The Abu-Remaileh Lab is interested in identifying novel pathways that enable cellular and organismal adaptation to metabolic stress and changes in environmental conditions. We also study how these pathways go awry in human diseases such as cancer, neurodegeneration and metabolic syndrome, in order to engineer new therapeutic modalities. To address these questions, our lab uses a multidisciplinary approach to study the biochemical functions of the lysosome in vitro and in vivo. Lysosomes are membrane-bound compartments that degrade macromolecules and clear damaged organelles to enable cellular adaptation to various metabolic states. Lysosomal function is critical for organismal homeostasis—mutations in genes encoding lysosomal proteins cause severe human disorders known as lysosomal storage diseases, and lysosome dysfunction is implicated in age-associated diseases including cancer, neurodegeneration and metabolic syndrome. By developing novel tools and harnessing the power of metabolomics, proteomics and functional genomics, our lab will define 1) how the lysosome communicates with other cellular compartments to fulfill the metabolic demands of the cell under various metabolic states, 2) and how its dysfunction leads to rare and common human diseases. Using insights from our research, we will engineer novel therapies to modulate the pathways that govern human disease.
I am interested in the application of computational technologies to problems in molecular biology of relevance to medicine. In particular, my laboratory focuses on drug response at the molecular level, working in three areas. First, we are building a comprehensive pharmacogenomics knowledge base (http://www.pharmgkb.org/) that provides access to information relating genotype to phenotype (in particular, how variation in genetics leads to variation in response to drugs). We are interested in collaboratively discovering and applying new pharmacogenomics knowledge. Second, we are interested in the analysis of three dimensional biological structures. We have methods for analyzing protein structures to recognize and annotate active sites and binding sites, particularly in the context of interactions with small molecule drugs. We are also interested in physics-based simulation of biological structures to understand how their dynamics impact their function (http://simbios.stanford.edu/). Finally, we are interested in computational methods for analyzing functional genomics information. We use natural language processing techniques for extracting and summarizing information in the literature, chemoinformatics methods for understanding small molecule function, and machine learning & data mining techniques to understand the molecular responses to drugs.
The Ashley lab is focused on precision medicine. We develop methods for the interpretation of whole genome sequencing data to improve diagnosis of genetic disease and to personalize the practice of medicine. We love big data questions and systems approaches to biology especially analysis of network graphs. The wet bench is where we test causality of key genes and investigate the biology of network modules. It is also the focus of our translational efforts. Therapeutic development is a near term goal and several of our discoveries are the focus of patents or are being actively pursued by pharmaceutical and biotechnology partners.
The observations that the p53 gene is mutated in at least half of all human cancers of a wide variety of types and that p53 null mice develop cancer at 100% frequency together underscore the critical role for p53 in tumor suppression. Wild-type p53 is a cellular stress sensor, responding to diverse insults such as DNA damage, hyperproliferative signals, and hypoxia by inducing growth arrest or apoptosis, responses thought to be important to tumor suppression. At the molecular level, p53 acts a transcription factor that activates gene expression programs to induce these different responses. Interestingly, in its capacity as a cellular stress sensor, p53 also plays physiological roles beyond tumor suppression as well as causing certain pathological effects. For example, p53 plays beneficial roles such as promoting fertility, and can promote detrimental phenotypes in certain situations such as the side effects of cancer therapies or developmental diseases. The overarching goal of our research is to better define the mechanisms by which the p53 protein promotes different responses in different settings, ranging from tumor suppression to responses to chemotherapeutics, using the mouse as an in vivo model system, with the ultimate goal of gaining insight that may facilitate clinical advances in diagnosis, prognostication and therapy. We utilize a combination of mouse genetic, cell biological, biochemical, and genomic approaches to address understand how p53 acts mechanistically. We hope to decipher the transcriptional networks responsible for mediating p53 functions in different contexts, an understanding that will help us understand how to best promote the beneficial and minimize the detrimental effects of p53 in the clinic.
We have a number of specific areas of investigation, which include:
- Defining the transcriptional networks responsible for tumor suppression, using CRISPR/Cas9 and shRNA high-throughput genetic screening approaches
- Identifying p53-interacting partners by mass spectrometry approaches
- Elucidating the genes activated and repressed by p53 in diverse settings using genomic technologies such as ChIP-sequencing and RNA-sequencing, to understand how p53 drives different responses
- Identifying p53 inhibitors to find strategies to suppress the detrimental effects of p53 activation, such as during cancer therapy
- Understanding p53’s role in developmental diseases such as CHARGE syndrome
- Characterizing p53-regulated noncoding RNAs and their roles in cancer
- Examining mechanisms of p53 gain-of-function properties in cancer
We examine how cells communicate and function during fetal development. The work in my laboratory focuses on the establishment of specific cell fates using genomics to decipher interactions between chromatin and developmental signaling cascades, between genomes and rapidly evolving cell types, and between genomic copy number variation and gene expression. In recent years we have focused on the vastly understudied biology of the trophoblast lineage, particularly how this lineage evolved.
Our lab studies how intricate control of gene expression and cell signaling is regulated on a minute-by-minute basis to give rise to the remarkable diversity of cell types and tissue morphology that form the living blueprints of developing organisms. Work in the Barna lab is presently split into two main research efforts. The first is investigating ribosome-mediated control of gene expression genome-wide in space and time during cellular differentiation and organismal development. This research is opening a new field of study in which we apply sophisticated mass spectrometry, computational biology, genomics, and developmental genetics, to characterize a ribosome code to gene expression. Our research has shown that not all of the millions of ribosomes within a cell are the same and that ribosome heterogeneity can diversify how genomes are translated into proteomes. In particular, we seek to address whether fundamental aspects of gene regulation are controlled by ribosomes harboring a unique activity or composition that are tuned to translating specific transcripts by virtue of RNA regulatory elements embedded within their 5’UTRs. The second research effort is centered on employing state-of-the-art live cell imaging to visualize cell signaling and cellular control of organogenesis. This research has led to the realization of a novel means of cell-cell communication dependent on a dense network of actin-based cellular extension within developing organs that interconnect and facilitate the precise transmission of molecular information between cells. We apply and create bioengineering tools to manipulate such cellular interactions and signaling in-vivo.
Endocytic Pathogens as Tools and Targets
Endocytic pathogens such as protein aggregates, viruses, protein toxins, and bacteria have evolved remarkable ways to enter the cell, disrupt homeostasis, and cause cell death. We use these agents both as probes to understand normal cellular trafficking and signaling events, and to find key targets for therapy.
Stress Signaling to the Cell Death Machinery
Cells have elaborate mechanisms of sensing diverse stresses (oxidative damage, nutrient deprivation, DNA breaks, etc), and must either repair damage or induce cell death. Misregulation of these pathways results in diseases such as cancer and Alzheimer’s. We would like to understand how these signals connect to the death pathway in health and disease in order to improve therapies.
Technology Development and Genetic Interaction Maps
Customized genome-scale gene perturbation libraries: Much of the work we do utilizes genetic screens enabled by novel high-coverage CRISPR/Cas9 libraries (10 sgRNAs/gene) and shRNA libraries (25 shRNAs/gene) we have developed. The high coverage greatly reduces false positive and false negative results. Our platform allows for easy creation of new library designs, and we use a pooled format that can be used to rapidly screen genome-scale libraries in ~1-2 weeks. Libraries can then be analyzed by deep sequencing to quantify changes in sgRNA/shRNA abundance.
Systematic comparison of gene perturbation technologies: We have systematically compared the ability of genome-wide RNAi and CRISPR/Cas9 deletion screens to identify drug targets and essential genes, and have found important differences in their outputs. For example, while both screens perform well in detecting a gold standard set of essential genes, they can identify distinct essential biological processes. Moreover, each technology exhibits its own set of off-target effects and limitations in on-target efficacy.
Using what we have learned about these technologies, we recently created new genome-wide CRISPR libraries that incorporate a number of improved sgRNA features and controls. With a novel statistical framework (casTLE) we can model on- and off-target effects accurately, markedly improve hit detection, and even combine results from screens to improve performance and further limit false positives and false negatives. These studies have demonstrated the utility of parallel screening approaches using complementary technologies to reveal a more complete biological picture.
Systematic genetic interaction maps: We have also developed strategies to systematically knock down/knock out pairs of genes. This has facilitated some of the first systematic genetic interaction maps in mammalian cells. Using these maps, we can understand coordinated gene functions and predict new functions for uncharacterized genes. They also allow us to quickly identify synergistic interactions under stress conditions that we hope to exploit for combination therapies.
Directed evolution using dCas9-targeted somatic hypermutation (CRISPR-X): More recently, we developed a strategy to re-purpose the somatic hypermutation machinery used in antibody diversification to create targeted populations of point mutations. Using dCas9 to recruit a hyperactive variant of the deaminase AID, we can target diverse point mutations within an ~100bp window centered on the sgRNA PAM site. These mutant populations can then be subjected to selection to evolve proteins with improved function or to map the sites of drug-protein interactions. For example, by tiling mutations across PSMB5, we could map known and novel mutations that affect binding to the chemotherapeutic bortezomib.
