertozzi, C. R., Gozani, O. 2011; 12 (2): 330-334

    Abstract

    Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome-wide identification of novel PKMT substrates. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts.

    View details for DOI 10.1002/cbic.201000433

    View details for Web of Science ID 000286433800014

    View details for PubMedID 21243721

  • TRIM24 links a non-canonical histone signature to breast cancer NATURE Tsai, W., Wang, Z., Yiu, T. T., Akdemir, K. C., Xia, W., Winter, S., Tsai, C., Shi, X., Schwarzer, D., Plunkett, W., Aronow, B., Gozani, O., Fischle, W., Hung, M., Patel, D. J., Barton, M. C. 2010; 468 (7326): 927-U320

    Abstract

    Recognition of modified histone species by distinct structural domains within 'reader' proteins plays a critical role in the regulation of gene expression. Readers that simultaneously recognize histones with multiple marks allow transduction of complex chromatin modification patterns into specific biological outcomes. Here we report that chromatin regulator tripartite motif-containing 24 (TRIM24) functions in humans as a reader of dual histone marks by means of tandem plant homeodomain (PHD) and bromodomain (Bromo) regions. The three-dimensional structure of the PHD-Bromo region of TRIM24 revealed a single functional unit for combinatorial recognition of unmodified H3K4 (that is, histone H3 unmodified at lysine 4, H3K4me0) and acetylated H3K23 (histone H3 acetylated at lysine 23, H3K23ac) within the same histone tail. TRIM24 binds chromatin and oestrogen receptor to activate oestrogen-dependent genes associated with cellular proliferation and tumour development. Aberrant expression of TRIM24 negatively correlates with survival of breast cancer patients. The PHD-Bromo of TRIM24 provides a structural rationale for chromatin activation through a non-canonical histone signature, establishing a new route by which chromatin readers may influence cancer pathogenesis.

    View details for DOI 10.1038/nature09542

    View details for Web of Science ID 000285344600039

    View details for PubMedID 21164480

  • The MBT Repeats of L3MBTL1 Link SET8-mediated p53 Methylation at Lysine 382 to Target Gene Repression JOURNAL OF BIOLOGICAL CHEMISTRY West, L. E., Roy, S., Lachmi-Weiner, K., Hayashi, R., Shi, X., Appella, E., Kutateladze, T. G., Gozani, O. 2010; 285 (48): 37725-37732

    Abstract

    The p53 tumor suppressor protein is regulated by multiple post-translational modifications, including lysine methylation. We previously found that monomethylation of p53 at lysine 382 (p53K382me1) by the protein lysine methyltransferase (PKMT) SET8/PR-Set7 represses p53 transactivation of target genes. However, the molecular mechanism linking p53K382 monomethylation to repression is not known. Here we show in biochemical and crystallographic studies the preferential recognition of p53K382me1 by the triple malignant brain tumor (MBT) repeats of the chromatin compaction factor L3MBTL1. We demonstrate that SET8-mediated methylation of p53 at Lys-382 promotes the interaction between L3MBTL1 and p53 in cells, and the chromatin occupancy of L3MBTL1 at p53 target promoters. In the absence of DNA damage, L3MBTL1 interacts with p53K382me1 and p53-target genes are repressed, whereas depletion of L3MBTL1 results in a p53-dependent increase in p21 and PUMA transcript levels. Activation of p53 by DNA damage is coupled to a decrease in p53K382me1 levels, abrogation of the L3MBTL1-p53 interaction, and disassociation of L3MBTL1 from p53-target promoters. Together, we identify L3MBTL1 as the second known methyl-p53 effector protein, and provide a molecular explanation for the mechanism by which p53K382me1 is transduced to regulate p53 activity.

    View details for DOI 10.1074/jbc.M110.139527

    View details for Web of Science ID 000284424000064

    View details for PubMedID 20870725

  • Methylation of the Retinoblastoma Tumor Suppressor by SMYD2 JOURNAL OF BIOLOGICAL CHEMISTRY Saddic, L. A., West, L. E., Aslanian, A., Yates, J. R., Rubin, S. M., Gozani, O., Sage, J. 2010; 285 (48): 37733-37740

    Abstract

    The retinoblastoma tumor suppressor (RB) is a central cell cycle regulator and tumor suppressor. RB cellular functions are known to be regulated by a diversity of post-translational modifications such as phosphorylation and acetylation, raising the possibility that RB may also be methylated in cells. Here we demonstrate that RB can be methylated by SMYD2 at lysine 860, a highly conserved and novel site of modification. This methylation event occurs in vitro and in cells, and it is regulated during cell cycle progression, cellular differentiation, and in response to DNA damage. Furthermore, we show that RB monomethylation at lysine 860 provides a direct binding site for the methyl-binding domain of the transcriptional repressor L3MBTL1. These results support the idea that a code of post-translational modifications exists for RB and helps guide its functions in mammalian cells.

    View details for DOI 10.1074/jbc.M110.137612

    View details for Web of Science ID 000284424000065

    View details for PubMedID 20870719

  • Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9 Regulation of lysine methyltransferases by physical interaction with their substrates EPIGENETICS Binda, O., Leroy, G., Bua, D. J., Garcia, B. A., Gozani, O., Richard, S. 2010; 5 (8): 767-775

    Abstract

    Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4(me3)) while heterochromatin contains the H3K9(me3) marks. The H3K9(me3) modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4(me3). In addition, we show that SETDB1 fails to methylate substrates containing the H3K4(me3) mark. Likewise, the functionally related H3K9 KMTs G9A, GLP, and SUV39H1 also fail to bind and to methylate H3K4(me3) substrates. Accordingly, we provide in vivo evidence that H3K9(me2)-enriched histones are devoid of H3K4(me2/3) and that histones depleted of H3K4(me2/3) have elevated H3K9(me2/3). The correlation between the loss of interaction of these KMTs with H3K4 (me3) and concomitant methylation impairment leads to the postulate that, at least these four KMTs, require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1?. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4(me3) and H3K9(me3). Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.

    View details for DOI 10.4161/epi.5.8.13278

    View details for Web of Science ID 000284263500013

    View details for PubMedID 21124070

  • Decoding Chromatin Goes High Tech CELL Levy, D., Gozani, O. 2010; 142 (6): 844-846

    Abstract

    Identifying proteins that recognize histone methylation is critical for understanding chromatin function. Vermeulen et al. (2010) now describe a cutting-edge strategy to identify and characterize several nuclear proteins and complexes that recognize five major histone trimethyl marks.

    View details for DOI 10.1016/j.cell.2010.08.032

    View details for Web of Science ID 000281855000007

    View details for PubMedID 20850007

  • Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans NATURE Greer, E. L., Maures, T. J., Hauswirth, A. G., Green, E. M., Leeman, D. S., Maro, G. S., Han, S., Banko, M. R., Gozani, O., Brunet, A. 2010; 466 (7304): 383-U137

    Abstract

    The plasticity of ageing suggests that longevity may be controlled epigenetically by specific alterations in chromatin state. The link between chromatin and ageing has mostly focused on histone deacetylation by the Sir2 family, but less is known about the role of other histone modifications in longevity. Histone methylation has a crucial role in development and in maintaining stem cell pluripotency in mammals. Regulators of histone methylation have been associated with ageing in worms and flies, but characterization of their role and mechanism of action has been limited. Here we identify the ASH-2 trithorax complex, which trimethylates histone H3 at lysine 4 (H3K4), as a regulator of lifespan in Caenorhabditis elegans in a directed RNA interference (RNAi) screen in fertile worms. Deficiencies in members of the ASH-2 complex-ASH-2 itself, WDR-5 and the H3K4 methyltransferase SET-2-extend worm lifespan. Conversely, the H3K4 demethylase RBR-2 is required for normal lifespan, consistent with the idea that an excess of H3K4 trimethylation-a mark associated with active chromatin-is detrimental for longevity. Lifespan extension induced by ASH-2 complex deficiency requires the presence of an intact adult germline and the continuous production of mature eggs. ASH-2 and RBR-2 act in the germline, at least in part, to regulate lifespan and to control a set of genes involved in lifespan determination. These results indicate that the longevity of the soma is regulated by an H3K4 methyltransferase/demethylase complex acting in the C. elegans germline.

