2006 — 2014 |
Gilad, Yoav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Natural Selection On Gene Regulation in Humans
[unreadable] DESCRIPTION (provided by applicant): Inter-species comparisons of gene expression levels will increase our understanding of the evolution of transcriptional mechanisms and help to identify targets of natural selection. This approach holds particular promise for apes, as many human-specific adaptations are thought to result from differences in gene expression rather than in coding sequence. Expression differences have also been associated with phenotypes of medical importance, including numerous diseases as well as differential drug response. [unreadable] [unreadable] To identify genes whose regulation is likely to be of functional importance in humans, we propose to compare gene expression levels in liver and kidney within and between five humans, five chimpanzees, five orangutans, and five rhesus macaques. To do so, we will develop and use multi-species cDNA arrays that enable the measurement of genes expression differences between species, while accounting for the effect of sequence divergence on hybridization intensity. We will focus on two sets of genes: first, those whose expression levels are approximately constant across individuals from all four species, and whose regulation is therefore likely to be under stabilizing selection. This set will represent promising candidates for disease-association studies. Using a new population genetic approach that we developed, we will use polymorphism and divergence data from these genes to infer the strength and mode of natural selection acting on their upstream regulatory regions. We will also identify genes whose expression levels are constant in the three non-human primates, but consistently elevated or reduced in humans. These regulatory changes are likely to underlie human-specific adaptations. Finally, to enable this type of study for any tissue, we will design and optimize genome-wide multi-species oligonucleotide arrays. [unreadable] [unreadable] In summary, the proposed research will lead to the identification of the first set of genes whose expression regulation is likely to evolve under natural selection in humans and will shed light on the relationship between gene expression patterns and the evolution of cis-regulatory regions. [unreadable] [unreadable] [unreadable]
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1 |
2009 — 2012 |
Gilad, Yoav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Integrating Genomics and Gene Expression Analyses to Map Cvd-Associated Loci
DESCRIPTION (provided by applicant): Despite advances in molecular and statistical genetics, identifying specific genes that contribute to the pathogenesis of common diseases has been challenging. This is due, at least in part, to the extensive genetic and phenotypic heterogeneity that characterize these diseases, the importance of non-genetic (e.g., environmental) factors that are rarely taken into account, and sample sizes that are often under powered to detect the likely modest effects of disease susceptibility genes. Here, we propose to integrate mapping and genome-wide expression profiling in order to find genes or regulatory regions that contribute to variability in susceptibility to and severity of cardiovascular diseases (CVD). To overcome the challenges described above, we propose to study the genetic basis for variation in physiological quantitative traits (QTs) that are associated with CVD susceptibility in a founder population with a remarkably uniform environment. By mapping genes for disease- associated physiological QTs, we will indirectly identify genes that influence susceptibility to or severity of the disease. Specifically, we plan to focus on four CVD-associated QTs, including a marker of general inflammation, for which associated genomic regions were previously identified in the Hutterites, a founder population of European descent. In order to hone in on the most promising candidate genes that underlie variation in these QTs, we will integrate several complementary approaches: (i) Use expression profiling in lymphoblastoid cell lines (LBL) from the Hutterites to identify candidate genes whose expression is associated with one or more of the QTs and that lie in genomic regions previously identified as linked to the QTs. (ii) Use a novel multi-species microarrays to compare expression profiles across primates and identify genes whose expression levels in the human liver, kidney, lymphocytes and heart evolved under natural selection and which lie in regions previously linked with the QTs, or whose expression is associated with variation in the QTs. (iii) Use an eQTL approach to map the genetic variants that influence the expression levels of the candidate genes identified in the first two approaches. PUBLIC HEALTH RELEVANCE: We propose a unique combination of genomics, evolutionary analyses of gene regulation, and genetic mapping to identify a set of genes that underlie variation in quantitative traits associated with cardiovascular diseases, including systolic blood pressure and a marker of general inflammation.
