Gregory E Crawford - US grants

[unreadable] DESCRIPTION (provided by applicant): Sequence analysis of the human genome has identified approximately 25,000 protein-coding genes, but little is known about how most of these genes are regulated in different tissues and stages of development. A number of approaches attempt to identify gene regulatory elements on a genome-wide scale, but there is yet to be a proven method that accurately accomplishes this goal. Only with the development of better experimental technologies will the rapid identification of regulatory elements be possible. Mapping DNase hypersensitive (HS) sites has been the gold-standard method for identifying the location of promoters, enhancers, silencers, and insulators. While this method has been proven invaluable for identifying the location of active regulatory elements for individual genes, the labor-intensive nature of this technique has limited its application to only a small number of human genes. We have developed a novel protocol to generate a genome-wide library of gene regulatory sequences by cloning DNase HS sites. As a pilot, we generated a library of DNase HS sites from quiescent primary human CD4+ T cells and analyzed 5,600 of the resulting clones. Compared to sequences from randomly generated in silico libraries, sequences from these clones were found to map more frequently to regions of the genome known to contain regulatory elements, such as regions upstream of genes, within CpG islands, and in sequences that align between mouse and human. Validation of putative regulatory elements was achieved by repeated recovery of the same sequence (clustering), and by real-time PCR. To distinguish all valid DNase HS sites from background, we estimate it is necessary to sequence approximately 1 million clones. To generate this number of sequences affordably and rapidly, we will employ massively parallel signature sequencing (MPSS), a bead-based technology capable of generating 1 million sequence tags per run. Preliminary data show that this technology is readily adaptable to our cloning protocol and can efficiently capture DNase HS sites. MPSS will be used to sequence DNase HS libraries from a number of different cell types, including human embryonic stem cells. Comparisons to comparative genomics will determine the degree to which human DNase HS sites are shared among different species. Functional characterization of a representative sample of DNase HS sites around interesting loci of the genome will identify how these regions of the genome are regulated. Characterizing different DNase HS sites libraries will allow for a better understanding of the chromatin differences that delineate tissue specificity, housekeeping function, cell activation, pluripotency, and early cell differentiation. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The goal of this proposal is to identify at high resolution all active gene regulatory elements in the human genome among cell types representative of most human tissues. We will accomplish this goal by identifying regions of open chromatin with two independent and complementary methods: DNasel hypersensitivity and Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE), combined with single-nucleosome mapping and chromatin immunoprecipitation (ChIP) for selected regulatory factors. The immediate benefit of success will be a high-quality public atlas of the human DNA regulatory elements that are likely to be active in each cell type. [unreadable] [unreadable] Identification of open chromatin regions has been one of the most accurate and robust methods to identify functional promoters, enhancers, silencers, insulators, and locus control regions in mammalian cells. A principal advantage of an open chromatin approach is that all potential sites in the genome are simultaneously assayed in an unbiased manner. DNasel hypersensitivity and FAIRE interrogate chromatin by entirely different underlying mechanisms, and therefore represent independent, cross-validating assessments of chromatin state. In addition, we will perform single-nucleosome mapping in selected cell types as an independent direct biochemical verification of open chromatin regions. For a selected subset of cell-types, we will further annotate open chromatin regions with respect to their biological activity by determining the binding location of proteins that mark transcription start sites, transcriptional units, insulators, or have broad regulatory function. [unreadable] [unreadable] The Aims of this proposal are 1) to identify all regions of open chromatin in 40 cell types by DNasel and FAIRE analyses, and 2) determine the biological function of open chromatin regions by ChIP and nucleosome mapping. To accomplish these Aims, we have assembled a team of five researchers who are leaders in their respective fields and who have contributed significantly to the ENCODE pilot project. Furthermore, we have developed a streamlined pipeline, which has been used to generate high quality whole-genome data from a number of cell types. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Genome sequencing and the identification of epigenetic marks by projects such as ENCODE and the Epigenomics Roadmap Project have transformed biomedical research. Technologies for targeted manipulation of these epigenetic properties are necessary to transform the knowledge gained from these projects into tangible scientific advances and benefits for human health, such as gene therapies that modify the epigenetic code at targeted regions of the genome and the engineering of epigenome-specific drug screening platforms. To address this technology gap, we are developing a suite of well-characterized tools for custom locus- and cell type-specific modification of any epigenomic property with precise spatiotemporal control. These tools consist of fusion proteins of programmable DNA-binding proteins and enzymes that control genome structure and function. These epigenetic modifiers (EGEMs) can be specifically targeted to nearly any site in the genome. Optimized EGEM designs will be tested on both proximal and distal regulatory elements that represent diverse chromatin states, including active, repressive, bivalent, and imprinted marks. The generality of EGEMs will be shown on additional high-value targets that have broad relevance to disease. Importantly, all of these tools function independent of cell- and species-type, and therefore are useful to all fields of biologic research. Comprehensive characterization of EGEM activity in human cells will be provided by targeted and genome-wide analysis of DNA-binding, chromatin structure, and gene regulation. A validated optogenetic approach for controlling protein localization with blue light will be used to achieve precise spatiotemporal control of EGEM activity. The utility of the tool set of epigenetic modifiers will b demonstrated by impacting gene regulation in a manner that is robust, specific, and heritable. We will test the working hypothesis that different genes will require a customized set of epigenetic modification(s) to achieve efficient changes in gene expression. The specificity and stability of epigenetic modifications will be of broad utility to the fields of genomics, epigenomis, imprinting, gene therapy, developmental biology, regenerative medicine, and drug development.

DESCRIPTION (provided by applicant): Cellular differentiation requires the precise orchestration of gene expression programs. Chromatin regulatory complexes coordinate this process by modulating the accessibility and activation state of gene regulatory elements. The end states of cellular differentiation can be readily visualized through the comparison of cell typ specific epigenome maps. Such analyses indicate that the differential regulation of distal enhancer elements is the primary determinant of cell-type specific programs of gene expression. However the dynamic chromatin regulatory events that sculpt the distribution of active enhancers and thus drive the differentiation of any single cell type have remained largely unknown. The goal of this proposal is to identify the chromatin regulatory events that control the differentiation of neurons in vivo. We will identify developmentally regulated chromatin changes that control contemporaneous changes in neuronal gene expression by comparing epigenomic profiles of chromatin harvested from discrete stages in the differentiation of a specific neuronal cell type. Cerebellar granule neurons (CGNs) provide an ideal in vivo model for this study because they represent a largely homogeneous neuronal population that can be obtained in very large numbers at discrete developmental stages directly from the postnatal mouse brain. Using the technique of DNaseI chromatin digestion followed by high-throughput sequencing (DNase-Seq) we have already identified substantial differences in the distribution of accessible chromatin over the course of CGN differentiation. Here we propose to characterize the developmental regulation of promoters and enhancers in these neurons by performing genome-wide chromatin immunoprecipitation (ChIP) for histone marks that denote the nature and activation state of these gene regulatory elements. We will then test the relationship between chromatin states and dynamic changes in gene expression during CGN differentiation through hidden Markov modeling of our combined DNase-Seq, ChIP-Seq, and RNA-Seq datasets. The outcome of this proposal will be the first identification of a comprehensive defined set of gene regulatory elements that control the dynamic changes in gene regulation that underlie neuronal differentiation.