    View details for DOI 10.1038/nature09195

    View details for Web of Science ID 000279867100052

    View details for PubMedID 20555324

  • Binding of the MLL PHD3 Finger to Histone H3K4me3 Is Required for MLL-Dependent Gene Transcription JOURNAL OF MOLECULAR BIOLOGY Chang, P., Hom, R. A., Musselman, C. A., Zhu, L., Kuo, A., Gozani, O., Kutateladze, T. G., Cleary, M. L. 2010; 400 (2): 137-144

    Abstract

    The MLL (mixed-lineage leukemia) proto-oncogene encodes a histone methyltransferase that creates the methylated histone H3K4 epigenetic marks, commonly associated with actively transcribed genes. In addition to its canonical histone methyltransferase SET domain, the MLL protein contains three plant homeodomain (PHD) fingers that are well conserved between species but whose potential roles and requirements for MLL function are unknown. Here, we demonstrate that the third PHD domain of MLL (PHD3) binds histone H3 trimethylated at lysine 4 (H3K4me3) with high affinity and specificity and H3K4me2 with 8-fold lower affinity. Biochemical and structural analyses using NMR and fluorescence spectroscopy identified key amino acids essential for the interaction with H3K4me3. Site-directed mutations of the residues involved in recognition of H3K4me3 compromised in vitro H3K4me3 binding but not in vivo localization of full-length MLL to chromatin sites in target promoters of MEIS1 and HOXA genes. Whereas intact PHD3 finger was necessary for MLL occupancy at these promoters, H3K4me3 binding was critical for MLL transcriptional activity. These results demonstrate that MLL occupancy and target gene activation can be functionally separated. Furthermore, these findings reveal that MLL not only "writes" the H3K4me3 mark but also binds the mark, and this binding is required for the transcriptional maintenance functions of MLL.

    View details for DOI 10.1016/j.jmb.2010.05.005

    View details for Web of Science ID 000279786900003

    View details for PubMedID 20452361

  • Molecular Mechanism of MLL PHD3 and RNA Recognition by the Cyp33 RRM Domain JOURNAL OF MOLECULAR BIOLOGY Hom, R. A., Chang, P., Roy, S., Musselman, C. A., Glass, K. C., Selezneva, A. I., Gozani, O., Ismagilov, R. F., Cleary, M. L., Kutateladze, T. G. 2010; 400 (2): 145-154

    Abstract

    The nuclear protein cyclophilin 33 (Cyp33) is a peptidyl-prolyl cis-trans isomerase that catalyzes cis-trans isomerization of the peptide bond preceding a proline and promotes folding and conformational changes in folded and unfolded proteins. The N-terminal RNA-recognition motif (RRM) domain of Cyp33 has been found to associate with the third plant homeodomain (PHD3) finger of the mixed lineage leukemia (MLL) proto-oncoprotein and a poly(A) RNA sequence. Here, we report a 1.9 A resolution crystal structure of the RRM domain of Cyp33 and describe the molecular mechanism of PHD3 and RNA recognition. The Cyp33 RRM domain folds into a five-stranded antiparallel beta-sheet and two alpha-helices. The RRM domain, but not the catalytic module of Cyp33, binds strongly to PHD3, exhibiting a 2 muM affinity as measured by isothermal titration calorimetry. NMR chemical shift perturbation (CSP) analysis and dynamics data reveal that the beta strands and the beta2-beta3 loop of the RRM domain are involved in the interaction with PHD3. Mutations in the PHD3-binding site or deletions in the beta2-beta3 loop lead to a significantly reduced affinity or abrogation of the interaction. The RNA-binding pocket of the Cyp33 RRM domain, mapped on the basis of NMR CSP and mutagenesis, partially overlaps with the PHD3-binding site, and RNA association is abolished in the presence of MLL PHD3. Full-length Cyp33 acts as a negative regulator of MLL-induced transcription and reduces the expression levels of MLL target genes MEIS1 and HOXA9. Together, these in vitro and in vivo data provide insight into the multiple functions of Cyp33 RRM and suggest a Cyp33-dependent mechanism for regulating the transcriptional activity of MLL.

    View details for DOI 10.1016/j.jmb.2010.04.067

    View details for Web of Science ID 000279786900004

    View details for PubMedID 20460131

  • Negative Regulation of Hypoxic Responses via Induced Reptin Methylation MOLECULAR CELL Lee, J. S., Kim, Y., Kim, I. S., Kim, B., Choi, H. J., Lee, J. M., Shin, H. R., Kim, J. H., Kim, J., Seo, S., Lee, H., Binda, O., Gozani, O., Semenza, G. L., Kim, M., Il Kim, K., Hwang, D., Baek, S. H. 2010; 39 (1): 71-85

    Abstract

    Lysine methylation within histones is crucial for transcriptional regulation and thus links chromatin states to biological outcomes. Although recent studies have extended lysine methylation to nonhistone proteins, underlying molecular mechanisms such as the upstream signaling cascade that induces lysine methylation and downstream target genes modulated by this modification have not been elucidated. Here, we show that Reptin, a chromatin-remodeling factor, is methylated at lysine 67 in hypoxic conditions by the methyltransferase G9a. Methylated Reptin binds to the promoters of a subset of hypoxia-responsive genes and negatively regulates transcription of these genes to modulate cellular responses to hypoxia.

    View details for DOI 10.1016/j.molcel.2010.06.008

    View details for Web of Science ID 000280139200009

    View details for PubMedID 20603076

  • Structural Insight into p53 Recognition by the 53BP1 Tandem Tudor Domain JOURNAL OF MOLECULAR BIOLOGY Roy, S., Musselman, C. A., Kachirskaia, I., Hayashi, R., Glass, K. C., Nix, J. C., Gozani, O., Appella, E., Kutateladze, T. G. 2010; 398 (4): 489-496

    Abstract

    The tumor suppressor p53 and the DNA repair factor 53BP1 (p53 binding protein 1) regulate gene transcription and responses to genotoxic stresses. Upon DNA damage, p53 undergoes dimethylation at Lys382 (p53K382me2), and this posttranslational modification is recognized by 53BP1. The molecular mechanism of nonhistone methyl-lysine mark recognition remains unknown. Here we report a 1. 6-A-resolution crystal structure of the tandem Tudor domain of human 53BP1 bound to a p53K382me2 peptide. In the complex, dimethylated Lys382 is restrained by a set of hydrophobic and cation-pi interactions in a cage formed by four aromatic residues and an aspartate of 53BP1. The signature HKKme2 motif of p53, which defines specificity, is identified through a combination of NMR resonance perturbations, mutagenesis, measurements of binding affinities and docking simulations, and analysis of the crystal structures of 53BP1 bound to p53 peptides containing other dimethyl-lysine marks, p53K370me2 (p53 dimethylated at Lys370) and p53K372me2 (p53 dimethylated at Lys372). Binding of the 53BP1 Tudor domain to p53K382me2 may facilitate p53 accumulation at DNA damage sites and promote DNA repair as suggested by chromatin immunoprecipitation and DNA repair assays. Together, our data detail the molecular mechanism of p53-53BP1 association and provide the basis for deciphering the role of this interaction in the regulation of p53 and 53BP1 functions.