Our lab seeks to exhaustively characterize the dynamics of the microbiome in patients with noncommunicable diseases (cancer, cardiometabolic disease), and to explore how changes in the microbiome are associated with clinical outcomes in this population.
Our Lab's Goals and Objectives:
a) We strive to better understand what microbial genes do, and how these genes are regulated
b) We hope to understand if shifts in the microbiome are associated with human disease phenotypes.
c) If alterations in the microbiome are associated with human disease phenotypes, develop methods to modify the composition of the microbiome or target specific microbial gene products with the hope of ameliorating these disease phenotypes.
My research aims to improve our understanding of how cells with the same genome can develop dramatically different behaviors. For example, consider the mechanical abilities of a muscle cell compared to the electrical excitability of a neuron, or the industrious activity of bone building cells in a youthful person compared to an elderly one. Each of these cells, (if taken from the same individual) has an identical genome — and yet each is “reading” a very distinct subset of that genome and consequently carrying out very different behaviors. The choice of what to read and what to hide away is made during development. An increasing body of data suggests this is accomplished by modifying the genome both in the nature of the proteins bound to different sequences and in the spatial organization of those sequences relative to each other. The spatial organization or folding of the genome may be particularly important in complex multicellular organisms, since many of the sequences known to interact based on genetic data are nonetheless substantially separated from each other along the linear genome. By regulating the folding of this linear sequence into a higher order structure, a cell might change which regulatory sequences have access to which genes, and achieve different behavioral states.
So far we have little imaging data on how the genome is folded within a cell on the length scale of individual genes, or whether this folding is regulated in any way relevant to the behavior of the cell. Our limited knowledge stems largely from want of a method that has both the resolution and specificity to visualize such genomic substructure. Conventional fluorescent microscopy has developed excellent tools for coloring specific regions of DNA and particular DNA-associated proteins with uniquely colored dyes — but lacks the resolution to turn these colored blurs into structures. Electron-microscopy has substantially greater resolution but lacks compatibility with specific labeling techniques to tell different gene clusters or different protein types apart. Super-resolution imaging approaches promise to address this balance by allowing the use of fluorescent labels while simultaneously resolving structures on the nano-scale. I have been adapting this approach to uncover the nano-scale structure of chromatin and determine to how this structure changes when bound by different types of nuclear proteins. While individual gene clusters appear as quite diverse structures, there appear to be a few general features, for example, linking structure with the epigenetic state.
The overarching goal of our lab is to understand the genetic mechanisms of aging and longevity. Aging is a highly plastic process regulated by a combination of genetic and environmental factors.
We have a long-standing interest in the genetic pathway that connects insulin to FOXO transcription factors, a central pathway to regulate lifespan from worms to humans. We use a combination of genetic, molecular, and cellular approaches to analyze the regulation and importance of FOXO transcription factors, and more generally 'longevity genes' in mammals. We are particularly interested in the role of longevity genes in the maintenance of the pool of adult neural stem cells and intact cognitive function during aging. We also use ultra-high throughput sequencing technologies to study epigenetic changes and transcriptional networks during aging.
In parallel, our goal is to identify novel longevity genes using short-lived animal models. Our lab uses unbiased approaches in the nematode C. elegans to identify novel pathways that control organismal longevity, particularly in response to dietary restriction. We are particularly interested in the role of chromatin modifiers in the regulation of lifespan and metabolism.
Finally, we are developing the extremely short-lived African killifish N. furzeri as a new vertebrate model for aging studies. We are taking advantage of this fish to explore the genetic architecture of longevity in vertebrates.
Nerve cells communicate by releasing the contents of neurotransmitter-bearing synaptic vesicles into the space between adjoining cells. This process depends on a handful of proteins that promote vesicle and nerve cell membrane fusion. The Brunger lab team uses structural and biophysical tools to capture this machinery at different stages of vesicle fusion. These structures (Figure 1) then provide the framework for further investigations, using microscopy and live neurons, into the functional and dynamic aspects of the system.
SNARE proteins, found in both nerve cell and vesicle membranes, set the stage for fusion by zipping together into a parallel, four-helix bundle that juxtaposes the two membranes. Brunger and his collaborators determined the first x-ray crystal structure of the neuronal SNARE complex, as well as the structures of other key components of the synaptic release machinery. Recently, the Brunger’s team visualized the SNARE complex bound to the Ca2+-sensor synaptotagmin-1 and to the regulator complexin, revealing two interfaces that are essential for fast synchronous release of neurotransmitters. The structure of this three-part complex suggests that it is in a primed and locked state. Action-potential-driven Ca2+ ions bind to the synaptotagmin proteins, unlock the complex, and trigger membrane fusion on a sub-millisecond timescale.
After fusion has occurred, SNARE complexes are recycled by the ATPase NSF, which breaks down the SNARE complex into its individual components. The Brunger team visualized this molecular machine at near-atomic level and obtained the first glimpses of how this SNARE-recycling machine works. The SNARE complex resembles a rope with a left-handed twist, and NSF uses adapter proteins called SNAPs to grasp the “rope” in multiple places. The SNAPs wrap around the SNARE complex with a right-handed twist, suggesting that the disassembly occurs via a simple unwinding motion that frees the zipped SNARE proteins.
The Brunger team is also using structural and functional studies to explore other machinery relevant to neurotransmitter release, such as factors involved in priming and pre-synaptic plasticity. Their research may one day provide new possibilities for targeting therapeutics to control neurotransmitter release.
My genetics research focuses on analyzing genome wide patterns of variation within and between species to address fundamental questions in biology, anthropology, and medicine. We focus on novel methods development for complex disease genetics and risk prediction in multi-ethnic settings. I am also interested in clinical data science and development of new diagnostics.I am also interested in disruptive innovation for healthcare including modeling long-term risk shifts and novel payment models.
The same genetic blueprint gives rise to thousands of cell types that make up the human body. Intricate mechanisms govern the choice to make skin, heart, or brain cells. These different cell types must be correctly arranged in spatial patterns to make functioning tissues and organs. In many organisms with continual turnover of cells, the genome faces the additional challenge of ensuring the faithful transmission of information throughout a lifetime-over decades in the case of humans. Thus, how one genome encodes thousands of patterns in space and time is of central importance to biology and medicine. Inappropriate activation of genes can give rise to birth defects, premature aging, or cancer, among many other diseases. Restoration of proper organ function often requires restoring homeostatic gene regulation.
Long Noncoding RNAs and Positional Identity
As a practicing dermatologist, I am fascinated by what makes human skin from different parts of the body different, a fact that guides the diagnosis and treatment of many skin diseases. Why do long hairs grow on the scalp but not on our palms or soles? How do cells know where they are located in the body, and how do they remember this information?
We discovered that one class of skin cells, the fibroblasts, encode the positional identity of skin via specific markings on their chromatin, the DNA-protein complex where genes reside. Based on the chromatin configurations of specific genes, most notably the HOX genes, fibroblasts differentially activate hundreds of genes based on their the cells location along three anatomic axes- anterior-posterior (head to tail), proximal-distal (close or far away from the trunk), and dermal-nondermal (surface or internal organ). This in effect creates a global positioning system for all cells to navigate.
These studies also revealed a surprising abundance of long intergenic long noncoding RNAs (also known as lincRNAs, a newly recognized type of genes that do not code forencode proteins) that are involved in programming chromatin states. We are particularly fascinated by HOTAIR, the first known lincRNA that can regulate the chromatin state of genes on distantly located chromosomes. We now appreciate that the genome is pervasively transcribed to give rise to thousands of lincRNAs, which are likely to play key roles in the gene regulation of diverse biological states and disease. We are interested in understanding how lincRNAs control gene activity, and in deciphering the rules that will allow the functions of thousands of lincRNAs to be predicted and studied.
Large-Scale Gene Regulatory Programs in Cancer Metastasis and Self-Renewal
In contrast to the orderly acquisition of positional identity, cancer progression is characterized by abrogation of normal positional boundaries, especially in metastasis, which is the leading cause of cancer death. We and many others have previously identified gene expression signatures (GES ), composed of dozens to hundreds of genes, that distinguish indolent human cancers from those prone to metastasis; these signatures can provide improved prognostic prediction for cancer patients. Furthermore, we have developed methods to pinpoint master regulators of GES singular control points that can toggle the activity of the entire genetic program. This allows complex gene programs observed in human cancers to be easily recapitulated in the laboratory as models for drug development. This has enabled the creation of faithful laboratory models of human cancer types, identified specific drugs that can target these cancers, and revealed the hierarchy of transcriptional programs involved in the generation of cancer stem cells the cells that continually repopulate a tumor or its metastases.