DESCRIPTION (provided by applicant): Genome-wide association (GWA) studies have identified >100 regions of the genome that contribute to risk for schizophrenia. As observed for other complex disorders, the identified regions are overwhelmingly non- coding, strongly suggesting that genetic variation in gene regulatory elements is a major mechanistic contributor. Further investigation of those regulatory mechanisms is precluded by a fundamental gap in the ability to identify disorder-specific regulatory elements in the brain, and limited understand of how genetic variation within those elements influences their function. To address that knowledge gap, this project will comprehensively identify, characterize, and validate non-coding functional regulatory elements in brain tissues relevant to schizophrenia. The central hypothesis of the proposal is that non-coding variation contributes to schizophrenia by directly altering the function of regulatory elements in the brain. The motivation for the proposed study is that identifying regulatory mechanisms of schizophrenia has the potential to translate into improved diagnosis and treatment of this common, chronically debilitating disorder. Powered by a team with strong interdisciplinary expertise in psychiatric disorders, functional genomics, comparative primate genomics, and statistical genetics, this hypothesis will be tested by completing three specific aims: 1) Comprehensively identify active gene regulatory elements in three brain regions from 100 schizophrenia cases and 100 controls using ATAC-seq; 2) Identify chromatin QTLs (cQTLs) that impact chromatin accessibility and gene expression, and perform targeted association tests using the most up to date PGC GWA mega analysis results; 3) Prioritize and quantify regulatory variant function using high-throughput reporter-gene expression assays, and validate by genome editing. The approach is innovative because it utilizes a highly complementary and diverse set of experimental approaches to drive targeted genetic and functional investigation into the regulatory mechanisms of schizophrenia. Ultimately, the data produced and the experimental and statistical approaches developed will enable related studies of other disorders and diseases. In doing so, the proposed research provides a much-needed path forward to understand how non- coding variation contributes to complex human phenotypes.

Project Summary/Abstract The goal of this proposal is to develop a novel high-throughput platform for understanding gene regulatory elements in order to identify new drug targets for common diseases. The human genome encodes approximately 50,000 genes. Understanding how those genes are regulated and how this correlates to complex cell phenotypes has long been a major focus of our team. Follow-up projects to the Human Genome Project, such as the NIH-funded Encyclopedia of DNA Elements (ENCODE) and the Roadmap Epigenomics Project, have identified millions of putative regulatory elements across the human genome for many human cell types and tissues. Importantly, genome wide association (GWA) studies have strongly indicated that non- coding regulatory elements determine the gene expression patterns responsible for most complex diseases including cancer, cardiovascular disease, diabetes, and neurological disorders. However, the function of these regulatory elements and their relationships to these disease phenotype are largely unknown. Additionally, conventional screening technologies for perturbing cellular processes, such as small molecules and RNA interference, cannot directly target genomic regulatory elements. To address this critical limitation and illuminate the fundamental genomic basis of these cell phenotypes, we have recently developed epigenome- editing technologies for directly and precisely activating and repressing genomic regulatory elements in their natural chromosomal location. More recently, we have developed a novel and robust method for using these tools for high-throughput identification and quantification of gene regulatory element activity. Here, we propose to apply these methods to the discovery and validation of regulatory elements associated with cancer and cardiovascular disease as demonstration of this novel platform technology for understanding the genetic basis of complex disease. This technology will be critical to translating modern advances in genetics and genomics into new drug targets, diagnostics, and personalized medicine catered to each patient genome.   