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1 |
2010 — 2013 |
Gilad, Yoav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Evolution of Human Specific Regulatory Pathways
DESCRIPTION (provided by applicant): A central goal of evolutionary biology is to elucidate the genetic architecture of adaptation. In humans, this question is of interest both for what it will reveal about our species-specific traits and because of the emerging links between adaptation and disease susceptibility. To date, however, there are only a handful of examples of human regulatory adaptations, such that many outstanding questions remain open. Among these: Which pathways have been remodeled in human evolution? Do adaptive changes in the regulation of entire pathways involve changes to many genes, or to few? What is the relative importance of changes in cis (e.g., promoter regions) vs. trans (e.g., transcription factors) regulatory elements? How prevalent are compensatory changes in regulatory pathways? As a first step towards answering these questions, we propose to identify transcriptional pathways that have been adaptively remodeled in humans and to examine their evolution across three primate species. Specifically, we plan to focus on five transcription factors that have been shown previously (in our work and by others) to be the target of positive selection in the human lineage. Through a combination of siRNA knockdowns, gene expression profiles, ChIP-seq, and reporter gene experiments, we will identify the genes that are directly regulated by these transcription factors, not only in humans but also in two close evolutionary relatives, chimpanzees and rhesus macaques. The proposed combination of approaches will lead to the reliable annotation of direct regulatory targets of five transcription factors in three species and facilitate the identification of transcriptional pathways that underlie human-specific adaptation. Comparison of regulatory networks in the three species will reveal the genetic basis for a large set of regulatory differences between humans and closely related species, enabling us to address many of the above questions. To our knowledge, this research represents the first genome-wide exploration of differences in regulatory pathways across species. In addition to identifying pathways that have been adaptively remodeled in the human lineage, it will yield unprecedented insights into the genetic basis of regulatory change at the transcription level. PUBLIC HEALTH RELEVANCE: The goal of the proposed study is to identify a first set of regulatory pathways that have been remodeled in humans, and learn about the genetic basis of gene regulatory changes in primates.
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1 |
2011 — 2015 |
Gilad, Yoav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mapping Eqtls That Affect Susceptibility to Tuberculosis
DESCRIPTION (provided by applicant): Tuberculosis is a major public health problem. One-third of the population of the world is estimated to be infected with Mycobacterium tuberculosis (Mtb), the etiological agent causing tuberculosis (TB), and active disease kills nearly 2 million individuals worldwide every year. Successions of treatments of TB have quickly become ineffective as the agent rapidly becomes resistant. However, strikingly, only 10% of infected individuals develop the disease. In other words, while Mtb quickly develops resistance to new drugs, roughly 90% of individuals are naturally resistant to infection (when not co-infected by agents, which compromise the immune system, such as HIV). Several lines of evidence indicate that genetic factors contribute to inter-individual differences in susceptibility to TB, including the observation that monozygotic twins have considerably higher concordance rates for tuberculosis morbidity than do dizygotic twins. In addition, multiple rare single-gene mutations with high penetrance have also been linked with susceptibility to mycobacteria. However, although genetic studies of TB have identified important pathways involved in protective immunity, very little is known about the underlying genetic determinants or mechanisms contributing for differences in susceptibility at the population level. Here, we propose to use a combination of empirical and statistical approaches to identify genes and regulatory pathways that contribute to inter-individual and inter-population variability in the immune response to Mycobacterium tuberculosis infection. Specifically, we will study inter- individual variation in the immune transcriptional response of dendritic cells following infection with Mtb, and map the genetic loci that are associated with such variation (eQTLs). To our knowledge, this will be the first genome-wide study of variation in molecular quantitative traits and associated genetic markers that underlie inter-individual variation in immune response to infection with Mtb, and ultimately variation in susceptibility to TB.
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1 |
2011 — 2013 |
Gilad, Yoav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mapping Qtls Associated With Variation in Rna Decay Rates
DESCRIPTION (provided by applicant): Expression quantitative trait loci (eQTL) mapping studies have become a widely-used tool for identifying genetic variants that affect gene regulation. In these studies, gene expression levels are viewed as quantitative traits, and expression phenotypes are mapped to particular genomic loci by combining estimates of gene expression levels across individuals with genome-wide genotyping data. Recent eQTL studies in humans, as well as in other species, have revealed substantial variation in gene expression levels within and between populations, and identified a large number of genetic factors that influence gene regulation. However, to date, all eQTL mapping studies, regardless of species, have considered variation in steady-state gene expression levels and thus could not determine the exact regulatory mechanism underlying the eQTL association. In particular, typical eQTL studies, as well as most other genome-wide studies of gene expression phenotypes, do not collect data that will allow one to distinguish between variation in transcriptional regulation and variation in RNA decay rates. In general, perhaps because transcription initiation rates are commonly assumed to be the major determinants of overall gene expression levels, RNA decay mechanisms are under-studied compared with the regulation of transcription. As a result, we know relatively little about variation in RNA decay rates across genes or between individuals, and the potential importance of such variation in determining ultimate physiological phenotypes such as human disease remains unclear. To address this issue, we propose to perform a genome-wide eQTL mapping study of RNA decay rates in HapMap lymphoblastoid cell lines (LCLs) for which genome-wide steady- state gene-expression data and genotype data are also available. The data we propose to collect will allow us to identify genetic factors underlying variation in RNA decay rates as well as to study the relative importance of variation in RNA decay rates to overall regulatory variation in gene expression levels.