Dr. Cong's research group at the Department of Pathology and Department of Genetics is developing technology for genome editing and single-cell genomics, using computational approaches inspired by data science. His group has a focus on using these tools to study immunological and infectious diseases. His work has led to one of the first FDA-approved clinical trials employing viral delivery of CRISPR/Cas9 gene-editing for in vivo gene therapy. More recently, his group has invited high-efficiency tools for large-scale genome engineering based on novel microbial proteins, and developed single-cell analysis approach with applications in cancer biology and immunology. Dr. Cong is a recipient of the NIH/NHGRI Genomic Innovator Award, a Baxter Foundation Faculty Scholar, and has been selected by Clarivate Web of Science as a Highly Cited Researcher.
We are particularly interested in elucidating tumor evolutionary dynamics, novel therapeutic targets, and the genotype to phenotype map in cancer. A unifying theme of our research is to exploit ‘omic’ data derived from clinically annotated samples in robust computational frameworks coupled with iterative experimental validation in order to advance our understanding of cancer systems biology. In particular, we employ advanced genomic techniques, computational and mathematical modeling, and powerful model systems in order to:
- Model the evolutionary dynamics of tumor progression and therapeutic resistance and metastasis
- Elucidate disease etiology and novel molecular targets through integrative analyses of high-throughput omic data
- Develop techniques for the systems-level interpretation of genotype-phenotype associations in cancer
Our research is funded by the NIH/NCI, NHGRI, Department of Defense, Breast Cancer Research Foundation, American Association for Cancer Research, Susan G. Komen Foundation, Emerson Collective and V Foundation for Cancer Research.
We are using Saccharomyces cerevisiae and Human to conduct whole genome analysis projects. The yeast genome sequence has approximately 6,000 genes. We have made a set of haploid and diploid strains (21,000) containing a complete deletion of each gene. In order to facilitate whole genome analysis each deletion is molecularly tagged with a unique 20-mer DNA sequence. This sequence acts as a molecular bar code and makes it easy to identify the presence of each deletion. The mixture of all such tag strains then allows for the analysis of the entire genome with the manipulation of a single culture. During growth under a variety of conditions the loss of a tag indicates the loss of a deletion from the population. The concentration of each tag is determined by PCR amplification of the tags and hybridization to an Affymetrix DNA chip that contains the complement to all of the DNA sequence tags. This approach is being applied to other microorganisms.
We have identified a number of wild isolates of yeast that grow at much higher temperatures than is typical for Saccharomyces cerevisiae and are pathogenic and can kill a mouse. Microarrays have been used to map complex genetic traits such as virulence traits in pathogenic Saccharomyces cerevisiae using hybridization to detect single nucleotide polymorphisms. We have developed a new technology termed Reciprocal Hemizygosity Scanning that allows the determination of the contribution to the phenotype of all pair wise alleles for the whole genome from 2 independent strains. Using this technology we can map and quantitate all of the alleles in the genome for any complex quantitative trait in a single tube assay. These technologies will allow us to explore allelic contributions in complex mixed culture real environments and to investigate ecology at the genome level.
We are conducting a whole genome analysis (transcriptome and proteome) from blood of Human trauma patients. In this large clinical study we are establishing the standards for clinical genomics. We have developed 2 new technologies, "Molecular Inversion Probes" (MIP) for massive multiplex analysis of SNP and DNA content in Human and, "Mismatch Repair Detection" for discovery of rare Human polymorphisms. Both technologies are being applied to numerous clinical investigations.
Jesse is an Assistant Professor at Stanford University in the Department of Genetics and the Children’s Heart Center Basic Sciences and Engineering (BASE) Initiative. The Engreitz Lab aims to map the regulatory wiring of the genome to understand the genetic basis of heart diseases. Previously, Jesse was currently a Junior Fellow at the Harvard Society of Fellows and the Broad Institute, where he developed large-scale CRISPR tools to map enhancer-gene regulation, and launched the Variants-to-Function (V2F) Initiative to connect genetic disease variants to their molecular and cellular functions. Jesse completed his PhD in the Harvard-MIT Division of Health Sciences and Technology, where he studied long noncoding RNAs with Eric Lander and Mitch Guttman.
Human genetic and cultural evolution, mathematical biology, demography of China
Our lab studies the mechanisms by which cells and organisms respond to genetic change.
The genetic landscape faced by a living cell is constantly changing. Developmental transitions, environmental shifts, and pathogenic invasions lend a dynamic character to both the genome and its activity pattern.We study a variety of natural mechanisms that are utilized by cells adapting to genetic change. These include mechanisms activated during normal development and systems for detecting and responding to foreign or unwanted genetic activity. At the root of these studies are questions of how a cell can distinguish "self" versus "nonself" and "wanted" versus "unwanted" gene expression.
We primarily make use of the nematode C. elegans in our experimental studies. C. elegans is small, easily cultured, and can readily be made to accept foreign DNA or RNA. The results of such experiments have outlined a number of concerted responses that recognize (and in most cases work to silence) the foreign nucleic acid. One such mechanism ("RNAi") responds to double stranded character in RNA: either as introduced experimentally into the organism or as produced from foreign DNA that has not undergone selection to avoid a dsRNA response. Much of the current effort in the lab is directed toward a molecular understanding of the RNAi machinery and its roles in the cell. RNAi is not the only cellular defense against unwanted nucleic acid, and substantial current effort in the lab is also directed at identification of other triggers and mechanisms used in recognition and response to foreign information.
The major investigative focus of this laboratory and translational research program is to explore the mammalian genetic determinants of the inducible response and cellular sensitivity to DNA damage, focusing particularly on the effects of the p53 and BRCA1 gene products on DNA repair and cancer susceptibility. We have found that loss of p53 and BRCA1 function results in defective repair of DNA damage, including effects on homologous recombination, nucleotide and base-excision repair. In addition, we are exploring ways to exploit the DNA repair deficiency of p53 and BRCA1 mutant cancer cells and to identify targeted therapeutic approaches for the treatment and prevention of related cancers.
Role of BRCA1 in base-excision DNA repair (BER): BRCA1 appears to have complex regulatory effects on multiple DNA repair pathways in addition to their shared role in homologous-recombination and DNA double strand break repair. We first described that breast cancer cell lines mutant for the BRCA1 gene exhibit sensitivity to oxidative DNA damage. We also developed a novel viral based “host-cell reactivation” assay to measure the repair of oxidative DNA damage in living cells using an adenoviral GFP reporter gene, and demonstrated that BRCA1 mutant cells were defective in BER.
Discovery of small molecules that activate BER and may prevent BRCA1-associated tumors: We designed and performed a high-throughput screen to identify small-molecules that enhance DNA repair in a BRCA1 mutant background, and thus may serve as candidate agents for prevention of cancer by enhancing DNA repair and interrupting multistep mutagenesis. Several of these drugs are potentially “repurposeable” and are currently or were previously used in humans for other indications. We have shown activity of two in preventing the development of BRCA1-associated breast cancers in mice and are developing plans for a clinical trial using the lead hit for prevention of BRCA1-associated premalignant changes in ovaries from women undergoing risk-reducing bilateral oopherectomies.
Clinical translation of Next-Generation Sequencing for hereditary cancer risk assessment: We recently led the first clinical study of next-generation gene panel DNA sequencing among women referred for breast cancer risk assessment using germline DNA samples from our large translational research biobank containing more than 2000 specimens, all donated by individuals tested for BRCA1/2 or other gene mutations. We found that >10% of patients had potentially pathogenic mutations in genes other than BRCA1/2, thus doubling the rate of identified germline cancer susceptibility gene alterations in this population, a discovery that has enabled early detection of cancers.
Targeting TNBC and other malignancies with DNA damaging drugs and PARP: We found through preclinical studies and clinical trials that nearly all BRCA mutation associated breast cancer, and approximately half of non-BRCA mutant TNBC exhibit clinical sensitivity to platinum chemotherapy and synthetic lethality with PARP inhibitors. As part of these efforts, we performed extensive correlative studies on tumor tissue and germline DNA samples obtained from patients enrolled in a large, multi-institutional neoadjuvant clinical trial, using gene expression microarrays, DNA copy-number analyses, and germline DNA sequencing. We described a bioinformatic measure of homologous recombination deficiency (HRD) that is highly predictive of clinical response in these patients. Our current and future research goals in this area is to leverage our expertise in germline and tumor genomics to identify patients with breast and other cancers harboring DNA repair gene defects and HRD for treatment using PARP inhibitors and other DNA repair directed therapies (ATR and DNA-PK inhibitors). We have also developed breast cancer cell lines resistant to PARP-inhibitors and are exploring the mechanism for this drug resistance.