There is a fundamental gap in understanding how the millions of known regulatory elements functionally contribute to gene regulation and phenotypes. Continued existence of that gap is an important problem because, until it is filled, it will remain extremely difficult to identify the genetic mechanisms underlying the thousands of observed genetic associations with disease phenotypes. Our long-term goal is to understand how and to what extent gene regulatory elements alter target gene expression and impact phenotypes. The objectives of this particular proposal are to functionally characterize all regulatory elements contributing to the differentiation of CD4+ T cells. In doing so, we will identify the causal regulatory mechanisms that modulate the immune system. The rationale for this work is that understanding those mechanisms will be the foundation for future efforts to therapeutically modulate the immune system, and will establish a discovery platform for determining the mechanisms underlying countless other model systems. Specifically, we will characterize three complementary components of regulatory element activity: (i) the capacity of regulatory elements to drive expression of a reporter gene, (ii) the effect of each regulatory element on the expression of one or more target genes, and (iii) the contributions of regulatory elements to phenotypic function, namely differentiation. We will accomplish those goals across three specific aims. In Aim 1, we will quantify the activity of all regulatory elements that have evidence of differential activity between subtypes of mouse CD4 T cells. We will do so using a capture-based high-throughput reporter assay that allows us to assay larger (>500 bp) fragments from specific genomic regions of interest. In Aim 2, we will quantify the effects of regulatory elements on target genes using a novel strategy that combines high-throughput CRISPR/ Cas9-based epigenome editing screens and targeted high-throughput single-cell RNA-sequencing. In Aim 3, we will determine which regulatory elements are necessary or sufficient for CD4 T cell differentiation using high-throughput CRISPR/Cas9-based epigenome-editing screens combined differentiation into particular CD4 T cell subtypes. Each aim will provide functional characterization of all of the regulatory elements implicated in CD4 T cell differentiation. Together, the aims will provide a comprehensive, multi-layered, and systematic understanding of the ways that gene regulatory elements modulate the immune system. The result will be an actionable set of targets for designing strategies to modulate immune system activity for therapeutic benefit. Because the approach is general to any model system, the same strategy can be readily transferred to diverse systems including differentiation and disease models. Therefore, we expect that this project will have both immediate and long-term benefit for determining the ways that regulatory elements contribute to health and disease.

Abstract Autism spectrum disorder (ASD) is a developmental disorder that emerges in the prenatal period, likely during the first weeks of brain development. Chromatin regulatory events in early brain development have been repeatedly implicated in ASD. Chromatin regulation in prenatal development differs in fundamental ways from chromatin regulation in adulthood, which has been an obstacle to understand ASD pathogenesis. Here, we will use telencephalic organoids derived from human iPSCs to assess the functional activity of regulatory elements we identified through the PsychENCODE project to begin to unravel chromatin and gene regulation during early stages of cortical development, including stages that are not commonly accessible using postmortem brain tissue. We will longitudinally map the activity of these elements at critical developmental transitions in both normal organoids and ASD organoids, fractioned in different cell types (progenitors and neurons), examine their functional disruption in ASD by assessing their enrichment in disease-associated variants and determine their target genes from chromatin conformation capture experiments. In Aim 1, we will use STARR- seq to map the activity of H3K27ac histone-associated putative enhancers in organoids mimicking early cortical development and will compare the STARR-seq enhancers with histone-based enhancers active in stem cells, prenatal and adult postmortem brain identified through PsychENCODE and Epigenome Road map projects. In Aim 2, we will use ATAC-seq and STARR-seq to identify and compare enhancer activity in organoids from ASD patients and controls across early development and in different cell types. For this, we will use a collection of iPSC lines we generated from families with ASD. In Aim 3, we will use capture Hi-C and RNA-seq to study the 3D chromatin organization and promoter-enhancer interactions and their effect on gene expression in ASD neural cells. We will then explore whether ASD-implicated enhancers harbor disease- associated mutations by intersection with Simons and MSSNG whole genome public databases sequence variants. Finally, in Aim 4, we will carry out detailed functional analyses on ASD-associated mutations found in the implicated enhancers. We will engineer mutations in control iPSC lines, compare pairs of isogeneic organoids with or without the mutations, and perform capture Hi-C to identify their target genes and RNA-seq to confirm their effect on gene expression. These studies will chart gene regulation in human prenatal forebrain, across stages and cell types, map enhancers that are differentially active in early neural development in autism and identify mutations that are putatively responsible for these alterations. The end results will be the identification of a network of interacting genes involved in the pathophysiology of ASD, and the genetic/epigenetic mechanism responsible for their altered function in the disorder.

