Kevin P. White - US grants

[unreadable] DESCRIPTION (provided by applicant): We propose to continue our analyses of the Drosophila genome during development. In the next five year period we will focus on two technological challenges in the context of Drosophila developmental and evolutionary change. The overarching theme is to study transcript diversity and variation. First we will develop new methods for assessing transcript diversity due to splicing in Drosophila. We will then apply these methods to annotate and analyze the patterns of splicing during the Drosophila life cycle. Second we will address the problem of transcript level variation between individuals and closely related species. We will measure this variation in terms of splicing and in terms of transcriptional regulation. We will focus on the developmental process of metamorphosis that is controlled by the hormone ecdysone via transcriptional mechanisms. We will then map variation in the ecdysone response at the level of transcriptional regulation, and we will determine the contribution of transcriptional variation due to variation of the in vivo binding sites of known transcription factors such as the Ecdysone Receptor. To accomplish the goals of this proposal we will use maskless photolithography for in situ synthesis of high density long oligonucleotide (36-60 n.t.) microarrays. This work will help to develop several applications for the study of complex eukaryotic genomes that were previously difficult, expensive or unfeasible using other technologies. The genome of Drosophila is representative of a complex animal but an order of magnitude smaller than the human genome, making it ideal for the proposed studies. In addition to providing technological proof-of-principle studies, the proposed research will make a contribution to our understanding of why different individuals exhibit different responses when exposed to steroids. The methods we develop should be applicable and relevant to the human genome as well, which could be the subject of similar future studies. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] We propose a strategy for systematic functional annotation of cis-regulatory sequences in a metazoan genome. We will combine automated comparative DNA sequence analysis and comparative gene expression analysis to identify regulatory elements in Drosophila. Our laboratory is engaged in a project to provide functional annotation of the Drosophila melanogaster genome using gene expression profiling during development. Here we propose to utilize the D. pseudoobscura genome for comparative gene expression profiling and regulatory motif analysis. We will test whether combining comparative sequence analysis with comparative gene expression analysis will allow us to systematically decode regulatory information in the genome. Regulatory elements identified in our studies will be tested experimentally in vivo. Our results will also identify sets of genes that have evolved expression. Regulatory elements associated with gene expression evolution will be examined in order to better understand the functional consequences of cis-regulatory sequence evolution. Principles derived from the proposed studies will likely be useful for interpreting the functional significance for conserved and diverged regulatory sequences in other genome comparisons, including those of primates and other vertebrates. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by the applicant): Drosophila presents an ideal model organism for the study of human cis-regulation. Its genome shares the structure and major features of the human genome; all major families of transcription factors, both the basal machinery and site-specific factors; the overall regulatory structure of developmental genes such as the Hox clusters; many of the "master regulatory" transcription factor proteins such as PAX6/eyeless and Distalless that control organ or tissue identity. In many cases, human proteins function in Drosophila just as well as Drosophila proteins. In addition to the shared biology, the fly also offers powerful experimental tools not yet available for mammalian species. Its small genome size, at 130 Mb, is amenable to genome-wide systematic experimentation at a resolution not yet achievable in the human. Additionally, a total of 12 fully sequenced species provide one of the richest comparative genomic datasets, powerful enough to identify individual functional binding sites of regulators across the entire genome. Finally, the wealth of high throughput experimental techniques enables us to validate functional predictions in vivo. In this proposal, we exploit these unique features to produce a comprehensive cis-regulatory map of the Drosophila genome, using a combined experimental and computational approach. We will identify fly promoter regions, short and long range enhancers, insulator and repressor regions, and their defining characteristics based on cis-regulatory motifs and grammars, chromatin state, and transcription factor binding. [unreadable] [unreadable] This project serves as a pilot for the full-scale mapping of functional regulatory elements in the human genome. The methods and strategies developed will be crucial for informing the human genome. In addition, the body of knowledge gained will be invaluable in the understanding of many human diseases and disorders due to regulatory malfunction of gene regulation. Drosophila is an ideal model system for this project, for its compact genome size, experimental resources, and comparative genomics power, while retaining all the major characteristics of the human genome for the study of gene regulation. [unreadable] [unreadable] [unreadable] [unreadable]