Cellular function and organismal homeostasis are governed by molecular interactions. Protein-DNA binding interactions are essential for regulating gene transcription and translation, dense networks of protein-protein and protein-peptide interactions further regulate cellular function, and enzymes make possible all of the chemical transformations essential to metabolism and signaling. Our goal is to understand, and eventually engineer, these complex processes by building and testing biophysical models of how the molecules that drive these processes work. To do so, an essential first step is to obtain the necessary quantitative measurements of the fundamental kinetic and thermodynamic constants of these molecular interactions and catalytic processes—the “universal language” needed to describe and ultimately predict function. In our lab, we use microfluidics and extensive hardware automation to perform these quantitative measurements at an unprecedented scale via 3 main platforms:
1. Array-based multiplexing experiments (MITOMI and HT-MEK) employ microfluidic devices containing 1,568 valved reaction chambers aligned to printed DNA arrays. We are currently using these devices to better understand how transcription factors find and bind their genomic targets to regulate gene expression, as well as to understand how enzymes achieve their extraordinary catalytic efficiency and substrate specificity.
2. MRBLEs (Microspheres with Ratiometric Barcode Lanthanide Encoding) rely on spectral multiplexing to track analytes throughout an experiment. We can create microspheres containing > 1,000 distinct ratios of lanthanide nanophosphors that can be uniquely identified via imaging alone, and are now using these MRBLEs in a variety of downstream assays.
3. Dropception is a microfluidic platform for creating double emulsion (water-in-oil-in-water) droplets that can be sorted in high-throughput using standard flow cytometers (FACS machines). We recently demonstrated the ability to generate and sort double emulsion droplets without breakage, isolate individual rare droplets of interest in wells of a multiwell plate, and recover all encapsulated nucleic acids, enabling a wide range of novel single-cell multi-omic techniques.
We study the evolution of complex traits by developing new experimental and computational methods.
Our work brings together quantitative genetics, genomics, epigenetics, and evolutionary biology to achieve a deeper understanding of how genetic variation shapes the phenotypic diversity of life. Our main focus is on the evolution of gene expression, which is the primary fuel for natural selection. Our long-term goal is to be able to introduce complex traits into new species via genome editing.
The long term goal of our research is to understand how proteins fold in living cells. My lab uses a multidisciplinary approach to address fundamental questions about molecular chaperones, protein folding and degradation. In addition to basic mechanistic principles, we aim to define how impairment of cellular folding and quality control are linked to disease, including cancer and neurodegenerative diseases and examine whether reengineering chaperone networks can provide therapeutic strategies.
My laboratory uses the Drosophila male germ line as a model to investigate how self-renewal, proliferation and differentiation are regulated in adult stem cell lineages. The central characteristic of adult stem cells is their long-term capacity to divide as relatively undifferentiated precursors while also producing daughter cells that initiate differentiation. Understanding the mechanisms that regulate stem cell specification and the choice between stem cell self-renewal and differentiation is crucial for realizing the potential of stem cells for regenerative medicine. We are using the Drosophila male germ line as a powerful genetic system to identify both the cell autonomous determinants and the extrinsic cell-cell interactions that govern stem cell specification, self-renewal, and differentiation. One of the great advantages of this system is that stem cells can be studied in situ, in the context of their normal support cells. Our results indicate that signals from surrounding somatic support cells specify asymmetric division of male germ line stem cells by inducing one daughter cell to self-renew stem cell identity while directing the other daughter cell to differentiate. A second focus of our work concerns how the developmental program directs cellular differentiation. Fundamental cellular functions like the cell cycle, the cytoskeleton, and the general transcription machinery are remodeled during development to give rise to specialized cell types. Several lines of research in our laboratory have recently converged on the molecular mechanisms underlying the developmentally programmed switch from proliferation to differentiation, a key regulatory point in the adult stem cell lineages that underlie tissue maintenance and repair. Failure to cleanly execute this switch may contribute to genesis of cancer. Our results implicate a number of molecular and cellular mechanisms in regulating this critical switch. We find that RNA binding proteins involved in translational control and alternative splicing act cell autonomously to regulate the cessation of proliferation and that progression of differentiation requires communication from associated somatic support cells. We discovered that a developmentally regulated alternate choice of site at which certain nascent transcripts are cut to form 3’ ends, leading to production of novel mRNA isoforms with shortened 3’UTRs, controls dramatic changes in the suite of proteins expressed in differentiating spermatocytes compared to proliferating spermatogonia. We found that dramatic changes in chromatin open over 2000 new promoters with novel core sequence structure to turn on the new cell type specific transcription program when cells initiate spermatocyte differentiation. Some of the earliest genes turned on in this differentiation program encode chromatin associated proteins that prevent spurious opening of normally cryptic promoters, thus preventing massive misexpression of genes associated with the wrong cell type. Other transcripts upregulated with differentiation onset encode cell type-specific translational regulators that delay production of core G2/M cell cycle machinery to program the extended G2 phase of meiotic prophase. Our goal over the next 5 years is to map how these processes collaborate to form the regulatory circuitry that initiates then executes the switch from proliferation to differentiation.
We use the baker’s yeast, Saccharomyces cerevisiae, as a model system to study the cell biology underpinning protein-misfolding diseases like Parkinson's disease and ALS. Since dealing with misfolded proteins is an ancient problem, we hypothesize that the mechanisms employed to cope with them are likely conserved from yeast to man. Our long-term goal is to identify the critical genes and cellular pathways affected by misfolded human disease proteins.
C9orf72 in ALS and FTD: Disease models and mechanisms
Mutations in the C9orf72 gene are the most common cause of ALS and frontotemporal dementia (FTD). The mutation is a massive hexanucleotide repeat (GGGGCC) expansion in the intron of C9orf72. The mechanism by which C9orf72 mutations cause disease has remained unclear and of intense interest. In collaboration with the Petrucelli laboratory we have recently identified a way to selectively inhibit the expression of both sense and antisense mutant C9orf72 transcripts, which could offer therapeutic potential (Kramer et al., Science 2016).
New yeast models of neurodegenerative diseases
Encouraged by the power of the yeast system to gain insight into α-synuclein biology, we are creating new yeast models to study additional protein-misfolding disorders, including Alzheimer’s disease and ALS. We recently developed a yeast model to study the ALS disease protein TDP-43 (Johnson et al., Proc Natl Acad Sci USA 2008).
We have used yeast and in vitro biochemistry (in collaboration with Jim Shorter at PENN) to analyze the effects of ALS-linked TDP-43 mutations on aggregation and toxicity (Johnson et al., J Biol Chem 2009). We are now using these models to perform high-throughput genetic and small molecule screens to elucidate the molecular pathways that regulate the function of these disease proteins and control their conversion to a pathological conformation. We are currently analyzing hits from recent high-throughput screens that identified potent modifiers of TDP-43 toxicity. We are validating these hits in cell culture, animal models (mouse, fly, and zebrafish), and human patient samples.
These TDP-43 modifier screens are providing insight in two main ways:
1. The genes and pathways that are able to modify TDP-43 toxicity in yeast are now good candidates for evaluation as genetic contributors to ALS and related disorders in humans (e.g., see ataxin 2 below).
2. The yeast hits and their homologs are candidate therapeutic targets, especially gene deletions (Armakola et al., Nat Genet 2012; Kim et al., Nat Genet 2014).
Ataxin-2 and ALS
Interestingly, one of the hits from our yeast TDP-43 genetic modifier screen, PBP1, is the homolog of a human neurodegenerative disease protein, ataxin 2. We have validated this genetic interaction in the fly nervous system (in collaboration with Nancy Bonini at PENN), used biochemistry to show the proteins physically associate in an RNA-dependent manner.
We analyzed the ataxin 2 gene in 915 individuals with ALS and 980 healthy controls and found mutations in this gene as a common geneticrisk factor for ALS in humans. Long polyglutamine (polyQ) expansions (>34Q) in ataxin 2 cause spinocerebellar ataxia type 2 (SCA2). We found intermediate-length polyQ expansions in ataxin 2 (27-33Q) significantly associated with increased risk for ALS (Elden et al., Nature 2010). A role for polyQ expansions in ataxin 2 in ALS and related diseases is being evaluated by us and others in independent patient populations worldwide. Click here for an updated summary of these results.
We found that lowering levels of ataxin 2 in mouse, either by knockout or with antisense oligonucleotides (ASOs) can markedly extend survival and reduce pathology in TDP-43 transgenic mice (Becker et al., Nature 2017). We are extending these studies to additional mouse models and testing effects of ataxin 2 lowering in human cell models.
The consistent focus of Anna’s research has been using naturally occurring mutations in humans as tools to identity critical regulatory pathways and insights into normal physiology. Her early post-doctoral research led to the identification a new genetic aetiology for permanent and transient neonatal diabetes due to KCNJ11 mutations and resulted in one of the first examples of precision medicine, where the determination of the molecular genetic aetiology lead to improved treatment options for patients. Whilst in Oxford, Anna's team discovered a novel genetic cause of constitutive insulin sensitivity in humans due to mutations in the PTEN gene highlighting the complex interplay between pathways involved in cell-growth and metabolism.