ABSTRACT Determining new causes for rare and common disease would have major and immediate benefits for patients and their families by improved genetic testing, genetic counseling, insurance reimbursement, and ultimately more effective treatment options. Our long term goal is to disruptively improve and expand genetic testing for rare and common disease. Current diagnostic tests only consider pathogenic variants in protein-coding genes. However, we now have evidence that a substantial fraction of rare disease is due to unknown non-coding genetic variants that influence the regulation of those genes. The goal of this proposal is to identify and quantify the effect of pathogenic non-coding genetic variants on the function and expression of genes that cause rare disease. This initial step will enable treatment early in life when it is still possible to stop the most severe consequences of disease, including death. We will focus on severe early-onset pediatric disorders, including glycogen storage diseases (GSD I, II, III, IV, and IX), and the fatty acid oxidation disorders, very long-chain acyl-CoA dehydrogenase deficiency (VLCAD), and multiple acyl-CoA dehydrogenase deficiency (MADD). To date, genetic tests for these and other diseases are limited to protein-coding mutations. However, our clinical team has collected numerous cases that have a single pathogenic coding variant on only one of the two alleles that must be both affected in these recessive disorders. We also have biochemical and biomarker evidence that supports the diagnosis. Those cases are an ideal opportunity to identify additional disease-causing variants. Our hypothesis is that the genetic causes of recessive disorders include novel genetic variants that can alter either protein sequence (Aim 1), splicing (Aim 2), or gene expression (Aim 3) of disease genes. We have assembled a team of Pediatric clinicians who are experts in GSDs, VLCAD, and MADD, as well as researchers who are experts in genetics, genomics, epigenetic regulation, biomedical engineering, and statistics. This team has obtained patient samples and received Duke IRB approval to begin immediately. We expect this study will identify and validate novel genetic variants that influence disease. While we propose to study a relatively small subset of rare disorders, these strategies will be immediately generalizable to any patient sample with any recessive disorder that has inconclusive genetic testing results. That outcome will provide comprehensive genetic testing, better understanding of disease mechanisms, and ultimately better treatment options.

Understanding the genetic causes of human disease has immense potential to benefit human health. The human genetics community has devoted tremendous resources to identifying those causes, including, most recently, whole genome sequencing of patient cohorts. Those studies have found genetic variation in non-coding regions of the genome to be most often associated with diseases and drug responses. Unfortunately, since the effects of genetic variation on gene regulation remain poorly understood and difficult to study at the genome-wide scale, the full benefit of most of those studies has yet to be realized. Our long-term goal is to understand how non-coding genetic variants act through gene regulatory elements to influence phenotypes. The objective of this proposal, a step towards that long-term goal, is to develop a platform of empirical and statistical methods to reliably and systematically determine the regulatory mechanisms underlying human traits and diseases. Specifically, in Aim 1, we will use high- throughput reporter assays to quantify the effects of millions of human genetic variants on regulatory element activity. Those variants will represent diverse human ancestries, and will cover over 60% of all regions associated with a trait or disease via GWAS. The outcome will be the most extensive catalog of human regulatory variation every created. In Aim 2, we will develop new technologies to systematically relate those changes in regulatory element activity to changes in gene expression. That technology will combine our previous work developing CRISPR-Cas9- based epigenome editing screens with targeted single-cell RNA-seq. In Aim 3 we will develop statistical analyses to integrate the effects of regulatory variants to infer changes in gene expression and differences in phenotypes between individuals. The resulting method will be analogous to gene based association tests, but for the noncoding genome. The expected outcomes of this project are (i) dramatically improved ability to establish mechanisms underlying non-coding associations with human traits and diseases; (ii) better understanding of the genetic architecture of regulatory element activity and gene regulation that will guide the design and interpretation of future genetic association studies; and (iii) novel reagents, protocols, and software that other labs can use to complete similar investigations of their own model systems of interest. Taken together, we expect that this project will be a major step towards fully realizing the potential of genome wide and whole genome association studies.