We propose to develop a Center for Systems Biology to promote interdisciplinary scientific investigation and education in Chicago. Faculty in the Institute for Genomics and Systems Biology at the University of Chicago will take a leadership role and together with collaborators at other Chicago institutions will create a broad outreach to the community. The Center's scientific program will focus on the robustness of transcriptional networks in physiological, developmental and evolutionary time scales. We propose to go beyond mapping network topologies to develop dynamical models of the behavior of transcriptional regulatory networks during physiological stress, during cellular and organismal development, and during the evolution of species. These goals will be achieved by bringing together experts in genomics, developmental biology, evolutionary biology, stress and physiology, network modeling, high performance and grid computing, chemistry, and physics. The overarching aim of the Center's research is to uncover the organizational principles that transcriptional regulatory networks share as they respond to physiological, development and evolutionary inputs and pressures. These principles are expected to reveal structure-function relationships in networks that lead to physiological and evolutionary robustness or, its complement, flexibility.

We propose to develop a Center for Systems Biology to promote interdisciplinary scientific investigation and education in Chicago. Faculty in the Institute for Genomics and Systems Biology at the University of Chicago will take a leadership role and together with collaborators at other Chicago institutions will create a broad outreach to the community. The Centers scientific program will focus on the robustness of transcriptional networks in physiological, developmental and evolutionary time scales. We propose to go beyond mapping Network topologies to develop dynamical models of the behavior of transcriptional regulatory networks during physiological stress, during cellular and organismal development, and during the evolution of species. These goals will be achieved by bringing together experts in genomics, developmental biology, evolutionary biology, stress and physiology, network modeling, high performance and grid computing, chemistry, and physics. The overarching aim of the Centers research is to uncover the organizational principles that transcriptional regulatory networks share as they respond to physiological, development and evolutionary inputs and pressures. These principles are expected to reveal structure-function relationships in networks that lead to physiological and evolutionary robustness or, its complement, flexibility.