Anna's current research projects are focused on the translation of genetic association signals for type 2 diabetes and glycaemic traits into cellular and molecular mechanisms for beta-cell dysfunction and diabetes. Her group uses a variety of complementary approaches, including human genetics, functional genomics, physiology and islet-biology to dissect out the molecular mechanisms driving disease pathogenesis.
Anna is an active member of multiple internal genetic discovery efforts including: NIH/Pharma funded Accelerated Medicines Partnership, DIAGRAM (Diabetes Genetics Replication and Meta-analysis), MAGIC (Meta-analysis of Glucose and Insulin traits Consortium), Type 2 Diabetes Genetic Exploration by Next-generation sequencing in multi-Ethnic Samples (T2D-GENES) and the Genetics of Type 2 Diabetes (GoT2D). She was also involved in the IMI funded STEMBANCC project which focused on delivering human IPS cell derived beta-cell models for drug discovery efforts.
Anna is also involved in several initiatives under the Human Islet Research Network (HIRN): the NIDDK funded Human Pancreas Atlas Programme (HPAP) for Type 2 Diabetes, and the Integrated Islet Phenotyping Programme (IIPP).
My current interests in neuroscience involve the consequences of advances in neurosciences for 1) predicting future diseases or traits; 2) reading minds to allow detection of subjective mental states such as pain, recognition, bias, or memory; 3) "treating" non-disease traits; 4) cognitive enhancement; 5) detecting consciousness and handling issues around disorders of consciousness; and 6) issues of responsibility.
My current interests in human genetics focus on 1) prenatal genetic diagnosis, 2) the effects of widespread adoption of clinical whole genome sequencing, and 3) ethical, legal, and social issues in genomic biobanks.
In stem cell research, I am currently interested in 1) legal challenges to stem cell research and 2) issues around human/non-human chimeras.
My interests in human research protections, biological enhancement, and the future of reproduction draw on, and involve, all three of the substantive fields described above.
Our lab focuses on developing methods to probe both the structure and function of molecules encoded by the genome, as well as the physical compaction and folding of the genome itself. Our efforts are split between building new tools to leverage the power of high-throughput sequencing technologies and cutting-edge optical microscopies, and bringing these technologies to bear against basic biological questions by linking DNA sequence, structure, and function.
Livnat Jerby is an Assistant Professor of Genetics at Stanford University. Her research focuses on multicellular dynamics, as a disease driver and therapeutic avenue, particularly in the context of cancer immunology. In her work, she aims to identify the drivers, molecular underpinnings, and causal structure of multifactorial immune evasion mechanisms, and use this information to identify new and more effective ways to augment and unleash targeted immunity via combinatorial interventions. To address this challenge at scale, she develops integrative approaches, fusing single-cell sequencing and imaging with machine learning, genetic and environmental perturbations.
Thus far, her research provided new perspectives to key facets of tumor biology, encompassing metabolism, genetics, and immunology. As a postdoctoral fellow at the Broad Institute of MIT and Harvard, she identified regulators of T cell exclusion and dysfunction with Levi Garraway and Aviv Regev. She holds a B.Sc. in Computer Science and Biology and obtained her PhD in 2016 from Tel Aviv University, where she worked with Eytan Ruppin and developed new ways to interrogate cancer metabolism and genetics.
This fall Livnat joined Stanford Genetics to establish a multidisciplinary lab that will harness machine learning in combination with clinical data and extensive functional testing to dissect and target immune dysregulation in cancer, aiming to leverage the versatile, interconnected, and non-linear function of genes, cells, and tissues for disease detection, prevention, and treatment.
Her research has been generously supported by the Schmidt Family Foundation, Rothschild Foundation, the Cancer Research Institute (CRI), the Burroughs Wellcome Fund (BWF), and Chan Zuckerberg Biohub initiative.
The goal of the Program in Human Gene Therapy is to develop gene transfer technologies and use them for hepatic gene therapy for the treatment of genetic and acquired diseases. The general approach is to develop new vector systems and delivery methods, test them in the appropriate animal models, uncover the mechanisms involved in vector transduction, and use the most promising approaches in clinical trials. Specifically, we work on a variety of viral and non-viral vector systems. Our major disease models are hemophilia, hepatitis C and B viral infections, and diabetes. The second major focus includes the role that small RNAs play in mammalian gene regulation.
Our experimental focus is on the mammalian setting, including mouse genetics, human genetics, single cell studies, and new human tissue platforms. The latter encompass human skin regenerated on immune deficient mice as well as organotypic constructs with epithelial and stromal cells embedded within architecturally faithful mesenchyma in vitro. These new models, which we term Multi-Functional Human Tissue Genetics, allow up to 10 alleles or more to be altered simultaneously, permitting genetic experiments with an unprecedented degree of rapidity and complexity.
Stem cell biology and differentiation
In stratified epithelia proliferative basal cells adherent to the underlying basement membrane undergo cell cycle arrest then outward migration and terminal differentiation. This process is mediated by 2 mutually exclusive programs of gene expression: 1) an undifferentiated program supporting proliferation by stem cells within the basal layer and 2) a differentiation program instructing growth arrest and differentiation-associated programmed cell death in suprabasal layers. The control of this transition from epithelial stem cell to differentiated corneocyte, which is abnormal in epidermal cancers, is not well understood. We are currently pursuing studies of the dominant signaling and gene regulatory networks that control this process, including the Ras/MAPK cascade, which is required for stem cell-mediated self-renewal and the p53 transcription factor family member, p63, which is required for epidermal differentiation.
Epigenetic regulation by histone modifying proteins and noncoding RNA
In addition to classical gene regulatory networks noted above, we have recently identified a central role for additional biologic mechanisms, namely gene regulation by chromatin regulators and by noncoding RNAs. Epigenetic control of gene expression lasts through multiple cell divisions without alterations in primary DNA sequence and can occur via mechanisms that include histone modification and DNA methylation. Noncoding RNA sequences can regulate gene expression via interactions with epigenetic and other control mechanisms. The function of histone modifying epigenetic regulators and noncoding RNA as central mediators of epithelial stem cell renewal and differentiation represent major emerging areas of study in the lab.
Skin malignancies, including epidermal squamous cell carcinoma (SCC), alone account for nearly as many cancers as all other tissues combined. Progress in understanding epithelial carcinogenesis has been hindered in the past by a lack of models that faithfully recapitulate the 3-dimensional architecture of tumor-stroma co-evolution. To address this and to also study the oncogenic potential of unregulated function of dominant regulators of epithelial homeostasis noted above, we developed Multi-Functional Human Tissue Genetics noted above which, when combined with skin tissue regeneration on immune deficient mice, has permitted the molecular reconstruction of events sufficient to trigger human cancer. These models are being used to systematically elucidate proteins required for cutaneous carcinogenesis and to test their potential role as therapeutic targets.
Epithelial tissues in general and skin in particular offer an attractive site for development of new approaches in molecular therapeutics. A family of human genetic skin diseases is characterized by defective epithelial gene expression. Among the most severe of these are subtypes of epidermolysis bullosa (EB) and lamellar ichthyosis (LI). We have developed approaches for high efficiency gene transfer to EB and LI patient skin tissue that are corrective at biochemical, histologic, clinical and functional levels. In addition to EB subtypes and LI, similar corrective efforts have also been undertaken with a number of other genetic skin disorders.
Naturally occurring species show spectacular differences in morphology, physiology, lifestyle, and behavior. They also differ in disease susceptibility and life span. Although the genomes of many organisms have now been completely sequenced, we still know relatively little about the specific DNA sequence changes that underlie interesting species-specific traits. My laboratory is using a combination of genetic and genomic approaches to identify the detailed molecular mechanisms that control evolutionary change in vertebrates, with a focus on five fundamental questions:
1. Are new evolutionary traits controlled by countless genetic differences of small effect, or by a few genetic changes with large effects?
2. What specific genes have changed to produce interesting evolutionary differences seen in nature?
3. What kinds of mutations have occurred in these genes (e.g., dominant or recessive, coding or regulatory, preexisting or de novo)?
4. How predictable is evolution? If you know how evolution has occurred in one population, is it possible to predict the genes and mutations that also underlie the same trait in different populations?
5. How has evolution produced the unique characteristics of humans?
We study these questions using a variety of methods in mice, sticklebacks, and people.
Mice are often the best system available for asking detailed mechanistic questions in mammals, or testing the phenotypic effects of particular sequence changes seen in other species. We have used classical genetics in mice to identify fundamental pathways that control formation and patterning of cartilage, bone, and joints. We also make extensive use of mice identifying the regulatory mechanisms that lay out expression of key developmental control genes, with the ultimate aim of identifying how vertebrate morphology itself is encoded in the genome.