    View details for DOI 10.1016/j.jmb.2010.03.024

    View details for Web of Science ID 000277895500003

    View details for PubMedID 20307547

  • A Functional Link between the Histone Demethylase PHF8 and the Transcription Factor ZNF711 in X-Linked Mental Retardation MOLECULAR CELL Kleine-Kohlbrecher, D., Christensen, J., Vandamme, J., Abarrategui, I., Bak, M., Tommerup, N., Shi, X., Gozani, O., Rappsilber, J., Salcini, A. E., Helin, K. 2010; 38 (2): 165-178

    Abstract

    X-linked mental retardation (XLMR) is an inherited disorder that mostly affects males and is caused by mutations in genes located on the X chromosome. Here, we show that the XLMR protein PHF8 and a C. elegans homolog F29B9.2 catalyze demethylation of di- and monomethylated lysine 9 of histone H3 (H3K9me2/me1). The PHD domain of PHF8 binds to H3K4me3 and colocalizes with H3K4me3 at transcription initiation sites. Furthermore, PHF8 interacts with another XMLR protein, ZNF711, which binds to a subset of PHF8 target genes, including the XLMR gene JARID1C. Of interest, the C. elegans PHF8 homolog is highly expressed in neurons, and mutant animals show impaired locomotion. Taken together, our results functionally link the XLMR gene PHF8 to two other XLMR genes, ZNF711 and JARID1C, indicating that MR genes may be functionally linked in pathways, causing the complex phenotypes observed in patients developing MR.

    View details for DOI 10.1016/j.molcel.2010.03.002

    View details for Web of Science ID 000277598100005

    View details for PubMedID 20346720

  • The Target of the NSD Family of Histone Lysine Methyltransferases Depends on the Nature of the Substrate JOURNAL OF BIOLOGICAL CHEMISTRY Li, Y., Trojer, P., Xu, C., Cheung, P., Kuo, A., Drury, W. J., Qiao, Q., Neubert, T. A., Xu, R., Gozani, O., Reinberg, D. 2009; 284 (49): 34283-34295

    Abstract

    The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target.

    View details for DOI 10.1074/jbc.M109.034462

    View details for Web of Science ID 000272165200059

    View details for PubMedID 19808676

  • Epigenome Microarray Platform for Proteome-Wide Dissection of Chromatin-Signaling Networks PLOS ONE Bua, D. J., Kuo, A. J., Cheung, P., Liu, C. L., Migliori, V., Espejo, A., Casadio, F., Bassi, C., Amati, B., Bedford, M. T., Guccione, E., Gozani, O. 2009; 4 (8)

    Abstract

    Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.

    View details for DOI 10.1371/journal.pone.0006789

    View details for Web of Science ID 000269335000031

    View details for PubMedID 19956676

  • Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6 CELL CYCLE Michishita, E., McCord, R. A., Boxer, L. D., Barber, M. F., Hong, T., Gozani, O., Chua, K. F. 2009; 8 (16): 2664-2666

    View details for Web of Science ID 000268983900036

    View details for PubMedID 19625767

  • NMR assignments and histone specificity of the ING2 PHD finger MAGNETIC RESONANCE IN CHEMISTRY Pena, P. V., Musselman, C. A., Kuo, A. J., Gozani, O., Kutateladze, T. G. 2009; 47 (4): 352-358

    Abstract

    The ING2 plant homeodomain (PHD) finger is recruited to the nucleosome through specific binding to histone H3 trimethylated at lysine 4 (H3K4me3). Here, we describe backbone and side chain assignments of the ING2 PHD finger, analyze its binding to the unmodified and modified histone and p53 peptides, and map the histone H3 and H3K4me3 binding sites based on chemical shift perturbation analysis.

    View details for DOI 10.1002/mrc.2390

    View details for Web of Science ID 000264514200011

    View details for PubMedID 19184981

  • 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

  • HBO1 HAT Complexes Target Chromatin throughout Gene Coding Regions via Multiple PHD Finger Interactions with Histone H3 Tail MOLECULAR CELL Saksouk, N., Avvakumov, N., Champagne, K. S., Hung, T., Doyon, Y., Cayrou, C., Paquet, E., Ullah, M., Landry, A., Cote, V., Yang, X., Gozani, O., Kutateladze, T. G., Cote, J. 2009; 33 (2): 257-265

    Abstract

    The HBO1 HAT protein is the major source of histone H4 acetylation in vivo and has been shown to play critical roles in gene regulation and DNA replication. A distinctive characteristic of HBO1 HAT complexes is the presence of three PHD finger domains in two different subunits: tumor suppressor proteins ING4/5 and JADE1/2/3. Biochemical and functional analyses indicate that these domains interact with histone H3 N-terminal tail region, but with a different specificity toward its methylation status. Their combinatorial action is essential in regulating chromatin binding and substrate specificity of HBO1 complexes, as well as cell growth. Importantly, localization analyses on the human genome indicate that HBO1 complexes are enriched throughout the coding regions of genes, supporting a role in transcription elongation. These results underline the importance and versatility of PHD finger domains in regulating chromatin association and histone modification crosstalk within a single protein complex.

    View details for DOI 10.1016/j.molcel.2009.01.007

    View details for Web of Science ID 000263204500016

    View details for PubMedID 19187766

  • SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair AGING-US McCord, R. A., Michishita, E., Hong, T., Berber, E., Boxer, L. D., Kusumoto, R., Guan, S., Shi, X., Gozani, O., Burlingame, A. L., Bohr, V. A., Chua, K. F. 2009; 1 (1): 109-121

    Abstract

    The Sir2 chromatin regulatory factor links maintenance of genomic stability to life span extension in yeast. The mammalian Sir2 family member SIRT6 has been proposed to have analogous functions, because SIRT6-deficiency leads to shortened life span and an aging-like degenerative phenotype in mice, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor with chromatin impacts on the efficiency of repair, and establish a link between chromatin regulation, DNA repair, and a mammalian Sir2 factor.