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1 |
2012 — 2015 |
Gilad, Yoav Pritchard, Jonathan K (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Analysis and Interpretation of Noncoding Regulatory Variation
DESCRIPTION (provided by applicant): One of the central problems in modern human genetics is to understand the functional impact of genetic variation. Of the millions of DNA positions that vary among humans, which sites actually impact human phenotypes and disease? It is becoming increasingly clear that noncoding variants that impact gene regulation play central roles in the genetics of disease. Yet we have limited understanding of the precise pathways by which these variants act, and it remains extremely difficult to predict which variants in the genome have regulatory effects. In this projec we propose to use detailed functional characterization of a variety of aspects of gene regulation, using 70 human lymphoblastoid cell lines as a model system, along with new computational methods, to dissect in detail the mechanisms by which genetic variation impacts gene expression levels.
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1 |
2014 — 2017 |
Gilad, Yoav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Eqtl Mapping in Ipsc-Derived Differentiated Cardiomyocytes - Renewal 01 - Resubmi
DESCRIPTION (provided by applicant): Genome-wide association studies (GWAS) have identified many variants associated with cardiovascular-related diseases, some of which are novel. However, similar to other common diseases, these identified risk-associated variants fail to explain a significant portion of the genetic heritability of cardiovascular disease (CVD). Moreover, many associated variants are non-coding with no obvious function, though putatively, these are involved in gene regulation. By combining results of GWAS with expression quantitative trait locus (eQTL) mapping one can identify functional variants that influence gene expression and are also associated with disease risk. Using such combination of approaches one can identify true weak associations that are otherwise difficult to distinguish from statistica noise using a GWAS approach alone, as well as develop an immediate intuition regarding both the function of associated variants and knowledge of the implicated genes. However, for this method to be most effective, eQTL studies should be performed in cells that are relevant to the phenotype of interest, which are often not easily accessible in population samples. Indeed, nearly all eQTL mapping studies in humans to date (including the studies we performed in the first term of this grant) used gene expression measurements from blood cell types, fibroblasts, or lymphoblastoid cell lines (LCLs). In that sense, induced pluripotent stem cells (iPSCs) can change human genetics in a profound way. The ability to differentiate iPSCs can allow us to perform functional studies in the most relevant cell types. We thus propose to map eQTLs and investigate the genetic basis of cardiovascular disease in cardiomyocytes, which will be differentiated from induced pluripotent stem cells (iPSCs) of 120 Hutterite individuals. The Hutterites are a founder population of European descent that practices a communal, farming lifestyle. The Hutterites of South Dakota, the subjects of our studies, live on communal (15-25 families) farms (called colonies), where all meals are prepared and eaten in a communal kitchen, smoking is prohibited (and rare), and early life environments are extremely uniform. Our specific aims are to reprogram iPSCs from the LCLs of 120 Hutterites and obtain differentiated cardiomyocytes from each individual (aim 1), map eQTLs in differentiated cardiomyocytes (aim 2), and integrate eQTL mapping with GWAS results to identify variants associated with CVD-related phenotypes (aim 3). At the conclusion of this work we expect to gain important insight on the genetic basis of gene regulation in the heart in general, as well as on gene regulatory variation that is associated with CVD risk.