Sticklebacks offer an unusually powerful system for studying the molecular basis of evolutionary change in naturally occurring species. Our lab has pioneered the development of a large number of new genetic and genomic resources for the fish, and has worked with Hudson Alpha Institute and the Broad Institute to develop a high-quality whole genome sequence assembly for sticklebacks. Using these new tools, we have now successfully identified both the molecular mechanisms that control repeated evolution of armor plate patterning, pelvic reduction, and spine and skin color changes in nature. Our studies show that big evolutionary changes can be controlled by single chromosome regions. The big changes are controlled by alterations in major developmental control genes (key signaling molecules and transcription factors). Although null mutations in these genes are typically deleterious or lethal, sticklebacks have made regulatory alterations in these genes that produce large morphological effects in particular tissues, while preserving overall viability. Interestingly, the same genes are used repeatedly when similar phenotypes evolve in different populations, revealing a surprising commonality to the molecular mechanisms that control rapid evolutionary change in diverse organisms.
Although many of our studies have begun in mice or sticklebacks, the genes and mechanisms that we have also turn out to control major differences in human morphology, hair color, arthritis susceptibility, and incidence of major psychiatric diseases in billions of people around the world. Building on this work, we have now begun a variety of projects to identify other mechanisms responsible for key evolutionary traits and diseases in humans. Although we are still far from knowing the detailed molecular basis of most human traits, we are optimistic that many aspects of this problem can now be studied both computationally and experimentally, and will provide new insights into both human origins and human medicine.
For many subcellular viruses and parasites, RNA, not DNA, is the carrier of genetic information. This has several interesting consequences for the genetics and biology of the virus. Poliovirus serves as a model to increase our understanding of positive-strand RNA viruses for which no vaccine is available and which remain a significant health hazard: examples include other picornaviruses, such as rhinoviruses, coxsackieviruses and the deadly enterovirus 71 as well as more distantly related positive-strand RNA viruses such as hepatitis C and Dengue fever.
Questions currently under scrutiny are posed below, and discussed in greater detail in our web site.
1. How does the biochemistry of RNA-dependent RNA polymerases affect the biology of RNA viruses?
2. How are the membranous structures on which viral RNA replication complexes assemble form, and
from what intracellular organelles do they derive?
3. Why are the genetic properties of many RNA genomes different from DNA genomes? How does the error-prone nature of RNA-dependent RNA replication and the membrane association of the RNA replication complexes affect these genetic properties?
4. How does the inhibition of the protein secretory apparatus by the 3A and 2B proteins of picornaviruses such as poliovirus affect their pathogenesis? What would happen to the secretion of interferons, and to the presentation of antigens in the context of MHC class I molecules, if the host secretory pathway were not inhibited during infection by polioviruses, rhinoviruses and coxsackieviruses?
Our research focusses on development of statistical and machine learning methods for integrative analysis of diverse functional genomic and genetic data to learn models of gene regulation. We have led the analysis efforts of the Encyclopedia of DNA Elements (ENCODE) and The Roadmap Epigenomics Projects with the development of novel methods for
- Adaptive thresholding and normalization of massive collections of functional genomic data (e.g. ChIP-seq and DNase-seq)
- Dissecting combinatorial transcription factor co-occupancy within and across cell-types
- Predicting cell-type specific enhancers from chromatin state profiles
- Exploiting expression and chromatin co-dynamics with to predict enhancer-target gene links
- Jointly modeling sequence grammars at regulatory elements and their chromatin state dynamics, expression changes of regulators and functional interaction data to learn unified multi-scale gene regulation programs
- Elucidating the heterogeneity of chromatin architecture at regulatory elements
- Improving the detection and interpretation of potentially causal disease-associated variants from Genome-wide association studies
More recently, we have also been developing methods to
- Decipher the functional heterogeneity of transcription factor binding
- Learn long-range, three-dimensional regulatory interactions
- Infer causal regulatory mechansisms by integrating diverse functional genomic data from temporal (e.g. differentiation/reprogramming) and perturbation (e.g. drug response, knockdown, genome-editing) experiments
- Model the complex relationships between genetic variation, regulatory chromatin variation and expression variation in healthy and diseased individuals
- Deep learning frameworks for genomics
The Li Lab is primarily interested in RNA editing mediated by ADAR enzymes. We co-discovered that the major function of RNA editing is to label endogenous dsRNAs as "self" to avoid being recognized as "non-self" by MDA5, a host innate immune dsRNA sensor, leading us to pursue therapeutic applications in cancer, autoimmune diseases, and viral infection. The other major direction of the lab is to develop technologies to harness endogenous ADAR enzymes for site-specific transcriptome engineering.
We focus on understanding the effects of genome variation on cellular phenotypes and cellular modeling of disease through genomic approaches such as next generation RNA sequencing in combination with developing and utilizing state-of-the-art bioinformatics and statistical genetics approaches. See our website at http://montgomerylab.stanford.edu/
Our research interests are to elucidate the contribution of chromatin to mechanisms that promote genomic integrity. The regulation of chromatin is a crucial component of DNA metabolism and processing in eukaryotic organisms. Chromatin-remodeling complexes, modified histones, and higher order chromatin structure are all factors influencing genome stability. We utilize an integrated approach of genetic, biochemical, and molecular techniques, in both yeast and mammalian systems, to examine the involvement of chromatin in processes that prevent genome instability and the pathogenesis of disease.
Hiro Nakauchi obtained a M.D. from Yokohama City University School of Medicine and a Ph.D. in immunology from University of Tokyo Graduate School of Medicine. He isolated CD8 genes during his post-doc period at the Laboratory of Prof. Leonard Herzenberg at Stanford University. After returning to Japan, he started working on hematopoietic stem cells in his laboratory at RIKEN. In 1994, he became Professor of Immunology at the University of Tsukuba where he demonstrated that a single hematopoietic stem cell could reconstitute the entire hematopoietic system, a definitive experimental proof for the “stemness”. Since April 2002, he has been a Professor of Stem Cell Therapy in the Institute of Medical Science at The University of Tokyo (IMSUT). In 2008, he was appointed Director of newly established Center for Stem Cell Biology and Regenerative Medicine at IMSUT. Just recently, he returned to Stanford University as a faculty to continue his stem cell research at the Institute of Stem Cell Biology and Regenerative Medicine. Goals of his work are to translate discoveries in basic research into practical medical applications.
Evolution of genomes and population genomics of adaptation and variation
Much of the research in the Pringle laboratory exploits the power of yeast as an experimentally tractable model eukaryote to investigate fundamental problems in cell and developmental biology such as the mechanisms of cell polarization and cytokinesis. In regards to cell polarization, the major current foci are the roles of cortical marker proteins and of a GTPase-based signal-transduction cascade in the selection of the polarization axes (as defined by the bud sites). Interestingly, the marker proteins appear to be delivered to polarized sites in the cell surface by an unconventional arm of the secretory pathway. In regards to cytokinesis, the major current foci are the roles of the septin proteins and the interactions among the actomyosin contractile ring, the enzymes of extracellular-matrix (cell-wall) synthesis, and proteins that appear to be involved in plasma-membrane reorganization. Our working hypothesis is that the conserved core mechanism is the rearrangements of the membrane during cleavage-furrow formation and that the actomyosin ring and extracellular matrix play accessory roles.
In a departure from our many years of yeast work, a major new project involves developing the small sea anemone Aiptasia pallida as a model system for study of the molecular and cellular biology of the dinoflagellate-cnidarian symbiosis, which is critical for the survival of most reef-building corals but still very poorly understood. Processes to be investigated include the recognition and signaling events involved in symbiosis establishment, the temporal and spatial coordination of symbiont and host cell cycles during symbiosis maintenance, and the signaling and cellular processes involved in symbiosis breakdown under stress. Currently much of our effort is directed at genomic analysis and method development that will underpin later studies.
My group has expertise in the development of new statistical methods for genetic analysis and in their application to genomic data from humans and other organisms. We focus on questions relating to genetic variation and evolution: How does genetic variation impact phenotypic traits and evolution, both at the organismal and cellular level? What can we learn from genome sequences of modern and ancient humans about the relationships among human populations, and the the nature of adaptation in these populations?
We often work on problems where there are no off-the-shelf statistical methods. Thus, an important part of our work is in developing appropriate statistical and computational approaches that can yield new insights into biological data. In the past, we have made important contributions to a variety of problems in human population genetics, including methods for complex trait mapping, inference of population structure and history, and studies of natural selection. We have a strong track record of producing user-friendly resources that are widely used in the community, and in applied data analysis to tackle important biological questions. Notably, our Structure algorithm and software package for inferring population structure from genetic data have received >30,000 total citations spread across several papers.