DESCRIPTION (provided by applicant): Previous studies in rats have provided evidence that brief, early-life exposures to bisphenol A (BPA) at environmentally relevant doses results in developmental reprogramming of the prostate gland via epigenetic modifications that enhance carcinogenic susceptibility later in life. While dose-response profiles, BPA pharmacokinetics and route of exposure studies in rodent models are underway to validate these findings, there is a current and compelling need for research on BPA effects in the developing human prostate gland. The National Toxicology Program 2008 report summarily stated that "studies in laboratory animals provide only limited evidence for adverse effects on development and more research is needed to better understand their implications for human health". In response to this need, novel models have been developed with human prostate progenitor cells that permit a direct examination of the impact of low-dose BPA exposures as they form prostate-like structures in vitro and in vivo. The goals of the proposed studies are to determine if exposure to environmentally relevant levels of BPA during the early stages of human prostate development increases susceptibility to prostate carcinogenesis later in life and to identify the underlying mechanism of this reprogramming event. It is hypothesized that prostate epithelial progenitor cells are the direct targets of BPA action during early gland formation. Further, it is predicted that BPA-induced reprogramming is mediated through a combination of altered DNA methylation and histone modifications that are heritable as progenitor cells self renew, transmitting altered epigenomic information throughout the lifespan of the individual. The proposed research thus represents a new paradigm that human prostate carcinogenesis may begin early in life in response to adverse environmental influences that epigenetically alter progenitor cells. An innovative approach will be exploited to test this hypothesis and directly examine BPA effects on human prostate progenitor cells using two model systems: 1) a 3-D co-culture system with human primary prostate epithelial/stromal cells to form prostaspheres in vitro, and 2) as recombinants with rat urogenital sinus mesenchyme grafted to murine kidney capsules for 1-3 months to form chimeric prostate-like tissues in vivo. These novel approaches will permit an examination of differentiation defects (Aim 1) and carcinogenesis (Aim 2) in the human prostate epithelial cells as a function of developmental BPA exposures. These studies will be informed by genome-wide studies of DNA methylation patterns and heritable chromatin modifications using human gene promoter arrays and Solexa ChIP-seq analysis (Aim 3). Using integrative bioinformatics, it is expected to identify BPA reprogrammed gene candidates that may serve as biomarkers for early-life BPA exposures in human epidemiology studies. To determine the potential relevance of BPA-reprogrammed candidate genes to human prostate cancer, tissue microarrays (TMAs) of human prostate cancer will be utilized to screen for misexpression of gene candidates as a function of disease stage, progression and ethnicity. The information gained from the proposed studies will be of high impact on the scientific, medical and regulatory communities in terms of 1) providing strong and compelling evidence for negative effects of BPA in humans, 2) establishing a mechanistic framework for developmental reprogramming, 3) identifying BPA-reprogrammed candidate genes for use as biomarkers, 4) ascertaining relevance of BPA-genes to human prostate cancer, 5) validating a useful model system for screening other endocrine disrupting chemicals, and 6) establishing a basis for studies on BPA in other organ systems and diseases. PUBLIC HEALTH RELEVANCE: There is increasing evidence in rodent models that brief, early-life exposures to bisphenol A (BPA) at dose levels typically found in humans results in developmental reprogramming of the prostate gland and increases susceptibility to prostate cancer later in life. The present proposal will test for this possibility in human prostate tissue using newly developed model systems with human prostate progenitor cells. Identification of epigenetic marks and BPA-reprogrammed genes may serve as biomarkers for developmental BPA exposures and provide molecular insight into the epigenomic plasticity that predisposes to prostate cancer with aging. The findings will be of high value to the medical and regulatory communities and serve as a model for human exposures to prevalent environmental endocrine disruptors with suspected carcinogenic potential.

DESCRIPTION (provided by applicant): We propose to use bacterial artificial chromosome (BAC) recombineering to epitope tag 40 transcription factors per year for chromatin immunoprecipitation (ChIP) followed by sequencing to map binding sites genome wide. A major hurdle for the ENCODE project is the availability of ChIP-grade antibodies for each factor to be analyzed. Epitope tagging of chromatin-associated proteins presents an alternative approach for ChIP, using the same epitope-specific antibody for each factor. Expressing epitope tagged factors from BACs ensures that the factors are expressed at near-physiological levels due to the presence of endogenous regulatory sequences that drive each tagged factor from its native local genomic context. We propose to analyze a diversity of transcription factors using this method, which we have already demonstrated for more than 20 nuclear receptor class proteins, a forkhead domain protein, Jun and Fos, and several other types of factors (Poser et al. 2008;Hua, Kittler and White 2009). The goal of this project is to integrate our approach with the ENCODE project, testing it for a wider diversity of transcription and chromatin-associated factors and scaling the approach to production levels necessary for ENCODE. The proposed project involves a formal collaboration between the White and Snyder labs, as well as integration with other funded ENCODE and human epigenome projects. PUBLIC HEALTH RELEVANCE: Using a BAC recombineering approach, we propose to systematically epitope tag transcription and chromatin associated factors for ChIP-seq to speed current large-scale mapping projects such as the Encyclopedia of DNA Elements (ENCODE) project by eliminating the laborious step of antibody production and testing. The technology presented here has the potential to facilitate the large-scale identification of the binding sites of mammalian transcription factors and other chromatin-binding proteins. This technology will also enable the ChIP analysis of proteins that are recalcitrant to ChIP grade antibody production and thus impractical to map using the conventional factor-specific antibody ChIP approach for mammalian cells.