    View details for Web of Science ID 000276347200013

    View details for PubMedID 20157594

  • Role for 53BP1 Tudor Domain Recognition of p53 Dimethylated at Lysine 382 in DNA Damage Signaling JOURNAL OF BIOLOGICAL CHEMISTRY Kachirskaia, I., Shi, X., Yamaguchi, H., Tanoue, K., Wen, H., Wang, E. W., Appella, E., Gozani, O. 2008; 283 (50): 34660-34666

    Abstract

    Modification of histone proteins by lysine methylation is a principal chromatin regulatory mechanism (Shi, Y., and Whetstine, J. R. (2007) Mol. Cell 25, 1-14). Recently, lysine methylation has been shown also to play a role in regulating non-histone proteins, including the tumor suppressor protein p53 (Huang, J., and Berger, S. L. (2008) Curr. Opin. Genet. Dev. 18, 152-158). Here, we identify a novel p53 species that is dimethylated at lysine 382 (p53K382me2) and show that the tandem Tudor domain of the DNA damage response mediator 53BP1 acts as an "effector" for this mark. We demonstrate that the 53BP1 tandem Tudor domain recognizes p53K382me2 with a selectivity relative to several other protein lysine methylation sites and saturation states. p53K382me2 levels increase with DNA damage, and recognition of this modification by 53BP1 facilitates an interaction between p53 and 53BP1. The generation of p53K382me2 promotes the accumulation of p53 protein that occurs upon DNA damage, and this increase in p53 levels requires 53BP1. Taken together, our study identifies a novel p53 modification, demonstrates a new effector function for the 53BP1 tandem Tudor domain, and provides insight into how DNA damage signals are transduced to stabilize p53.

    View details for DOI 10.1074/jbc.M806020200

    View details for Web of Science ID 000261469100021

    View details for PubMedID 18840612

  • Aire employs a histone-binding module to mediate immunological tolerance, linking chromatin regulation with organ-specific autoimmunity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Koh, A. S., Kuo, A. J., Park, S. Y., Cheung, P., Abramson, J., Bua, D., Carney, D., Shoelson, S. E., Gozani, O., Kingston, R. E., Benoist, C., Mathis, D. 2008; 105 (41): 15878-15883

    Abstract

    Aire induces ectopic expression of peripheral tissue antigens (PTAs) in thymic medullary epithelial cells, which promotes immunological tolerance. Beginning with a broad screen of histone peptides, we demonstrate that the mechanism by which this single factor controls the transcription of thousands of genes involves recognition of the amino-terminal tail of histone H3, but not of other histones, by one of Aire's plant homeodomain (PHD) fingers. Certain posttranslational modifications of H3 tails, notably dimethylation or trimethylation at H3K4, abrogated binding by Aire, whereas others were tolerated. Similar PHD finger-H3 tail-binding properties were recently reported for BRAF-histone deacetylase complex 80 and DNA methyltransferase 3L; sequence alignment, molecular modeling, and biochemical analyses showed these factors and Aire to have structure-function relationships in common. In addition, certain PHD1 mutations underlying the polyendocrine disorder autoimmune polyendocrinopathy-candidiases-ectodermaldystrophy compromised Aire recognition of H3. In vitro binding assays demonstrated direct physical interaction between Aire and nucleosomes, which was in part buttressed by its affinity to DNA. In vivo Aire interactions with chromosomal regions depleted of H3K4me3 were dependent on its H3 tail-binding activity, and this binding was necessary but not sufficient for the up-regulation of genes encoding PTAs. Thus, Aire's activity as a histone-binding module mediates the thymic display of PTAs that promotes self-tolerance and prevents organ-specific autoimmunity.

    View details for DOI 10.1073/pnas.0808470105

    View details for Web of Science ID 000260240900044

    View details for PubMedID 18840680

  • Histone H3K4me3 binding is required for the DNA repair and apoptotic activities of ING1 tumor suppressor JOURNAL OF MOLECULAR BIOLOGY Pena, P. V., Hom, R. A., Hung, T., Lin, H., Kuo, A. J., Wong, R. P., Subach, O. M., Champagne, K. S., Zhao, R., Verkhusha, V. V., Li, G., Gozani, O., Kutateladze, T. G. 2008; 380 (2): 303-312

    Abstract

    Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair, and apoptosis. Mutations within the ING1 gene and altered expression levels of ING1 are found in multiple human cancers. Here, we show that both DNA repair and apoptotic activities of ING1 require the interaction of the C-terminal plant homeodomain (PHD) finger with histone H3 trimethylated at Lys4 (H3K4me3). The ING1 PHD finger recognizes methylated H3K4 but not other histone modifications as revealed by the peptide microarrays. The molecular mechanism of the histone recognition is elucidated based on a 2.1 A-resolution crystal structure of the PHD-H3K4me3 complex. The K4me3 occupies a deep hydrophobic pocket formed by the conserved Y212 and W235 residues that make cation-pi contacts with the trimethylammonium group. Both aromatic residues are essential in the H3K4me3 recognition, as substitution of these residues with Ala disrupts the interaction. Unlike the wild-type ING1, the W235A mutant, overexpressed in the stable clones of melanoma cells or in HT1080 cells, was unable to stimulate DNA repair after UV irradiation or promote DNA-damage-induced apoptosis, indicating that H3K4me3 binding is necessary for these biological functions of ING1. Furthermore, N216S, V218I, and G221V mutations, found in human malignancies, impair the ability of ING1 to associate with H3K4me3 or to induce nucleotide repair and cell death, linking the tumorigenic activity of ING1 with epigenetic regulation. Together, our findings reveal the critical role of the H3K4me3 interaction in mediating cellular responses to genotoxic stresses and offer new insight into the molecular mechanism underlying the tumor suppressive activity of ING1.

    View details for DOI 10.1016/j.jmb.2008.04.061

    View details for Web of Science ID 000257630000004

    View details for PubMedID 18533182

  • 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

  • RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination NATURE Matthews, A. G., Kuo, A. J., Ramon-Maiques, S., Han, S., Champagne, K. S., Ivanov, D., Gallardo, M., Carney, D., Cheung, P., Ciccone, D. N., Walter, K. L., Utz, P. J., Shi, Y., Kutateladze, T. G., Yang, W., Gozani, O., Oettinger, M. A. 2007; 450 (7172): 1106-U18

    Abstract

    Nuclear processes such as transcription, DNA replication and recombination are dynamically regulated by chromatin structure. Eukaryotic transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent post-translational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here we show that RAG2--an essential component of the RAG1/2 V(D)J recombinase, which mediates antigen-receptor gene assembly--contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the mouse RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together, our results identify a new function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence indicating that disrupting the read-out of histone modifications can cause an inherited human disease.

    View details for DOI 10.1038/nature06431

    View details for Web of Science ID 000251579900092

    View details for PubMedID 18033247

  • The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ramon-Maiques, S., Kuo, A. J., Carney, D., Matthews, A. G., Oettinger, M. A., Gozani, O., Yang, W. 2007; 104 (48): 18993-18998

    Abstract

    Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymphocytes as the first step in the assembly and maturation of antigen receptors. RAG2 contains a plant homeodomain (PHD) near its C terminus (RAG2-PHD) that recognizes histone H3 methylated at lysine 4 (H3K4me) and influences V(D)J recombination. We report here crystal structures of RAG2-PHD alone and complexed with five modified H3 peptides. Two aspects of RAG2-PHD are unique. First, in the absence of the modified peptide, a peptide N-terminal to RAG2-PHD occupies the substrate-binding site, which may reflect an autoregulatory mechanism. Second, in contrast to other H3K4me3-binding PHD domains, RAG2-PHD substitutes a carboxylate that interacts with arginine 2 (R2) with a Tyr, resulting in binding to H3K4me3 that is enhanced rather than inhibited by dimethylation of R2. Five residues involved in histone H3 recognition were found mutated in severe combined immunodeficiency (SCID) patients. Disruption of the RAG2-PHD structure appears to lead to the absence of T and B lymphocytes, whereas failure to bind H3K4me3 is linked to Omenn Syndrome. This work provides a molecular basis for chromatin-dependent gene recombination and presents a single protein domain that simultaneously recognizes two distinct histone modifications, revealing added complexity in the read-out of combinatorial histone modifications.