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1 |
2015 |
Crawford, Gregory E Furey, Terrence S. [⬀] Gilad, Yoav Rusyn, Ivan (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genes, Genomes, and Genotoxicity: in Vivo Epigenetic Toxicology of 1,3-Butadiene @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): Epigenetic eprogramming has been proposed as an integral part of the genome instability enabling characteristic of cancer cells. Chemical-induced epigenetic changes may be a consequence of DNA damage, or may be part of the non-genotoxic mechanisms of carcinogenesis. Our recent studies provide critical additional insights into linkages between genotoxic and epigenetic mechanisms of carcinogenesis. First, using a multi-strain mouse model of the human population, we showed that important inter-individual (e.g., inter- strain) differences exist in both genotoxic and epigenotoxic effects of the classic genotoxic carcinogen 1, 3-butadiene and other chemicals. Second, we confirmed the hypothesis that the chromatin remodeling response is an underlying mechanism for the inter-strain differences in butadiene-induced DNA damage. These novel findings shaped this project's overall objective to uncover the mechanistic linkages between the genome (e.g., DNA sequence variants), epigenome (e.g., chromatin status), and molecular initiating events (e.g., DNA damage) elicited by a genotoxic carcinogen butadiene in an in vivo mouse model. Two Specific Aims will test the hypothesis that genetic variability-associated chromatin remodeling events affect the genotoxic potential of butadiene. In Specific Aim 1, we will extend our exciting finding that major differences in the extent of butadiene- induced DNA damage between inbred mouse strains are the result of epigenetically-controlled chromatin status. We will utilize deep sequencing-based DNaseI hypersensitivity mapping and chromatin immunoprecipitation analyses of representative histone modifications that regulate chromatin status, coupled with RNA sequencing-enabled gene expression analysis and measurements of butadiene-specific DNA damage. This data will permit deeper understanding of the toxicant-induced changes in chromatin in butadiene-sensitive and resistant strains. We will probe these events in both sexes and in target and non- target tissues for butadiene-induced carcinogenesis. In Specific Aim 2, using similar experimental techniques we will connect chromatin variation and genotoxic effects of butadiene with DNA sequence variation. To do so, we will use a large panel of recombinant inbred mouse lines from the Collaborative Cross resource, a unique and powerful tool for population genetics studies in experimental animals. In summary, this proposal not only will use the most novel tools to investigate carcinogen effects on genome biology, but it also will offer experimental proof to a paradigm-shifting concept that genetically-determined chromatin status modulates disease risk from genotoxic exposures.
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0.921 |
2016 — 2020 |
Gilad, Yoav Kotton, Darrell N. [⬀] Morrisey, Edward E (co-PI) [⬀] Raby, Benjamin Alexander Wilson, Andrew A |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A National Ips Cell Network With Deep Phenotyping For Translational Research @ Boston University Medical Campus
Project Summary/Abstract The discovery of iPSCs provides an unprecedented opportunity for any scientist to derive an inexhaustible supply of patient-derived primary cells. These cells containing each patient's own genetic background can now be applied for in vitro human disease modeling, drug screening of personalized therapeutics, and the development of future regenerative cell-based therapies. The most valuable human clones already generated by the CTSA investigators collaborating on this proposal not only carry common disease-associated mutations and polymorphisms, but also carry knock-in fluorochrome reporters targeted to specific loci through state-of-the-art gene editing technologies. The goal of this proposal is the establishment of a CTSA network of induced pluripotent stem cell (iPSC) repositories and iPSC cores that will enable advanced disease modeling using >1000 existing normal and disease specific human cell lines and banking 6,000 additional samples procured from the 2nd and 3rd generation participants of the Framingham Study. A concerted effort for curation, sharing, and distribution of this vital resource across all CTSAs does not exist. This proposal thus creates a CTSA iPSC Network led by teams who have championed an `Open Source Biology' approach, freely sharing iPSC lines and their reprogramming reagents with more than 500 labs to date across the globe. Its goals are to make patient-derived iPSCs together with the tools and expertise for their genetic manipulation available to the greater research community on a large scale to realize their promise for extending understanding of disease and developing potential therapies. To achieve these goals, it proposes: a) national sharing of >1000 iPSC lines already derived by the CTSA teams collaborating in this proposal, representing a critical resource in high demand by both basic and clinical researchers, b) development and support of formalized education and training programs able to nationally disseminate the expertise required to fully harness these new tools and differentiate them into the wide diversity of human cell lineages, c) maintenance and sharing of open source gene-editing tools and gene edited iPSC lines that will enable CTSA investigators to manipulate the human genome at will, and d) derivation for national sharing of additional iPSC lines generated from the most densely clinically and genetically phenotyped cohort of individuals currently followed in the USA today: the ~6,000 participants of the second and third generations of the Framingham Study.