DESCRIPTION (provided by applicant): We request the Illumina Solexa GAIIx system, which will be an essential "next generation" sequencing platform for a wide range of NIH funded projects at the greater Chicago metropolitan area. This new machine will be housed at the High-Throughput Genomic Analysis Core (HGAC) of the Joint Institute of Genomics and Systems Biology (IGSB) at the University of Chicago and Argonne National Laboratory (ANL) http://www.igsb.org/services/index.php?p=hgac. Importantly, the addition of this capacity will enable NIH funded investigators at universities such as Loyola University and University of Illinois at Chicago to gain access to state-of-the-art technology. Investigators at The University of Chicago and Northwestern University already make use of existing facility, but our capacity is limited due to major NIH funded grants such as an NHGRI modENCODE project (U01HG004264) already occupying existing machines at 100% capacity (two Solexa GAII machines and one Roche GS-FLX 454 sequencer). Specifically, multiple NIH funded investigators from the University of Chicago Cancer Center and who are part of the IGSB-led "1000 Chicago Cancer Transcriptomes Initiative" will be enabled by the requested instrument. Also, investigators in the Chicago Center for Systems Biology (CCSB), one of ten National Centers for Systems Biology funded by NIGMS, would be supported with this instrument. Funding this instrument request would thus leverage not only the existing HGAC infrastructure, but also multiple NIH research funded projects of dozens of investigators in the Chicago area who require whole-genome sequencing and re-sequencing, transcriptome sequencing, de novo sequencing, SNP discovery, gene expression, small RNA discovery, or ChIP-seq. Critically important for success and immediate impact, the machine would be integrated with an existing production service pipeline that includes trained technical staff with experience in all aspects of nucleic acid isolation, library construction, cluster generation and quality control for Solexa;as well as a LIMS, computing, primary analysis and data distribution infrastructure that has been developed with a team of bioinformatics experts in IGSB (www.igsb.org) over the last 18 months. This will allow the machine to have immediate impact on NIH investigators at four universities in Chicago.

The major objective of this core is to provide wet-lab technological support and expertise to the investigators in the Center. We anticipate that results from network reverse engineering modeling in Core 1 will make predictions about not only the role of individual genes but also the state of particular suites of genes and of networks. To identify the relationship between multiple phenotypes, stimuli, and genotypes, high-throughput biochemical, proteomic, and genetic screening is essential. Simultaneous molecular profiling of large numbers of gene products to assess genetic status, expression level or protein modification state can rapidly provide an overview of the state of a cellular system.

DESCRIPTION (provided by applicant): It is now well recognized that African Americans experience a disproportionate burden of pre-menopausal breast cancer and higher mortality rates in comparison to other racial/ethnic groups. Recent studies demonstrate that African Americans are more likely to develop triple-negative breast cancer (TNBC) or basal- like breast cancer in particular. We recently showed that indigenous West Africans, the founder population of African Americans had even higher proportions of TNBC than do African Americans. We have recruited 1233 breast cancer cases and 1101 controls in Phase 1 of the Nigerian Breast Cancer Study (NBCS) and recruitment of additional 1500 cases and ethnicity & age-matched 1500 controls is ongoing. In addition, we are conducting a genome-wide association study (GWAS) of breast cancer in women of African Ancestry to study common variants for breast cancer and results will be available in autumn 2011. Here, we propose a high- throughput whole genome DNA sequencing and computational biology approach to examine rare, moderate- penetrance variants for breast cancers and expand the analysis of ethnic diversity in breast cancer genomes. Our specific aims are to: 1) fully sequence genomes (WGS) of normal blood and matched primary breast tumors from 200 well-phenotyped cases and 200 controls to identify germline and somatic variants for triple negative breast cancer. We will distinguish inherited from somatic variants by comparing variants identified in tumors with the paired normal blood samples and the healthy controls to evaluate the etiologic effect of the inherited variants; 2) Validate selected genes/variants in >5000 breast cancer cases and >5000 controls of African and non-African ancestry. We will first impute rare genotypes identified by whole genome sequencing into all the GWAS samples to conduct an in silico replication. Then, we will perform replication in the African American Breast Cancer Consortium, which includes Black Women's Health Study and the Triple Negative Breast Cancer Consortium. Our access to other Consortia including BCAC, CIMBA and Post GWAS U19 will provide other cohorts for replicating our studies. This integrative approach will increase our power to identify associations between rare inherited variants and the most aggressive form of breast cancer in an understudied but unique population. The replication of our study findings in other populations and our data sharing plans will bring enormous public health benefit by harnessing genomics and biotechnology to improve global health equity and reduce health disparities.