    View details for DOI 10.1073/pnas.0709170104

    View details for Web of Science ID 000251498700024

    View details for PubMedID 18025461

  • Modulation of p53 function by SET8-mediated methylation at lysine 382 MOLECULAR CELL Shi, X., Kachirskaia, L., Yamaguchi, H., West, L. E., Wen, H., Wang, E. W., Dutta, S., Appella, E., Gozani, O. 2007; 27 (4): 636-646

    Abstract

    Reversible covalent methylation of lysine residues on histone proteins constitutes a principal molecular mechanism that links chromatin states to diverse biological outcomes. Recently, lysine methylation has been observed on nonhistone proteins, suggesting broad cellular roles for the enzymes generating and removing methyl moieties. Here we report that the lysine methyltransferase enzyme SET8/PR-Set7 regulates the tumor suppressor protein p53. We find that SET8 specifically monomethylates p53 at lysine 382 (p53K382me1). This methylation event robustly suppresses p53-mediated transcription activation of highly responsive target genes but has little influence on weak targets. Further, depletion of SET8 augments the proapoptotic and checkpoint activation functions of p53, and accordingly, SET8 expression is downregulated upon DNA damage. Together, our study identifies SET8 as a p53-modifying enzyme, identifies p53K382me1 as a regulatory posttranslational modification of p53, and begins to dissect how methylation may contribute to a dynamic posttranslational code that modulates distinct p53 functions.

    View details for DOI 10.1016/j.molcel.2007.07.012

    View details for Web of Science ID 000249050200014

    View details for PubMedID 17707234

  • Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene repression NATURE Lan, F., Collins, R. E., De Cegli, R., Alpatov, R., Horton, J. R., Shi, X., Gozani, O., Cheng, X., Shi, Y. 2007; 448 (7154): 718-U14

    Abstract

    Histone methylation is crucial for regulating chromatin structure, gene transcription and the epigenetic state of the cell. LSD1 is a lysine-specific histone demethylase that represses transcription by demethylating histone H3 on lysine 4 (ref. 1). The LSD1 complex contains a number of proteins, all of which have been assigned roles in events upstream of LSD1-mediated demethylation apart from BHC80 (also known as PHF21A), a plant homeodomain (PHD) finger-containing protein. Here we report that, in contrast to the PHD fingers of the bromodomain PHD finger transcription factor (BPTF) and inhibitor of growth family 2 (ING2), which bind methylated H3K4 (H3K4me3), the PHD finger of BHC80 binds unmethylated H3K4 (H3K4me0), and this interaction is specifically abrogated by methylation of H3K4. The crystal structure of the PHD finger of BHC80 bound to an unmodified H3 peptide has revealed the structural basis of the recognition of H3K4me0. Knockdown of BHC80 by RNA inhibition results in the de-repression of LSD1 target genes, and this repression is restored by the reintroduction of wild-type BHC80 but not by a PHD-finger mutant that cannot bind H3. Chromatin immunoprecipitation showed that BHC80 and LSD1 depend reciprocally on one another to associate with chromatin. These findings couple the function of BHC80 to that of LSD1, and indicate that unmodified H3K4 is part of the 'histone code'. They further raise the possibility that the generation and recognition of the unmodified state on histone tails in general might be just as crucial as post-translational modifications of histone for chromatin and transcriptional regulation.

    View details for DOI 10.1038/nature06034

    View details for Web of Science ID 000248598000050

    View details for PubMedID 17687328

  • Stabilized phosphatidylinositol-5-phosphate analogues as ligands for the nuclear protein ING2: Chemistry, biology, and molecular modeling JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Huang, W., Zhang, H., Davrazou, F., Kutateladze, T. G., Shi, X., Gozani, O., Prestwich, G. D. 2007; 129 (20): 6498-6506

    Abstract

    The interaction of PtdIns(5)P with the tumor suppressor protein ING2 has been implicated in the regulation of chromatin modification. To enhance the stability of PtdIns(5)P for studies of the biological role in vivo, two phosphatase-resistant moieties were used to replace the labile 5-phosphate. The total asymmetric synthesis of the 5-methylenephosphonate (MP) and 5-phosphothionate (PT) analogues of PtdIns(5)P is described herein, and the resulting metabolically stabilized lipid analogues were evaluated in three ways. First, liposomes containing either the dioleoyl MP or PT analogues bound to recombinant ING2 similar to liposomes containing dipalmitoyl PtdIns(5)P, indicating that the replacement of the hydrolyzable 5-phosphate group does not compromise the binding. Second, the dioleoyl MP and PT PtdIns(5)P analogues were equivalent to dipalmitoyl PtdIns(5)P in augmenting cell death induced by a DNA double-strand break in HT1080 cells. Finally, molecular modeling and docking of the MP or PT analogues to the C-terminus PtdInsP-binding region of ING2 (consisting of a PHD finger and a polybasic region) revealed a number of complementary surface and electrostatic contacts between the lipids and ING2.

    View details for DOI 10.1021/ja070195b

    View details for Web of Science ID 000246535300040

    View details for PubMedID 17469822

  • Proteome-wide analysis in Saccharomyces cerevisiae identifies several PHD fingers as novel direct and selective binding modules of histone H3 methylated at either lysine 4 or lysine 36 JOURNAL OF BIOLOGICAL CHEMISTRY Shi, X., Kachirskaia, I., Walter, K. L., Kuo, J. A., Lake, A., Davrazou, F., Chan, S. M., Martin, D. G., Fingerman, I. M., Briggs, S. D., Howe, L., Utz, P. J., Kutateladze, T. G., Lugovskoy, A. A., Bedford, M. T., Gozani, O. 2007; 282 (4): 2450-2455

    Abstract

    The PHD finger motif is a signature chromatin-associated motif that is found throughout eukaryotic proteomes. Here we have determined the histone methyl-lysine binding activity of the PHD fingers present within the Saccharomyces cerevisiae proteome. We provide evidence on the genomic scale that PHD fingers constitute a general class of effector modules for histone H3 trimethylated at lysine 4 (H3K4me3) and histone H3 trimethylated at lysine 36 (H3K36me3). Structural modeling of PHD fingers demonstrates a conserved mechanism for recognizing the trimethyl moiety and provides insight into the molecular basis of affinity for the different methyl-histone ligands. Together, our study suggests that a common function for PHD fingers is to transduce methyl-lysine events and sheds light on how a single histone modification can be linked to multiple biological outcomes.