Since 2008 an important emphasis of my group has focused on understanding gene regulation, and in particular how genetic variation may impact regulation. Ultimately, we would like to be able to predict which noncoding variants in the genome are likely to have regulatory effects in any given cell type, and how these link to phenotypic variation and disease. My lab has been deeply involved in developing new computational methods to interpret various types of modern genomic assays and in linking these to genetic variation.
Secondly, we have had a major focus on understanding the genetic architecture of complex traits, and the implications for understanding evolution. We have argued that much--if not most--evolution in humans likely proceeds through a process that we call "polygenic adaptation" in which populations evolve through small allele frequency shifts at many loci.
We have also written extensively about conceptual models for understanding the genetic architecture of trait variation (Boyle et al, 2017). We have argued that the data are consistent with a model in which essentially every regulatory variant in disease-relevant cell types can affect risk, and proposed that most of these effects act through trans-regulatory networks. Testing this model is an ongoing focus of our work.
Cardiovascular developmental biology
Research in the lab addresses problems in evolutionary biology and human genetics through a combination of mathematical modeling, computer simulations, development of statistical methods, and inference from population-genetic data. Our current work covers topics such as human genetic variation, inference of human evolutionary history, the role of population genetics in the search for disease-susceptibility genes, the relationship of gene trees and species trees, and mathematical properties of statistics used for analyzing genetic variability.
Our research program focuses on the mechanisms that control the proliferation of mammalian cells under normal and pathological conditions (regeneration, cancer), with a particular emphasis on stem cells and gene regulatory networks. We combine genetic, genomics, and proteomics approaches to identify and investigate genes and pathways involved in cancer initiation and progression. We use genome-editing strategies to develop and study genetically-engineered mouse models for human cancers, including lung cancer, pancreatic cancer, and liver cancer. Our work spans the investigation of fundamental biological processes to the implementation of clinical trials based on our findings in pre-clinical models.
- Assistant Professor, Genetics
- Member, Bio-X
- Postdoctoral Fellow, University of California, San Francisco, Pharmaceutical Chemistry (2020)
- Ph.D., Pierre and Marie Curie University, Curie Institute (Paris, France), Life Science Complexity (2013)
- M.S., University of Bologna (Italy), Biotechnology (2008)
- B.S., University of Bologna (Italy), Biotechnology (2006)
Adaptive Evolution and the Fitness Landscape
When yeast are evolved under various selective pressures in a chemostat, mutations that arise and provide an adaptive advantage will expand within the population. We have pioneered the use of high throughput sequencing to determine the identity of such mutations, as well as to understand the dynamics of the mutations within the populations, and the interactions between the mutations (such as epistasis). Further, we have developed a DNA barcode based lineage tracking system to determine the distribution of fitness effects (DFE) for newly arising beneficial mutations. We have also characterized what we call the genotype-fitness map for beneficial mutations, and have investigated why beneficial mutations provide a positive fitness effect. We are also interested in how beneficial mutations trade-off for different traits, and how those trade-offs constrain adaptive evolution.
We have a highly collaborative research program in the evolutionary genomics of cancer. We apply well-established principles of phylogenetics to cancer evolution on the basis of whole genome sequencing and functional genomics data of multiple tumor samples from the same patient. Introductions to our work and the concepts we apply are best found in the Newburger et al paper in Genome Research (2013) and the Sidow and Spies review in TIGS (2015).
More information can be found here: http://www.sidowlab.org
We are presently in an omics revolution in which genomes and other omes can be readily characterized. Our laboratory uses a variety of approaches to analyze genomes and regulatory networks. Our research focuses on yeast, an ideal model organism ideally suited to genetic analysis, and humans.
To annotate genomes, we developed RNA sequencing for annotation the yeast and human transcriptomes. We discovered that the eukaryotic transcriptome is much more complex than previously appreciated and that embryonic stem cells have more transcript isoforms than differentiated cells.
2) Transcription Factor Binding Networks
We have also developed methods for mapping transcription factor binding sites through the genome. We used this to develop regulatory maps and have been using this to help decipher the combinatorial regulatory code which factors work together to regulate which genes. Using this approach we have mapped out pathways crucial for metabolism and inflammation.
3) Integrated Regulatory Networks
In addition to transcriptional factor binding networks we have also been mapping phosphorylation and metabolite-protein interaction networks. These studies have revealed novel global regulators and key points in integrated regulatory networks.
We have been analyzing differences between individuals and species at two levels: DNA sequence variation and regulatory information variations. We developed paired end sequencing for humans and found that humans have extensive structural variation (SV), i.e. deletions, insertions and inversions. This is likely to be a major cause of phenotypic variation and human disease. In addition, by mapping binding sites difference among different yeast strains and humans, we have found that individuals differ much more in their regulatory information than in coding sequence differences. We can correlate these differences with those in SNPS and SVs, thereby associating noncoding DNA differences with regulatory information.
5) Human Disease
Finally, we are applying omics approaches of genome sequencing, transcriptomics proteomics metabolomics, DNA methylation and microbiome assays to the analysis of human disease. These integrative omics approaches are being applied to help understand the molecular basis of disease and the development of diagnostics and therapeutics.
We are interested in the basic principles that govern interactions within the intestinal microbiota and between the microbiota and the host. To pursue these aims, we colonize germ-free (gnotobiotic) mice with simplified, model microbial communities, apply systems approaches (e.g. functional genomics), and use genetic tools for the host and microbes to gain mechanistic insight into emergent properties of the host-microbial super-organism.
Tim Stearns holds the Frank Lee and Carol Hall Professorship in the Department of Biology at Stanford University and is Senior Associate Vice Provost of Research. He also holds appointments in the Department of Genetics, is a member of the Stanford Cancer Institute, and Bio-X, is a Faculty Fellow in Chem-H, and is an affiliated faculty member of the Center for International Security and Cooperation (CISAC). He is a member of JASON, a national organization that advises the government on matters of science, technology and national security. He has also been an advisor to the National Academies of Science and the President's Council of Advisors on Science and Technology (PCAST). Dr. Stearns received a B.S. from Cornell University, a Ph.D. from MIT, and did his postdoctoral fellowship at the University of California, San Francisco. His research concerns the mechanism and regulation of cell division, the organization of signaling pathways within cells, and cell biology of fungal pathogens. Stearns was named an HHMI Professor in 2002, for his work in science education, and has taught international laboratory workshops in South Africa, Chile, Ghana, and Tanzania. He is the chair of the NCSD Study Section at the NIH, and has served on the editorial boards of several journals.
One of the most daunting challenges in medicine is the complex nature of most diseases (including cancer, diabetes, heart disease and some forms of rare disease) due to interactions between multiple genetic variants and environmental influences. Our research is directed at understanding such complex traits; to do so, we develop novel genomic approaches to investigate the molecular processes that link genotype to phenotype, identify the causal underlying factors, and quantify their contributions. We investigate variation at the level of the genome, transcriptome and proteome, which we integrate with higher-level phenotypes. We also use the resulting molecular networks to predict and evaluate intervention points that enable modulation of phenotype. In particular, our projects are in the following areas:
1) Functions and mechanisms of gene regulation
We have developed several technologies to characterize and quantify pervasive transcription at the genome-wide level as well as its functional impact. In particular, we are interested in the function and regulation of non-coding RNAs, antisense transcription, and the molecular phenotypes that arise from transcriptome complexity.
2) Systems genetics
We have also piloted new technologies to dissect the genetic basis of complex, multifactorial phenotypes. We are interested in studying how genetic variation is inherited through recombination, the consequences of genetic variation, learning to predict phenotype from genotype, and integrating multiple layers of molecular data in order to define intervention points that can be targeted to modulate phenotypes of interest.
3) Disease models
We have used multiple model systems, primarily yeast and human cells, to characterize the genetic and cellular systems affected in particular diseases and assess potential therapeutic strategies. We are studying diseases in patient-derived cells using systematic, followed by mechanistic approaches, to unravel mechanisms and discover novel treatments.
Notably, we place a strong emphasis on the development of new technologies, leveraging the Stanford Genome Technology Center's strengths in this area. Some engineering applications we aim to develop include novel biosensors for detection of minute quantities of biological material and single-cell approaches to investigate genome and transcriptome complexity. Through developing such technologies, we will work with the Center to reduce the cost and increase the accuracy and throughput of biomedical research and health care. Ultimately, we strive to develop approaches that will enable personalized, preventive medicine.
Our research focuses on the development and function of glial cells in the vertebrate nervous system. Glia are non-neuronal cells with many essential functions, ranging from forming the myelin sheath to defending the brain against infection.
One of our goals is to use genetic approaches in zebrafish to discover new genes with essential functions in the glial cells that form the myelin sheath, which allows for rapid axonal conduction in vertebrates. Disruption of myelin underlies important human diseases, including Multiple Sclerosis and peripheral neuropathies. The formation of myelin, which involves reciprocal signaling between neurons and glial cells, a dramatic morphological transformation of the glial cells, and organization of the axon into different specialized domains, is fascinating but nonetheless poorly understood.