The major objective of this core is to provide wet-lab technological support and expertise to the investigators in the Center. We anticipate that results from network reverse engineering modeling in Core 1 will make predictions about not only the role of individual genes but also the state of particular suites of genes and of networks. To identify the relationship between multiple phenotypes, stimuli, and genotypes, high-throughput biochemical, proteomic, and genetic screening is essential. Simultaneous molecular profiling of large numbers of gene products to assess genetic status, expression level or protein modification state can rapidly provide an overview of the state of a cellular system.

DESCRIPTION (provided by applicant): We request a custom built Digital Scanned Laser Light Sheet Microscope (DSLM), which will enable a broad group of investigators in Chicago to visualize proteins in live tissues and organisms with high resolution and speed and without sample damage associated with other fluorescence microscopy techniques. A major user group will be the investigators participating in at the Chicago Center for Systems Biology (CCSB) (://www.chicago-center-for-systems- biology.org/; grant P50GM081892). The Center's scientific program focuses on the dynamics of transcriptional networks on physiological, developmental and evolutionary scales. The new system will particularly enable us to generate and analyze extensive libraries of the expression networks in development, norm and disease progression. Because of the unique capabilities of this instrument, we have also received requests from groups outside The University of Chicago and we will reserve 25% time on the instrument for these users (minor user group). The DSLM is an innovative live-imaging fluorescence microscopy system developed by Dr. Stelzer's group at the European Molecular Biology Laboratory (EMBL) (Keller et al., 2008. Science, 322:1065-9). In comparison to other advanced fluorescence microscopy techniques (confocal and two-photon) the DSLM provides more than 50 times higher imaging speeds with 10 times higher signal to noise ratio, while exposing the specimens to at least two orders of magnitude less light. It presents a further improvement of a related technology, also introduced by the Stelzer group, known as Selective Plane Illumination Microscopy (SPIM) (Huisken et al., 2004. Science, 305:1007-9). The new equipment will be housed at the Institute for Genomics and Systems Biology (IGSB) (://www.igsb.org/) and will have an immediate impact on CCSB's advanced imaging platform for three important reasons. First, it would largely complement our existing confocal system coupled with a microfluidics device developed at The University of Chicago. Second, it will allow us for the first time to dynamically image fluorescently tagged transcription and other factors in model organisms (worm, fly, frog, and zebrafish) as well as in tissues and cell cultures under different physiological conditions over long periods of time, with the highest spatiotemporal resolution. Finally, the DSLM would leverage not only the CCSB research and infrastructure, but also other multiple NIH funded projects, including those presented in this application, bringing live imaging microscopy studies to the next level of speed and resolution. PUBLIC HEALTH RELEVANCE: The newly developed fluorescence Digital Scanned Laser Light Sheet Microscope (DSLM) dramatically minimizes photo damage to the specimen at the same time increasing both the speed and quality of live imaging. Such combination of features is unique to this instrument, which will enable investigators at The University of Chicago and collaborating institutions to perform in-vivo imaging experiments over long durations (e.g. several days of development)and at cellular resolution. This will greatly enhance our capabilities in cellular and developmental genomic research, particularly through visualizing and studying spatiotemporal networks of gene expression in norm and disease.

