Jason Lieb, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): DNA is transformed into a living genome by processes that occur in the cells nucleus, such as transcription, replication, recombination, and DNA repair. These hallmarks of life are controlled by regulatory networks that ultimately modulate, as a function of time and environment, just two aspects of DNA-dependent enzymes: the level of their activity, and where they act. Therefore, a critical part of understanding the mechanism and logic of cellular regulatory networks is a comprehensive understanding of where enzymes and their regulatory proteins interact with the genome in vivo. High-resolution, genome-wide maps of protein-DNA interactions can be made by immunoprecipitating specific protein-DNA complexes and determining the genomic location of the lP-enriched DNA by microarray hybridization. With this information, we can identify the genomic features that specify protein binding, and simultaneously identify genes or other chromosomal elements whose function is affected by the binding. The broad goal of this proposal is to use this and other methods to map the regulatory functions of non-coding sequences onto the entire genome of the yeast Saccharomyces cerevisiae, the most tractable eukaryote, and then to extend this approach to a model metazoan, Caenorhabditis elegans. [unreadable] [unreadable] How do DNA-binding proteins attain their genome-wide target specificity in vivo? This question arises from previous work showing that the transcription factor Rap1 binds to non-coding regions upstream of genes in preference to the coding regions, even though both regions contain strong consensus Rap1 binding sites. The proposed yeast experiments use Rap1 and its associated DNA-binding proteins as a model system to investigate the unaccounted-for determinants of in vivo binding specificity. In addition, by correlating the genome-wide position of Rap1 and its cofactors with expression level of downstream genes, we aim to discover how combinations of proteins assembled at promoters code for specific transcriptional outputs. [unreadable] [unreadable] Currently, I am a third-year Helen Hay Whitney postdoctoral fellow in the laboratory of Patrick Brown at Stanford University. I recently accepted an assistant professor position (tenure-track) at the University of North Carolina in Chapel Hill in the Department of Biology and Carolina Center for the Genome Sciences, starting in June, 2002. Therefore, this application is for only the Faculty Transition Phase of this award. UNC offers an excellent research environment for the proposed work, providing 1000 sq. ft. of wet bench lab space, a state-of-the-ad DNA microarrayer, and generous start-up funding. The close alignment between my research interests and those of colleagues at UNC ensures a rich environment for collaboration and the exchange of knowledge. The award would greatly facilitate the establishment of a productive laboratory engaged in cutting-edge genomics research.

DESCRIPTION (provided by applicant): DESCRIPTION (provided by applicant): Despite the availability of precise consensus DMA-binding sequences for hundreds of proteins, and the complete genome sequence of dozens of organisms, it is not possible to predict where a given DNA-binding protein will associate with a genome in vivo. How DNA-binding proteins recognize and bind to a subset of genomic DNA sequences, while at the same time not binding to thousands of computationally indistinguishable sequences, remains a major unsolved problem in biology. Using Saccharomyces cerevisiae as a model system, we have developed a unique set of genetic, biochemical, and genomic tools to attack this problem. . Aim one: Our previous work has shown that in vivo, transcription factors bind to DNA upstream of genes in preference to coding regions, even though both regions contain strong consensus binding sites. Cooperative protein-protein-DNA interactions and differential chromatin accessibility are hypothesized to mediate context-dependent binding. To quantitate the degree of specificity dependent on in vivo factors, and how much is inherent to the protein and DNA, the genome-wide specificities of Raplp and LeuSp will be determined in vitro using purified proteins and naked yeast genomic DNA, and compared to specificity in vivo. Changes in the distribution of Raplp in response to changes in environmental conditions will also be determined. Aim two: Our previous work has shown that nucleosome occupancy throughout the genome is heterogeneous in living yeast cells. We propose experiments to determine the molecular basis for differential nucleosome occupancy, and how it is established, regulated, and maintained in yeast. Aim three: Our data and data from other groups suggest an intimate relationship between target selection by DMA binding proteins (addressed in Aim 1), global chromatin organization (addressed in Aim 2), and transcriptional activity. The third aim specifically tests relationships between these three processes. We will assay Rap1p target selection in strains in which (i) the context of Raplp binding sites has been changed (ii) transcription at specific loci has been disabled, and (iii) the RNA Pol II CTD is mutated. We will map transcription-coupled chromatin modifications and perform high-throughput site-directed mutagenesis to link histone structure and modification to biological outcomes. Human Health: Transcription factors, when missexpressed or mutated, are a prevalent cause of human disease. Better prediction of their in vivo targets may lead to therapies that inhibit binding to inappropriate targets. FAIRE, a new chromatin assay we have developed, has potential as a prognostic or diagnostic tool for diseases (including cancer) that affect, or arise from defects in, chromatin or transcription.