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0.948 |
2017 — 2019 |
Gilad, Yoav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Using Single Cell Rna-Seq to Study Regulatory Noise and Robustness
Abstract The importance of robustness and the regulation of noise as mechanisms that maintain high evolutionary fitness is obvious. Yet, we still have a relatively poor understanding of how robustness is achieved and how is noise being regulated at the molecular level. In model organisms, robustness and evolvability can be studied using experimental evolution approaches. Relative robustness is typically quantified with respect to the change in variation of a trait when the experimental perturbation is applied. In such experiments, the phenotypic outcomes rather than the underlying mechanisms of robustness are measured. With few exceptions, experimental evolution studies have always considered population-average measurements of phenotypes using entire organisms, tissues, or cell cultures. However, as was often suggested in the literature, to truly understand how robustness is established and encoded in the genome, one needs to consider variation in phenotypes across individual cells. To take first steps towards understanding how robustness is regulated in humans, we propose to focus a molecular phenotype, namely gene expression levels. We will characterize the loci in which genetic variation is associated with inter-individual differences in gene expression robustness, validate a subset of such loci, and develop an understanding of the underlying mechanics. To do so, we propose to apply a QTL approach to map inter-individual variation in regulatory noise using single-cell gene expression level phenotypes measured in human induced pluripotency stem cells (iPSCs). Our preliminary results indicate that the level of gene-specific regulatory noise is a trait whose largest component of variation is associated with the individual origin of the sample. Other studies, mostly in yeast, have shown that heterogeneity across single cells in the expression of certain genes is highly heritable and placed under complex genetic control, suggesting that the level of noise in gene regulation may also differ between individuals of multicellular organisms depending on their genetic background. Follow-up studies further demonstrated that gene expression noise mediated by promoter variants could provide a fitness benefit at times of environmental stress in yeast, highlighting the direct role of genetically controlled stochastic cell-cell variation in evolutionary robustness. We will therefore test the hypothesis that the regulation of gene expression noise and robustness is genetically encoded. Specifically, we will collect single cell RNA-seq from iPSCs of 70 Yoruba individuals (Aim 1), map genetic loci that are associated with inter-individual variation in gene expression noise (robustness QTLs; Aim 2), and study the mechanisms underlying robustness QTLs (Aim 3), by analyzing the single cell data in combination with functional genomic data from the same samples.
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1 |
2018 |
Gilad, Yoav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterizing and Mapping Gene Regulatory Robustness in Cardiomyoctes
Abstract We propose to study gene regulatory noise in differentiated cardiomyocytes using single cell technology. The importance of robustness and the regulation of noise as mechanisms that maintain high evolutionary fitness is obvious. Yet, we still have a relatively poor understanding of how robustness is achieved and how is noise being regulated at the molecular level. In model organisms, robustness and evolvability can be studied using experimental evolution approaches. Relative robustness is typically quantified with respect to the change in variation of a trait when the experimental perturbation is applied. In such experiments, the phenotypic outcomes rather than the underlying mechanisms of robustness are measured. With few exceptions, experimental evolution studies have always considered population-average measurements of phenotypes using entire organisms, tissues, or cell cultures. However, as was often suggested in the literature, to truly understand how robustness is established and encoded in the genome, one needs to consider variation in phenotypes across individual cells. To take first steps towards understanding how robustness is regulated in humans, we propose to focus a molecular phenotype, namely gene expression levels. We will characterize the loci in which genetic variation is associated with inter-individual differences in gene expression robustness, validate a subset of such loci, and develop an understanding of the underlying mechanics. To do so, we propose to apply a QTL approach to map inter-individual variation in regulatory noise using single-cell gene expression level phenotypes measured in human differentiated cardiomyocytes. Our preliminary results indicate that the level of gene-specific regulatory noise is a trait whose largest component of variation is associated with the individual origin of the sample. Other studies, mostly in yeast, have shown that heterogeneity across single cells in the expression of certain genes is highly heritable and placed under complex genetic control, suggesting that the level of noise in gene regulation may also differ between individuals of multicellular organisms depending on their genetic background. Follow-up studies further demonstrated that gene expression noise mediated by promoter variants could provide a fitness benefit at times of environmental stress in yeast, highlighting the direct role of genetically controlled stochastic cell-cell variation in evolutionary robustness. We will therefore test the hypothesis that the regulation of gene expression noise and robustness is genetically encoded. Specifically, we will collect single cell RNA-seq from in differentiated cardiomyocytes of 70 Yoruba individuals (Aim 1), map genetic loci that are associated with inter-individual variation in gene expression noise (robustness QTLs; Aim 2), and study the mechanisms underlying robustness QTLs (Aim 3), by analyzing the single cell data in combination with functional genomic data from the same samples.