DESCRIPTION (provided by applicant): Mental illnesses are some of the most devastating diseases affecting human populations, placing a huge burden on individuals, families and society. Genome-wide association studies (GWAS) have identified dozens of common single nucleotide polymorphisms (SNPs) that are associated with psychiatric diseases, but a majority of those SNPs have been mapped to intergenic or intronic regions and are functionally unclassified. The overall goal of this proposed study is to use genetic mapping of quantitative trait loci (QTL), including expression QTLs (eQTLs), protein QTLs (pQTLs), and DNase I sensitivity QTLs (dsQTLs), to map non-coding regulatory elements in human brain, then to use the QTL SNPs to uncover regulatory mechanisms underlying GWAS findings and to discover novel risk genes. Our previous studies have shown that psychiatric GWAS signals are enriched with brain eQTL SNPs (eSNPs), and these brain eSNPs are likely to be functional and contribute to disease susceptibilities. We hypothesize that other QTLs will similarly represent other levels of regulation. So, using QTL mapping, we will identify SNPs affecting chromatin accessibility in brain (dsQTLs), and downstream gene and protein level variations (eQTLs and pQTLs). We will use RNA-seq, micro-western arrays (MWAs), reverse phase protein arrays (RPPAs), and DNase-seq to profile prefrontal cortex and cerebellum of 432 postmortem brains, along with sorted NeuN+ and NeuN- nuclei. Using the optimal deconvolution method, all brain measures will be partitioned into neuronal and non-neuronal measures for QTL mapping. We will thereafter re-analyze existing GWAS data for seven psychiatric diseases, plus three non- psychiatric diseases/traits as controls, to understand the contributions of neuronal- and non- neuronal QTL SNPs to disease risks. We will also look for differential expressions of transcripts and proteins, as well as for differential DNA sensitivities, in patient brains, and use these molecular measures to construct novel regulatory networks. This integrative study represents a timely, novel and powerful approach that will transform our understanding of brain genomics and the genetic risks of psychiatric diseases. It is positioned to create a new paradigm for integrating brain genomics and psychiatric genetics that are truly distinct from current approaches.

The proposed ?I/UCRC for Computing and Genomics - An Essential Partnership for Biology Breakthroughs? proposed by the U. of Illinois at Urbana-Champaign, the U. of Chicago, and Mayo Clinic will enhance the research, education, and entrepreneurship while performing the important technology transfer by bringing together an interdisciplinary team of industry partners from computer systems, health care/pharmaceuticals, and life sciences working in collaboration with genomic experts to address the colossal big-data challenge. The application of genomics across the life sciences industry is currently challenged by an inadequate ability to generate, interpret, and apply genomic data quickly and accurately for a wide variety of applications. One challenge has been that of integrating thought and market leaders across what had historically been orthogonal industries: those involved with computer sciences, and those involved with biological sciences. With the advent of Next Generation Sequencing technology, those industries are now interdependent and have a critical need to synthesize and coordinate activities at the interface of computing and genomics. The participating sites propose to establish a collaborative environment that improves the applicability, timeliness, efficiency, and accuracy of the computational infrastructure to address the pressing genome-based challenges. The CompGen consortium?s vision is to engineer and optimize computing systems needed by industry for genome analysis.

The CompGen Center will address the experimental process for genomic data. A variety of questions on health and social problems will be addressable, enabling much needed biological and healthcare breakthroughs. Outcomes will enrich research infrastructure, develop next generation of leaders in engineering and science, improve the quality of workforce, and involve international partners. Collaborations will produce artifacts such as new algorithms, optimizations, and statistical models, in turn driving the design of the computing enterprise. The goal is to generalize those artifacts to drive the design and evaluation of computational models and hardware/software co-designed architectures, tightly coupled with new memory and computing technologies for scalability and accuracy.