    View details for DOI 10.1074/jbc.C600286200

    View details for Web of Science ID 000243593200036

    View details for PubMedID 17142463

  • The Yng1p plant homeodomain finger is a methyl-histone binding module that recognizes lysine 4-methylated histone H3 MOLECULAR AND CELLULAR BIOLOGY Martin, D. G., Baetz, K., Shi, X., Walter, K. L., MacDonald, V. E., Wlodarski, M. J., Gozani, O., Hieter, P., Howe, L. 2006; 26 (21): 7871-7879

    Abstract

    The ING (inhibitor of growth) protein family includes a group of homologous nuclear proteins that share a highly conserved plant homeodomain (PHD) finger domain at their carboxyl termini. Members of this family are found in multiprotein complexes that posttranslationally modify histones, suggesting that these proteins serve a general role in permitting various enzymatic activities to interact with nucleosomes. There are three members of the ING family in Saccharomyces cerevisiae: Yng1p, Yng2p, and Pho23p. Yng1p is a component of the NuA3 histone acetyltransferase complex and is required for the interaction of NuA3 with chromatin. To gain insight into the function of the ING proteins, we made use of a genetic strategy to identify genes required for the binding of Yng1p to histones. Using the toxicity of YNG1 overexpression as a tool, we showed that Yng1p interacts with the amino-terminal tail of histone H3 and that this interaction can be disrupted by loss of lysine 4 methylation within this tail. Additionally, we mapped the region of Yng1p required for overexpression of toxicity to the PHD finger, showed that this region capable of binding lysine 4-methylated histone H3 in vitro, and demonstrated that mutations of the PHD finger that abolish binding in vitro are no longer toxic in vivo. These results identify a novel function for the Yng1p PHD finger in promoting stabilization of the NuA3 complex at chromatin through recognition of histone H3 lysine 4 methylation.

    View details for DOI 10.1128/MCB.00573-06

    View details for Web of Science ID 000241462400005

    View details for PubMedID 16923967

  • Molecular mechanism of histone H3K4me3 recognition by plant homeodomain of ING2 NATURE Pena, P. V., Davrazou, F., Shi, X., Walter, K. L., Verkhusha, V. V., Gozani, O., Zhao, R., Kutateladze, T. G. 2006; 442 (7098): 100-103

    Abstract

    Covalent modifications of histone tails have a key role in regulating chromatin structure and controlling transcriptional activity. In eukaryotes, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with active chromatin and gene expression. We recently found that plant homeodomain (PHD) finger of tumour suppressor ING2 (inhibitor of growth 2) binds H3K4me3 and represents a new family of modules that target this epigenetic mark. The molecular mechanism of H3K4me3 recognition, however, remains unknown. Here we report a 2.0 A resolution structure of the mouse ING2 PHD finger in complex with a histone H3 peptide trimethylated at lysine 4. The H3K4me3 tail is bound in an extended conformation in a deep and extensive binding site consisting of elements that are conserved among the ING family of proteins. The trimethylammonium group of Lys 4 is recognized by the aromatic side chains of Y215 and W238 residues, whereas the intermolecular hydrogen-bonding and complementary surface interactions, involving Ala 1, Arg 2, Thr 3 and Thr 6 of the peptide, account for the PHD finger's high specificity and affinity. Substitution of the binding site residues disrupts H3K4me3 interaction in vitro and impairs the ability of ING2 to induce apoptosis in vivo. Strong binding of other ING and YNG PHD fingers suggests that the recognition of H3K4me3 histone code is a general feature of the ING/YNG proteins. Elucidation of the mechanisms underlying this novel function of PHD fingers provides a basis for deciphering the role of the ING family of tumour suppressors in chromatin regulation and signalling.

    View details for DOI 10.1038/nature04814

    View details for Web of Science ID 000238724500045

    View details for PubMedID 16728977

  • PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection EMBO JOURNAL Pendaries, C., Tronchere, H., Arbibe, L., Mounier, J., Gozani, O., Cantley, L., Fry, M. J., Gaits-Iacovoni, F., Sansonetti, P. J., Payrastre, B. 2006; 25 (5): 1024-1034

    Abstract

    The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4-phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns5P). We show that PtdIns5P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri-induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short-chain penetrating PtdIns5P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns5P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD-expressing cells. Accordingly, IpgD and PtdIns5P production specifically activates a class IA PI 3-kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3-kinase/Akt activation via PtdIns5P production that plays an important role in host cell responses such as survival.

    View details for DOI 10.1038/sj.emboj.7601001

    View details for Web of Science ID 000236225400010

    View details for PubMedID 16482216

  • The fellowships of the INGs JOURNAL OF CELLULAR BIOCHEMISTRY Shi, X. B., Gozani, O. 2005; 96 (6): 1127-1136

    Abstract

    The inhibitor of growth (ING) family of proteins is an evolutionarily conserved family, with members present from yeast to humans. The mammalian ING proteins are candidate tumor suppressor proteins and accordingly can cooperate with p53 to arrest proliferation and induce apoptosis. ING proteins are also reported to function in the promotion of cellular senescence, the regulation of DNA damage responses and the inhibition of angiogenesis. At the molecular level, ING proteins are thought to function as chromatin regulatory molecules, acting as co-factors for distinct histone and factor acetyl-transferase (H/FAT) and deacetylase (HDAC) enzyme complexes. Further, ING proteins interact with a number of additional proteins involved in the regulation of critical nuclear processes, such as gene expression and DNA replication, and also function as nuclear phosphoinositide (PtdInsP) receptors. Despite the increasing number of known molecular interacting partners for ING proteins, the specific biochemical action of mammalian ING proteins and its relationship to tumor suppression remain elusive. In this Prospect, we summarize the present understanding of the binding partners and physiologic roles of ING proteins and propose a general molecular paradigm for how ING proteins might function to prevent cancer.

    View details for DOI 10.1002/jcb.20625

    View details for Web of Science ID 000233353000003

    View details for PubMedID 16167330

  • A PHD finger motif in the C terminus of RAG2 modulates recombination activity JOURNAL OF BIOLOGICAL CHEMISTRY Elkin, S. K., Ivanov, D., Ewalt, M., Ferguson, C. G., Hyberts, S. G., Sun, Z. Y., Prestwich, G. D., Yuan, J. Y., Wagner, G., Oettinger, M. A., Gozani, O. P. 2005; 280 (31): 28701-28710

    Abstract

    The RAG1 and RAG2 proteins catalyze V(D)J recombination and are essential for generation of the diverse repertoire of antigen receptor genes and effective immune responses. RAG2 is composed of a "core" domain that is required for the recombination reaction and a C-terminal nonessential or "non-core" region. Recent evidence has emerged arguing that the non-core region plays a critical regulatory role in the recombination reaction, and mutations in this region have been identified in patients with immunodeficiencies. Here we present the first structural data for the RAG2 protein, using NMR spectroscopy to demonstrate that the C terminus of RAG2 contains a noncanonical PHD finger. All of the non-core mutations of RAG2 that are implicated in the development of immunodeficiencies are located within the PHD finger, at either zinc-coordinating residues or residues adjacent to an alpha-helix on the surface of the domain that participates in binding to the signaling molecules, phosphoinositides. Functional analysis of disease and phosphoinositide-binding mutations reveals novel intramolecular interactions within the non-core region and suggests that the PHD finger adopts two distinct states. We propose a model in which the equilibrium between these states modulates recombination activity. Together, these data identify the PHD finger as a novel and functionally important domain of RAG2.