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1 |
2018 — 2021 |
Gilad, Yoav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Eqtl Mapping in Ipsc-Derived Differentiated Cardiomyocytes
We propose to study gene regulatory noise in differentiated cardiomyocytes using single cell technology and use these functional data to identidy novel CVD risk loci. Genome-wide association studies (GWAS) have identified many variants associated with cardiovascular- related diseases, some of which are novel. However, similar to studies of other common diseases, these identified risk-associated variants fail to explain a significant portion of the genetic heritability of cardiovascular disease (CVD). Moreover, many associated variants are non-coding without obvious function, though putatively, these are involved in gene regulation. By combining results of GWAS with functional genomic data (for example, eQTL mapping), one can identify variants that influence molecular functions and are also associated with disease risk. Individual cells are expected to tolerate uncertainties in the form of both external and internal perturbations arising from variable environments or mutations. This is especially critical in the context of cell fate transitions during differentiation. It has long been recognized that robustness is an inherent property of all biological systems and is strongly favored by evolution. Depending on their different roles, the regulation of subsets of genes is required to be particularly robust in order to maintain the phenotype or the identity of a cell. Many dynamic physiological processes must also be robust, and as a result, loss or grain of robustness is associated with certain clinically relevant phenotypes and complex genetic disease. In particular, inter-individual variation in penetrance of a mutation associated with a disease, or the variability in response to drug, can also be explained at times by different degrees of robustness. Despite the importance of robustness and the regulation of noise as mechanisms that maintain high fitness, we still have a relatively poor understanding of how robustness is achieved and how is noise being regulated at the molecular level. To take first steps towards understanding how robustness is regulated in humans, and to identify loci where mutations can affect robustness and underlie CVD risk, we will use single cell technology to collect gene expression data from differentiated cardiomyocytes. We will use a detailed time course design and high- resolution single cell gene expression data. Our approach will allow us to map variance QTLs (robustness QTLs) in addition to the more standard expression QTLs. Robustness QTLs may be of particular importance as contributors to CVD risk, a diseases in which threshold effects are predominant. Specifically, we will propose to collect single cell RNA-seq throughout cardiomyocyte differentiation of 70 Hutterite individuals (Aim 1), map eQTLs, as well as genetic loci associated with inter-individual variation in gene expression robustness (Aim 2), and Integrate eQTL and robustness QTL mapping with GWAS results to identify variants associated with CVD risk and with CVD-related phenotypes (Aim 3).
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1 |
2018 — 2020 |
Gilad, Yoav |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Genomics
ABSTRACT The University of Chicago Medicine Comprehensive Cancer Center (UCCCC) Genomics Core Facility (GCF) is tasked with providing state-of-the-art genomics data generation services to University of Chicago faculty in a fee-for-service model. UCCCC members? fees are subsidized by the Cancer Center Support Grant (CCSG) via a co-pay mechanism. At present, the GCF main services are next-generation sequencing, DNA microarray analysis, and Sanger sequencing. The GCF also provides data storage services to its clients and partners with the Bioinformatics Core Facility (BiCF) for genomic data analysis. The GCF is organized into two Subcores, Next-Generation Sequencing and Microarrays Subcore, and DNA Sequencing and Genotyping Subcore, and has been located on the first floor of the Knapp Center for Biomedical Discovery (KCBD) since 2009. The GCF is directed scientifically by Yoav Gilad, PhD, Professor of Human Genetics, and operationally by Pieter Faber, PhD, with the assistance of William Buikema, PhD, as Technical Director of the DNA Sequencing and Genotyping Subcore. In addition to the leadership, the GCF employs nine technologists (six in the Next- Generation Sequencing and Microarrays Subcore, and three in the DNA Sequencing and Genotyping Subcore). Core developments in the current funding period include increased emphasis on next-generation sequencing (NGS) services to meet demand and advancements in the field. The main operating instruments of the Facility include Applied Biosystems 3730xl DNA analyzers (DNA Sequencing and Genotyping Subcore), an Illumina HiScan, and an Affymetrix GeneScan3000 microarray scan system, as well as three Illumina next- generation sequencing instruments (HiSEQ4000, HiSEQ2500, and a NextSeq500; Next-Generation Sequencing and Microarrays Subcore). The GCF effectively serves users? needs, and the