DESCRIPTION (provided by applicant): Biospecimens are an invaluable resource to the biomedical community. The objective of this proposal is to employ a combination of micro-western arrays and reverse phase protein arrays to dramatically increase the throughput of antibody validation as well as the comprehensiveness of proteins that can be examined in biospecimens. We will use this platform to examine the relationship of about 500 protein abundances and modification states with sources of in- and ex-vivo peri-operative sources of preanalytical variability. We will record standard sources of per-operative variability during the normal course of surgical resection of head-and- neck tumors and relate these variables to differential protein expression and modification. In parallel, we will measure changes in protein expression and modification following defined times of ex-vivo ischemia to mimic a standard source of ex-vivo peri-operative pre-analytical variability. In summary, we will develop standard operating procedures for surgical biospecimen removal and test the efficacy of a platform employing micro- western arrays and reverse phase protein arrays for measuring changes in the expression of about 500 cell signaling proteins in approximately 140 biospecimens subjected either to standard perioperative handling variables or to defined and controlled sources of preanalytical variability. Following surrogate variable analysis to control for known biological covariates (such as age, sec, etc.), we will use linear mixed effects modeling to identify baseline protein levels associated with preanalytical variability. Two-step, cubic regression will be used to identify proteins with significant ex-vivo temporal expression changes following resection. Our model will be used to establish metrics of tissue quality and to identify protein signatures indicative of biospecimen quality.

Project Summary The purpose of each ENCODE Functional Characterization Center is ?to develop and apply generalizable approaches to characterize the role of candidate functional elements identified the ENCODE project in specific biological contexts?. Our proposed Center will focus on characterization of candidate enhancer elements. We will develop, refine and apply experimental methods for functional assays of enhancers. We will use two very different biological models chosen for their high potential to act as generalizable exemplars for the study of enhancers in the context of (i) inherited risk factors for disease, and (ii) somatic mutations involved in cancers. We will also develop and refine our experimental methods in ENCODE cell lines, and we will reserve 25% of our efforts for testing candidate genomic elements that will be studied in common across all of the ENCODE Functional Characterization Centers. Using STARR-seq, and variations thereof, we will test for sufficiency of candidate enhancer elements to modulate gene expression. Using CRISPR-Cas9 methods we will edit the human genome, testing for necessity of candidate enhancer elements in their endogenous context. We will utilize these methods to examine the effects of inherited DNA variation on enhancer function in models of coronary artery disease (CAD), and to examine the effects of acquired somatic DNA mutations on enhancer function in models of pancreatic cancer (Pancreatic Ductal Adenocarcinoma ? PDAC). While our approach necessarily requires a bioinformatics component to utilize ENCODE and other existing data sets in order to define the best candidate enhancer elements for testing in the specific biological models we will assay, our Center will be focused on experimental characterization of enhancers, testing different combinations of approaches in order to create extensible and generalizable protocols for systematic and accurate characterization of enhancer function.

In this project, we will develop novel methods for finding brain-specific enhancers, build regulatory networks, deconvolve brain-region-specific regulation, and relate differential enhancer signals to variations in the human population. We will then apply these analytical methods to the psychENCODE data corpus, integrating these data with GTEx, ENCODE, and CommonMind data, annotating GWAS SNPs associated with psych disease, prioritizing the discovered regulatory elements for validation, and visualizing all psychENCODE data in an integrated fashion. We will then validate these predicted regulatory elements using large-scale genomic assays in neuroblastoma cells, iPSC cells, and neuronal precursor cells differentiated into neuronal lineages, and using a microfluidics platform capable of culturing neuronal cells and neuronal organoids. The results from these studies will further our understanding of the genetic regulatory basis for neuronal function in both normal and neuropsychiatric disease states.

In this project, we will develop novel methods for finding brain-specific enhancers, build regulatory networks, deconvolve brain-region-specific regulation, and relate differential enhancer signals to variations in the human population. We will then apply these analytical methods to the psychENCODE data corpus, integrating these data with GTEx, ENCODE, and CommonMind data, annotating GWAS SNPs associated with psych disease, prioritizing the discovered regulatory elements for validation, and visualizing all psychENCODE data in an integrated fashion. We will then validate these predicted regulatory elements using large-scale genomic assays in neuroblastoma cells, iPSC cells, and neuronal precursor cells differentiated into neuronal lineages, and using a microfluidics platform capable of culturing neuronal cells and neuronal organoids. The results from these studies will further our understanding of the genetic regulatory basis for neuronal function in both normal and neuropsychiatric disease states.