    View details for DOI 10.1074/jbc.M504731200

    View details for Web of Science ID 000230857300061

    View details for PubMedID 15964836

  • Modification of protein sub-nuclear localization by synthetic phosphoinositides: Evidence for nuclear phosphoinositide signaling mechanisms ADVANCES IN ENZYME REGULATION, VOL 45 Gozani, O., Field, S. J., Ferguson, C. G., Ewalt, M., Mahlke, C., Cantley, L. C., Prestwich, G. D., Yuan, J. Y. 2005; 45: 171-185

    Abstract

    PtdInsPs are critical signaling molecules that regulate diverse cellular functions. One method to study PtdInsP biology involves using synthetic PtdInsP analogs to activate endogenous PtdInsP-mediated events in living cells. Such methodology has been successfully employed to explore the role of several PtdInsP-biological outcomes in the cytoplasm. However, this strategy has not previously been used to examine the function of PtdInsPs in the nucleus of live cells, primarily because there has not been a well-defined PtdInsP-binding protein to provide functional nuclear readouts. Here we have shown that synthetic PtdIns(5)P analogs access and function in the nucleus. We have found that these molecules modify the sub-nuclear localization of PHD finger-containing proteins in live cells and in real time. This work demonstrates that synthetic PtdInsPs and PtdInsP derivatives may be powerful tools for probing nuclear PtdInsP functions. Finally, our work supports a model that endogenous PtdInsPs regulate sub-nuclear localization and function of endogenous nuclear PtdInsP-binding proteins.

    View details for DOI 10.1016/j.advenzreg.2005.02.010

    View details for Web of Science ID 000235349900013

    View details for PubMedID 16199078

  • The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor CELL Gozani, O., Karuman, P., Jones, D. R., Ivanov, D., Cha, J., Lugovskoy, A. A., Baird, C. L., Zhu, H., Field, S. J., Lessnick, S. L., Villasenor, J., Mehrotra, B., Chen, J., Rao, V. R., Brugge, J. S., Ferguson, C. G., Payrastre, B., Myszka, D. G., Cantley, L. C., Wagner, G., Divecha, N., Prestwich, G. D., Yuan, J. Y. 2003; 114 (1): 99-111

    Abstract

    Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that the PHD fingers of ING2 and other diverse nuclear proteins bind in vitro to PtdInsPs, including the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we demonstrate that the ING2 PHD finger interacts with PtdIns(5)P in vivo and provide evidence that this interaction regulates the ability of ING2 to activate p53 and p53-dependent apoptotic pathways. Together, our data identify the PHD finger as a phosphoinositide binding module and a nuclear PtdInsP receptor, and suggest that PHD-phosphoinositide interactions directly regulate nuclear responses to DNA damage.

    View details for Web of Science ID 000184219900009

    View details for PubMedID 12859901

  • The Peutz-Jegher gene product LKB1 is a mediator of p53-dependent cell death MOLECULAR CELL Karuman, P., Gozani, O., Odze, R. D., Zhou, X. C., Zhu, H., Shaw, R., Brien, T. P., Bozzuto, C. D., Ooi, D., Cantley, L. C., Yuan, J. Y. 2001; 7 (6): 1307-1319

    Abstract

    Here, we investigate the mechanism and function of LKB1, a Ser/Thr kinase mutated in Peutz-Jegher syndrome (PJS). We demonstrate that LKB1 physically associates with p53 and regulates specific p53-dependent apoptosis pathways. LKB1 protein is present in both the cytoplasm and nucleus of living cells and translocates to mitochondria during apoptosis. In vivo, LKB1 is highly upregulated in pyknotic intestinal epithelial cells. In contrast, polyps arising in Peutz-Jegher patients are devoid of LKB1 staining and have reduced numbers of apoptotic cells. We propose that a deficiency in apoptosis is a key factor in the formation of multiple benign intestinal polyps in PJS patients, and possibly for the subsequent development of malignant tumors in these patients.

    View details for Web of Science ID 000169547400017

    View details for PubMedID 11430832

  • Characterization of a protein complex containing spliceosomal proteins SAPS 49, 130, 145, and 155 MOLECULAR AND CELLULAR BIOLOGY Das, B. K., Xia, L., Palandjian, L., Gozani, O., Chyung, Y., Reed, R. 1999; 19 (10): 6796-6802

    Abstract

    SF3b is a U2 snRNP-associated protein complex essential for spliceosome assembly. Although evidence that SF3b contains the spliceosomal proteins SAPs 49, 130, 145, and 155 has accumulated, a protein-mediated association between all of these proteins has yet to be directly demonstrated. Here we report the isolation of a cDNA encoding SAP 130, which completes the cloning of the putative SF3b complex proteins. Using antibodies to SAP 130 and other putative SF3b components, we showed that SAPs 130, 145, and 155 are present in a protein complex in nuclear extracts and that these proteins associate with one another in purified U2 snRNP. Moreover, SAPs 155 and 130 interact with each other (directly or indirectly) within this complex, and SAPs 49 and 145 are known to interact directly with each other. Thus, together with prior work, our studies indicate that SAPs 49, 130, 145, and 155 are indeed components of SF3b. The Saccharomyces cerevisiae homologs of SAPs 49 and 145 are encoded by essential genes. We show here that the S. cerevisiae homologs of SAPs 130 and 155 (scSAP 130/RSE1 and scSAP 155, respectively) are also essential. Recently, the SF3b proteins were found in purified U12 snRNP, which functionally substitutes for U2 snRNP in the minor spliceosome. This high level of conservation, together with the prior observation that the SF3b proteins interact with pre-mRNA very close to the branch site, suggest that the SF3b complex plays a critical role near or at the spliceosome catalytic core.

    View details for Web of Science ID 000082660200032

    View details for PubMedID 10490618

  • Cyclin E associates with components of the pre-mRNA splicing machinery in mammalian cells MOLECULAR AND CELLULAR BIOLOGY Seghezzi, W., Chua, K., Shanahan, F., Gozani, O., Reed, R., Lees, E. 1998; 18 (8): 4526-4536

    Abstract

    Cyclin E-cdk2 is a critical regulator of cell cycle progression from G1 into S phase in mammalian cells. Despite this important function little is known about the downstream targets of this cyclin-kinase complex. Here we have identified components of the pre-mRNA processing machinery as potential targets of cyclin E-cdk2. Cyclin E-specific antibodies coprecipitated a number of cyclin E-associated proteins from cell lysates, among which are the spliceosome-associated proteins, SAP 114, SAP 145, and SAP 155, as well as the snRNP core proteins B' and B. The three SAPs are all subunits of the essential splicing factor SF3, a component of U2 snRNP. Cyclin E antibodies also specifically immunoprecipitated U2 snRNA and the spliceosome from splicing extracts. We demonstrate that SAP 155 serves as a substrate for cyclin E-cdk2 in vitro and that its phosphorylation in the cyclin E complex can be inhibited by the cdk-specific inhibitor p21. SAP 155 contains numerous cdk consensus phosphorylation sites in its N terminus and is phosphorylated prior to catalytic step II of the splicing pathway, suggesting a potential role for cdk regulation. These findings provide evidence that pre-mRNA splicing may be linked to the cell cycle machinery in mammalian cells.

    View details for Web of Science ID 000074950000013

    View details for PubMedID 9671462

  • A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site MOLECULAR AND CELLULAR BIOLOGY Gozani, O., Potashkin, J., Reed, R. 1998; 18 (8): 4752-4760

    Abstract

    Base pairing between U2 snRNA and the branchpoint sequence (BPS) is essential for pre-mRNA splicing. Because the metazoan BPS is short and highly degenerate, this interaction alone is insufficient for specific binding of U2 snRNP. The splicing factor U2AF binds to the pyrimidine tract at the 3' splice site in the earliest spliceosomal complex, E, and is essential for U2 snRNP binding in the spliceosomal complex A. We show that the U2 snRNP protein SAP 155 UV cross-links to pre-mRNA on both sides of the BPS in the A complex. SAP 155's downstream cross-linking site is immediately adjacent to the U2AF binding site, and the two proteins interact directly in protein-protein interaction assays. Using UV cross-linking, together with functional analyses of pre-mRNAs containing duplicated BPSs, we show a direct correlation between BPS selection and UV cross-linking of SAP 155 on both sides of the BPS. Together, our data are consistent with a model in which U2AF binds to the pyrimidine tract in the E complex and then interacts with SAP 155 to recruit U2 snRNP to the BPS.