DESCRIPTION (provided by applicant): Eukaryotic genomes are packaged into chromatin, which regulates the function of proteins that mediate transcriptional activity and other essential processes, including recombination and the faithful segregation of the genome during mitosis and meiosis. The goal of this proposal is to identify discrete elements that regulate chromatin structure and function in the nematode C. elegans, a model metazoan of central importance in large-scale genomic research and gene function discovery. We will first use ChlP-chip and related methods to map the genomic distributions of selected histone modifications and chromosome-associated proteins, and then use that information, in combination with data from other modENCODE groups, to build quantitative models of chromatin function. Specifically, we will: 1. Identify and technically validate functional elements that control chromatin and chromosome behavior. The focus of our analysis will be elements that specify nucleosome positioning and occupancy, control domains of gene expression, induce repression of the X chromosome, guide mitotic segregation and genome duplication, govern homolog pairing and recombination during meiosis, and organize chromosome positioning within the nucleus. 126 strategically selected targets include key histone modifications, histone variants, RNA polymerase II isoforms, dosage-compensation proteins, centromere components, homolog-pairing facilitators, recombination markers, and nuclear-envelope constituents. An efficient pipeline design will facilitate identification and validation of the different classes of functional elements associated with these targets and will integrate the results with the well-annotated C. elegans genome. 2. Biologically validate identified functional elements and build integrated, quantitative models of chromosome function. We will integrate information generated in Aim 1 with existing knowledge on the biology of the targets, perform ChlP-chip analysis on mutant and RNAi extracts lacking selected target proteins, use extrachromosomal arrays to assess the ability of candidate identified sequence motifs to recruit targets in vivo, identify tissue-specific patterns of selected targets, and create integrated, quantitative models of transcription and whole-chromosome functions. Achieving these goals in the context of the ongoing expansion and rich history of C. elegans research will provide an important milestone in meeting the challenge of using genome sequence information to understand and predict biological functions.

DESCRIPTION (provided by applicant): The information stored in the DNA of every living thing must be read and interpreted, and this is accomplished chiefly by proteins. One class of regulatory proteins control the transcription of DNA into messenger RNAs, which are then translated into structural proteins and enzymes. Defects in the ability to properly regulate transcription are at the foundation of many human diseases, with some, such as cancer and many aging-related maladies, very clearly rooted in genomic dysfunction. To take part in development and to respond to their environment, cells respond extremely rapidly to their surroundings, in part by enacting specific transcriptional responses. Therefore transcriptional regulation is by necessity a fundamentally dynamic process. However, almost everything we know about the mechanisms underlying transcriptional regulation are derived from static assays like footprinting or Chromatin Immunoprecipitation (ChIP). The major thrust of this grant is to combine elements from distinct disciplines to explore in vivo binding dynamics, a fundamental parameter that is lost completely in standard ChIP experiments. We aim to (1) measure transcription factor binding dynamics for nearly every transcription factor in yeast, each at every position the genome simultaneously, (2) to create experimental systems in yeast amenable to both FRAP and sequential ChIP experiments, so that we and other expert laboratories can use their methods on the exact same system, and (3) to measure purified transcription factor targeting and dynamics on reconstituted chromatin templates. We can then use these systems to test specific hypotheses regarding competition between chromatin components and transcription factors, to test the biological function of turnover in regulating transcription, and to determine the cellular components required for proper regulation of turnover dynamics.