ABSTRACT Large scale genome annotation consortia such as ENCODE, Epigenomics Roadmap, and others have identified millions of putative regulatory elements. We now need to focus efforts on comprehensively characterizing and quantifying the function of those elements, and noncoding variants that map within these regions, on gene expression and cell phenotypes. Our long-term goal is to assign function to every regulatory element and noncoding variant in the human genome, understand how that function changes in different contexts, and use that information to better understand cell fitness, disease mechanisms, cell lineage specification, and tissue homeostasis. To accomplish this goal, we have developed multiple novel high-throughput CRISPR-based technologies for characterizing the function of putative gene regulatory elements by perturbing their activity in their endogenous, native context. We have coupled these methods with single-cell RNA-seq to identify the target gene(s) for each regulatory element. We have also developed dCas9 effector mice to characterize elements in their natural in vivo context. In addition, we have developed population-based high-throughput reporter assays (POP-STARR) to characterize the impact of noncoding genetic variation across the entire genome. The objective of this proposal is to apply and share our compendium of complementary, robust, scaleable, and well-characterized methods by working collaboratively to support the IGVF Consortium goals of understanding how genomes and genomic variation function and orchestrate complex phenotypes. Our track record in developing, applying, and sharing these high-throughput characterization methods, as well as providing access to all data, supports that we will be successful in accomplishing our objective via the following specific aims: Aim 1. Characterize all gene regulatory elements essential for cell survival. Aim 2. Characterize all gene regulatory elements essential to cell lineage specification. Aim 3. Characterize all gene regulatory elements in select eQTL regions. Aim 4. Characterize all non- coding elements essential to tissue homeostasis in a mouse model. We will make all data immediately available, as well as share comprehensive protocols, reagents, and analysis tools to the scientific community. Together, the diverse approaches of this Characterization Center will lead to transformative progress in understanding the role of regulatory elements and noncoding variants across many diverse phenotypes.

ABSTRACT Growing cognitive demands over the course of human evolution have shaped the adaptation of human brains for increasingly complex higher cognitive functions, like executive control, social cognition, attention, and language. Research on those higher cognitive functions has focused predominantly on parts of the neocortex and related subcortical areas that comprise forebrain networks linked to specific cognitive functions. Recent research makes it clear, however, that each of those forebrain networks is functionally connected to distinct regions of the cerebellum. Surprisingly, evolutionary studies show further that it is those parts of the cerebellum that show the most dramatic expansion in humans compared to non-human primates, and even in modern humans compared to Neanderthals. In humans living today, individual variation in the size or functional connectivity of those cerebellar regions has been linked to disorders affecting higher cognitive functions, such as autism spectrum disorder (ASD), attention-deficit/hyperactive disorder (ADHD), and schizophrenia. These converging results suggest strongly that molecular and cellular mechanisms controlling the development and functional organization of the human cerebellum have undergone systematic changes that have proven functionally important in modern humans. The proposed studies begin to map out those changes, beginning with a genome-wide association study (GWAS) using an existing dataset of structural MRI images of cerebellum from 30,000 genotyped human participants to identify genes and genomic variants associated with overall cerebellar volume and individual differences in relative size and gray matter thickness across different regions of the cerebellar cortex (Aim 1). A parallel study (Aim 2) will use single-cell genomics of human, macaque, and mouse cerebellum to investigate possible differences in gene expression FKURPDWLQ DFFHVVLELOLW\ and the cell type composition of intrinsic cerebellar circuits between humans and other animals (Aim 2). Together, those studies address an essential but unresolved issue, whether expansion of the cerebellum in humans represents a simple increase in capacity of a basic cerebellar circuit module that is otherwise unchanged in humans, or whether the local circuitry in expanded regions of the cerebellum has undergone functionally significant modifications. In the final part of this research (Aim 3), evolutionary analysis will identify specific regulatory elements within the genes identified in the first two aims that show accelerated rates of substitution in humans or evidence of positive, purifying, or balancing selection over the course of human evolution, and whether evolutionary selection has tended to increase or decrease diversity at these sites in since the divergence of modern humans from other primates. These studies will allow us to identify specific regulatory elements or other variants that have been targets of natural selection within the genes involved in cerebellar development or adult cerebellar functions, and to compare those targets of evolutionary selection to specific variants associated with individual variation or increased risk for major psychiatric disorders in modern human populations.