    View details for Web of Science ID 000074950000036

    View details for PubMedID 9671485

  • Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis GENES & DEVELOPMENT Wang, C. Y., Chua, K., Seghezzi, W., Lees, E., Gozani, O., Reed, R. 1998; 12 (10): 1409-1414

    Abstract

    The U2 snRNP component SAP 155 contacts pre-mRNA on both sides of the branch site early in spliceosome assembly and is therefore positioned near or at the spliceosome catalytic center. We have isolated a cDNA encoding human SAP 155 and identified its highly related Saccharomyces cerevisiae homolog (50% identity). The carboxy-terminal two-thirds of SAP 155 shows the highest conservation and is remarkably similar to the regulatory subunit A of the phosphatase PP2A. Significantly, SAP 155 is phosphorylated concomitant with or just after catalytic step one, making this the first example of a protein modification tightly regulated with splicing catalysis.

    View details for Web of Science ID 000073793500003

    View details for PubMedID 9585501

  • Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly MOLECULAR AND CELLULAR BIOLOGY Chiara, M. D., Gozani, O., Bennett, M., CHAMPIONARNAUD, P., Palandjian, L., Reed, R. 1996; 16 (7): 3317-3326

    Abstract

    We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly. This was achieved by site-specifically labeling individual nucleotides within the 5' and 3' splice sites, the branchpoint sequence (BPS), or the exons with 32P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex. Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region approximately 25 nucleotides upstream from the 5' splice site. At the 5' splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5(200), was detected in both the B and C complexes. As observed in yeast cells, U5(200), also cross-links to intron/exon sequences at the 3' splice site in the C complex and may play a role in aligning the 5' and 3' exons for ligation. With label at the branch site, we detected three distinct proteins, designated BPS72,BpS70, and BPS56, which replace one another in the E, A, and C complexes. Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3' splice site. In this case, a protein, AG100,cross-links in the A complex and is replaced by another protein, AG75, in the C complex. The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway.

    View details for Web of Science ID A1996UT08600010

    View details for PubMedID 8668147

  • Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A GENES & DEVELOPMENT Gozani, O., Feld, R., Reed, R. 1996; 10 (2): 233-243

    Abstract

    A critical step in the pre-mRNA splicing reaction is the stable binding of U2 snRNP to the branchpoint sequence (BPS) to form the A complex. The multimeric U2 snRNP protein complexes SF3a and SF3b are required for A complex assembly, but their specific roles in this process are not known. Saccharomyces cerevisiae homologs of all of the SF3a, but none of the SF3b, subunits have been identified. Here we report the isolation of a cDNA encoding the mammalian SF3b subunit SAP 145 and the identification of its probable yeast homolog (29% identity). This first indication that the homology between yeast and metazoan A complex proteins can be extended to SF3b adds strong new evidence that the mechanism of A complex assembly is highly conserved. To investigate this mechanism in the mammalian system we analyzed proteins that cross-link to 32P-site-specifically labeled pre-mRNA in the A complex. This analysis revealed that SAP 145, together with four other SF3a/SF3b subunits, UV cross-links to pre-mRNA in a 20-nucleotide region upstream of the BPS. Mutation of this region, which we have designated the anchoring site, has no apparent effect on U2 snRNP binding. In contrast, when a 2'O methyl oligonucleotide complementary to the anchoring site is added to the spliceosome assembly reaction, A complex assembly and cross-linking of the SF3a/SF3b subunits are blocked. These data indicate that sequence-independent binding of the highly conserved SF3a/SF3b subunits upstream of the branch site is essential for anchoring U2 snRNP to pre-mRNA.

    View details for Web of Science ID A1996TT48900010

    View details for PubMedID 8566756

  • ACCUMULATION OF A NOVEL SPLICEOSOMAL COMPLEX ON PRE-MESSENGER-RNAS CONTAINING BRANCH SITE MUTATIONS MOLECULAR AND CELLULAR BIOLOGY CHAMPIONARNAUD, P., Gozani, O., Palandjian, L., Reed, R. 1995; 15 (10): 5750-5756

    Abstract

    Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of ATP. U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E* complex. Significantly, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E*-->A). In the first step, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.

    View details for Web of Science ID A1995RV77200059

    View details for PubMedID 7565727

  • A NOVEL SET OF SPLICEOSOME-ASSOCIATED PROTEINS AND THE ESSENTIAL SPLICING FACTOR PSF BIND STABLY TO PRE-MESSENGER-RNA PRIOR TO CATALYTIC STEP-II OF THE SPLICING REACTION EMBO JOURNAL Gozani, O., PATTON, J. G., Reed, R. 1994; 13 (14): 3356-3367

    Abstract

    We have isolated and determined the protein composition of the spliceosomal complex C. The pre-mRNA in this complex has undergone catalytic step I, but not step II, of the splicing reaction. We show that a novel set of 14 spliceosome-associated proteins (SAPs) and the essential splicing factor PSF are specifically associated with the C complex, implicating these proteins in catalytic step II. Significantly, immunodepletion and biochemical complementation studies demonstrate directly that PSF is essential for catalytic step II. Purified PSF is known to UV crosslink to pyrimidine tracts, and our data show that PSF UV crosslinks to pre-mRNA in purified C complex. Thus, PSF may replace the 3' splice site binding factor U2AF65 which is destabilized during spliceosome assembly. Finally, we show that SAPs 60 and 90, which are present in both the B and C complexes, are specifically associated with U4 and U6 snRNPs, and thus may have important roles in the functioning of these snRNPs during the splicing reaction.

    View details for Web of Science ID A1994NZ03600016

    View details for PubMedID 8045264

Conference Proceedings


  • Life and death in paradise. Gozani, O., Boyce, M., Yoo, L., Karuman, P., Yuan, J. 2002: E159-62

    Abstract

    Over 500 researchers participated in a recent American Association for Cancer Research special conference, entitled "Apoptosis and Cancer: Basic Mechanisms and Therapeutic Opportunities in the Post-Genomic Era" (February 13-17, 2002) in sunny Hawaii (Hilton Waikoloa village, Kona, Hawaii). The meeting participants presented the most recent findings on the mechanisms regulating cell death in cancer. In the past decade, apoptosis research has undergone a quantum leap, metamorphosing from a descriptive, phenomenological discipline into a molecularly defined, highly complex signalling field. This transformation was highlighted in the conference's opening talk by meeting co-organizer, John Reed (The Burnham Institute, La Jolla, CA). Reed and colleagues used published protein functional information and bio-informatic mining of the available human genome databases to tabulate the number of human proteins predicted to be involved in regulating apoptosis. The list includes 11 catalytically active caspases, 26 CARD (caspase associated recruitment domain)-, 32 DD (death domain)-, 12 DED (death effector domain)-, 8 BIR (baculovirus inhibitor of apoptosis protein region)-, 24 BH (Bcl-2 homology)-, and 34 PAAD/PYD (pyrin/PAAD)-containing sequences.

    View details for PubMedID 12042835

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