In genetic screens, we have identified mutations in more than 15 different genes that have specific functions in the development of myelinated axons. Among these are a novel G-protein coupled receptor that instructs Schwann cells to make myelin in peripheral nerves, receptors that control migration of glial cells along growing axons, a kinesin motor protein that is essential for mRNA localization and normal membrane compaction in myelinating oligodendrocytes, and a transcription factor that regulates the migration of the cells that form myelin in the brain and spinal cord.
Another goal of our research is to identify new genes that regulate microglia, which are specialized macrophages that are dedicated to the immune defense of the brain. Microglia also have critical roles in regulating synaptic connectivity and engulfing dead neurons to maintain homeostasis in the brain. Microglial dysfunction has been implicated a wide array of disorders, including autism and Alzheimer disease.
Starting with screens for mutants with abnormal microglia, we have identified novel genes regulate microglial development and function. Examples include a NOD-like receptor that suppresses inappropriate inflammation, a phosphate exporter that functions specifically in microglia and other tissue macrophages, and a regulator of lysosomal action that allows microglia to digest material that they engulf.
These projects provide new insights into glial cell development and function, generate new animal models of human disease, define pathways that may be disrupted in disease, and may provide new avenues toward therapies for diseases of glia.
Research in our laboratory develops and applies statistical methods for analyzing patterns of human genetic variation, which underlie the phenotypic diversity of our species. We are collaborating on various genome-wide studies focusing on stratified or recently admixed populations. These studies offer unique opportunities to elucidate the evolutionary forces that have shaped the patterns of genetic variation in humans, to uncover the genetic basis of complex traits, and to shed light on the mechanisms that lead to diverse phenotypes and disparate disease risks among populations.
The goal of our laboratory is to develop, scale up, and broadly disseminate molecular technologies for mapping cells and functional circuits. At the sub-cellular scale, maps document the spatial organization of proteins, RNA, DNA, and metabolites with nanometer precision and temporal acuity on the order of seconds. Maps also chart the connectivity between these molecules, elucidating the circuits and signaling processes that give rise to function.
Beyond the single cell, we also strive to map cellular ensembles, such as brain tissue. Can we create tools that contribute to the construction of cell and tissue atlases, and can we map the cellular circuits that give rise to function and behavior? To achieve these ambitious goals, our laboratory has focused on the development of scalable technologies to detect, measure, and manipulate molecules and circuits, both at the sub-cellular level, and at the level of cell populations.
I have over a decade’s worth of experience in developing and applying high-throughput and high-resolution genomics analysis tools and procedures, in particular in the context of studying genomic sequence variation in brain development and function.
I have been involved on numerous occasions in using a large-scale and high-throughput setup for genomics analyses as well as carrying out analyses over several levels of genomics and epigenomics information. This includes participation in the ENCODE and 1000 Genomes projects, for the latter as a member of both the analytical and structural variation groups.
I have experience with developing and applying state-of-the-art and emerging genomics and epigenomics technologies (array and next-generation-sequencing based) for the analysis of gene expression, genomic DNA sequence and structure, DNA methylation and chromatin modification, in human cells and human cell culture systems, including stem cell culture models. For example I was co-first author of the paper in Science (Korbel, Urban, Affourtit et al., 2007, PMID 17901297) on developing next-generation-sequencing based paired-end mapping of CNVs and SVs, an approach that is now a standard part of whole-human-genome sequencing projects such as the 1000 Genomes Project. Paired-end mapping is also a critical component of advanced RNA-Seq approaches, mapping of transposable elements and the study of long-range chromatin interactions using the HiC method.
Two main, and connected, directions of research in my laboratory are the investigation of the molecular effects of large genome variants during neuronal development using iPSC model systems and the study of the nature and effects of somatic genome variation in the brain using tissue culture models and primary tissue samples.
We investigate mechanisms underlying the faithful inheritance of eukaryotic chromosomes. Our primary focus is on elucidating the events required for orderly segregation of homologous chromosomes during meiosis, the crucial process by which diploid germ cells generate haploid gametes. These events are of central importance to sexually reproducing organisms, since errors in meiosis lead to chromosomal aneuploidy, one of the leading causes of miscarriages and birth defects in humans.
Diploid germ cells face several major challenges on the road to reducing their ploidy to generate haploid gametes: 1) Chromosomes must locate, identify and align with their appropriate homologous pairing partners. 2) Chromosomes must acquire a structural organization that will promote controlled breakage of DNA molecules and subsequent recombinational repair using the homologous chromosome as a repair partner to yield interhomolog crossovers. 3) Chromosomes must couple the events of recombination with further structural reorganization to yield an organization in which homologs are connected by chiasmata, yet oriented away from each other in a way that promotes their attachment to and segregation toward opposite poles of the meiosis I spindle. Moreover, the connections afforded by chiasmata must be coupled with a two-step loss of cohesion, such that partial loss of cohesion occurs at meiosis I to permit dissolution of chiasmata and homolog separation while maintaining the connections between sisters required to permit bipolar attachment on the meiosis II spindle. 4) During oocyte meiosis, a bipolar spindle must be assembled and function without the aid of centrosomes. All of these events must be tightly coordinated to achieve a successful outcome.
Despite the fundamental importance of meiosis, the mechanisms underlying many key events remain poorly understood. We are approaching the study of meiosis using the nematode C. elegans, a simple metazoan that is especially amenable to combining genetic, genomic and cytological approaches in a single system, and in which the events of meiosis are particularly accessible. The germ line accounts for more than half of the cell nuclei in the adult worm, with nuclei in all stages of meiosis present simultaneously in a temporal/spatial gradient along the distal-proximal axis of the gonad, so that each gonad represents a complete meiotic time course. These features facilitate visualizing chromosome organization using high-resolution microscopic imaging in the context of intact 3D nuclear architecture.
Metastasis is a major clinical challenge driven by poorly understood cell state alterations. The goal of our lab is to use unbiased genomic methods and in vivo models to better understanding the molecular and cellular changes that underlie tumor progression and each step of the metastatic cascade. We use genetically-engineered mouse models of metastatic cancer in which the resulting tumors recapitulate the genetic alterations and histological progression of the human disease.
In these models, tumors develop within their appropriate microenvironment and undergo changes in their gene expression programs that endow them with the ability to invade blood and lymphatic vessels, survive in circulation, enter various distant organs, and ultimately grow into new tumor lesions. Given the dearth of human tissue samples from metastatic disease, especially from primary tumors and metastases from the same patient prior to therapy, these models represent a unique opportunity to understand the molecular biography of the most prevalent tumor types.
By generating activating and inactivating germline and inducible alleles, and modulating gene expression using lentiviral vectors, these models allow us to characterize the function of candidate genes and pathways during tumor progression and metastasis in vivo. By incorporating increasingly quantitative methods and powerful in vivo methods, our work is focused on uncovering general rules that govern tumor progression and metastatic spread and discovering novel therapeutic targets across the continuum of cancer progression including the lethal metastatic stage.
Lab website: http://yehlab.stanford.edu/
The chemistry and biology of the unique plastid organelle, the apicoplast, in malaria parasites
Malaria caused by Plasmodium spp parasites has an enormous disease burden that disproportionately affects the worlds poorest and youngest. New anti-malarials with novel drug mechanisms are desperately needed in the face of existing or emerging drug resistance to all available therapies. Investigation of Plasmodium biology offers both the potential for important biomedical impact and an opportunity to explore fascinating eukaryotic biology. Given the challenges of genetic and other approaches to studying this complex organism, the development of chemical tools will be especially critical in pushing forward basic research.
My research focuses on the apicoplast, a prokaryotically-derived plastid organelle unique to Plasmodium (and other pathogenic Apicomplexa parasites) and a key anti-malarial drug target. My laboratory's goal is to elucidate apicoplast biology, function, and role in pathogenesis with the ultimate goal of realizing the potential of the apicoplast as a therapeutic target. In a major step toward this goal, my previous work has demonstrated that the sole essential function of the apicoplast in blood-stage P. falciparum parasites is the biosynthesis of isoprenoid precursors. As such, I was able to generate parasites completely devoid of this essential organelle but chemically rescued by supplementation of the growth media with isopentenyl pyrophosphate (IPP), the pathway product. Chemical rescue and apicoplast(-) parasites are innovative tools for investigating apicoplast biology and for advancing apicoplast-directed drug and vaccine development. Our research takes advantage of these new tools and our newfound understanding of apicoplast function to explore a variety of topics, including protein trafficking to the apicoplast and the protein "prenylome" in Plasmodium. We employ a variety of methods but have a particular focus on the use of chemical tools to overcome the current challenges in studying this organelle. Our exploration of the Plasmodium apicoplast are likely to reveal both unique biology and targets for anti-malarial drug development.