services provided using these instruments occupy 80-90% of the available instrument and/or personnel time, indicating that staffing and instrumentation are operating and maintained at an appropriate level. UCCCC members receive priority, and projects are prioritized based on sample reception date and project urgency. To direct operations, Drs. Gilad and Faber meet on a biweekly basis, discussing any operational issues, as well as short-term and long-term strategies. Additionally, Drs. Gilad and Faber meet regularly with the GCF Faculty Oversight Committee (FOC) to receive constructive feedback from expert users in the genomics field. To best serve users? needs and gauge user satisfaction, the University of Chicago Office of Shared Research Facilities under the leadership of George Langan, DVM, conducts annual on-campus user surveys. The most recent survey from August 2016 showed a high approval rating (approximately 80%), with 99% of responders predicting continued use of the Facility in the future. New services will be added as needed (e.g., the Facility intends to add single cell RNA-SEQ to its repertoire (DROP-SEQ protocol) in 2017).
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1 |
2019 — 2021 |
Gilad, Yoav |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Characterizing and Understanding Variation in Gene Regulatory Mechanisms Within and Between Species'
A central challenge in modern genomics is to decipher how DNA sequence encodes regulatory information, and how this is interpreted by trans-acting factors to produce cell type-specific programs of gene expression. We propose to use a variety of approaches and model systems to elucidate the genomic mechanisms that control spatially and temporally dynamic gene expression programs. To do so, we will use our recently established panels of induced pluripotent stem cells (iPSCs) from humans and chimpanzee, which allow us to perform dynamic and high-resolution studies of different gene regulatory phenotypes. We focus on addressing three complementary questions that allow us to study gene regulation using different perspectives: (i) Using evolutionary perspective, we ask what are the genetic and regulatory differences between humans and non-human apes? (ii) Using population perspective, we ask how do genetic changes in regulatory elements result in inter-individual differences in transcript and protein expression levels? (iii) Finally, focusing on a fundamental property of gene regulation, which until recently we were unable to study because of lack of suitable technology: We ask what is the genetic and mechanistic basis for the regulation of gene expression noise and robustness?
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1 |
2019 — 2021 |
Gilad, Yoav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Development of Ipscs For Comparative Genomics in Primates
Abstract This is a new proposal submitted in response to a funding opportunity focused on comparative genomics research (PAR-17-482). The FOA states that NHGRI invites applications for the ?development of new comparative genomics research approaches using genomic data types to understand biological systems, networks, and pathways.? And that ?high priority will be given to applications that propose innovative and promising approaches to genome-wide and multi-species comparisons.? Differences in gene regulation between humans and other primates may ultimately be used to explain the molecular basis for human-specific traits. While current comparative studies in primates have provided valuable insight into the genetic architecture of gene regulation, they do not provide a flexible framework to study inter-species variation in gene regulation in multiple cell types from the same individuals. In particular, frozen post-mortem tissues are not optimal templates for many functional genomic assays; as a result, we lack data sets that survey multiple dimensions of gene regulatory mechanisms and phenotypes from the same samples. Moreover, because it is rare to collect a large number of tissue samples from the same donor, we have never had the opportunity to study population-level patterns of gene regulation in multiple tissues or cell types derived from the same non-human ape genotype (same donor), and we have not been able to study population-level dynamics of gene regulation, for example, during perturbation. We propose to explore an alternative promising way to move forward. Recent extraordinary advances in molecular biology suggest a way forward. It has now become possible to reprogram somatic cells, such as fibroblasts and several types of blood cells, into a pluripotent state, in which the cells have the capability to both self-replicate indefinitely and to differentiate into any cell type in the body. These reprogrammed (or induced) pluripotent stem cells (iPSCs) can then be directed to differentiate into specific cell types, which can then be studied in detail. The availability of iPSC lines from multiple species could therefore change comparative primate genetics and genomics in a profound way, by allowing us to sidestep traditional limitations on research in primates.
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1 |