DESCRIPTION (provided by applicant): Highly parallel functional characterization of human regulatory elements PROJECT SUMMARY One of the most effective means of identifying human regulatory elements is by discovery of open chromatin using methods like DNase hypersensitivity or FAIRE. While there is ample evidence that open chromatin regions are functional and bound by sequence-specific regulatory factors, we typically do not know what function an individual element has, or how DNA sequence variation in human open chromatin regions affects that function. Traditionally, function has been measured experimentally in reporter assays, one functional element at a time. However, it is not feasible to characterize the ~100,000 open chromatin regions that exist in each cell type using low-throughput, serial methods. We propose to develop two complimentary approaches to overcome these obstacles. The first will test the function of tens of thousands of human regulatory elements in a single experiment, and the second will test the effect of natural human sequence variation within 10,000 of those elements in a single experiment, representing 1,000 to 10,000-fold improvements over existing methods. First, putative regulatory elements isolated by FAIRE will be cloned en masse into a Gateway-based entry vector, allowing us to easily swap the inserts into reporters that test promoter, enhancer, insulator, or silencer function. Cells containing inserts with biological activity can be isolated by cell sorting, and the corresponding inserts can be identified by next-generation sequencing. We also will develop a variant of this method that does not require cell sorting. A second major obstacle in discovering the effect of human sequence variation on the function of regulatory elements is the limited ability to measure the effect of a large number of designed DNA sequences in a highly controlled setting. Using Agilent array technology, we will synthesize 10,000 regulatory sequences ~200 bp in length that corresponds to alternate alleles of 5,000 putative regulatory regions. The 5,000 regions synthesized will be selected based on their linkage to human disease risk. After transfection into cells, we will use a flow cytometer to sort the resulting pool of transfected cells into 64 bins of reporter levels, amplify the inserted synthesized region from the cells of each bin using PCR, and measure the DNA content of each activity bin using next-generation sequencing. For every barcode (representing one tested element), the distribution of its next-generation sequencing reads across the expression bins provides a measure of both its mean and standard deviation of expression. Promoter, enhancer, insulator, and silencer function will be tested. Since all tested sequences are transfected to the same cell line, the trans-factor environment is held constant, allowing us to truly test whether the genetic variation among human individuals has a causal effect on expression. PUBLIC HEALTH RELEVANCE: Highly parallel functional characterization of human regulatory elements PROJECT NARRATIVE Public health relevance Defects in transcriptional regulation are at the foundation of many human diseases, including cancer and many aging-related maladies. Gene transcription programs are determined by regulatory elements encoded in DNA. However, it is currently difficult to test the function of more than a few of these DNA regulatory elements at the same time, and it is difficult to test the effect of sequence variation. We propose to develop two complementary technologies. The first will test the function of tens of thousands of human regulatory elements in a single experiment, and the second will test the effect of natural human sequence variation, including variants associated with human disease risk, in 10,000 DNA elements in a single experiment. These experiments may prove valuable in identifying non-coding DNA sequence variants that are causally linked to human disease.

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.

DESCRIPTION (provided by applicant): The three aims of this project address the function of molecular asymmetries that occur very early in animal development, and the mechanisms by which they arise. Asymmetric cell division is essential for generation of cell-type diversity during differentiation and development. Failure o properly undergo asymmetric cell divisions can lead to defects in stem-cell renewal, tissue regeneration, and can contribute to carcinogenesis. We propose to use a near-ideal natural system, the early C. elegans embryo, to characterize the mechanisms of asymmetric distribution of cellular contents in specific blastomeres with modern genomic tools and analysis, followed by tests of function. Specifically, this proposal aims (1) to use blastomere-specific isolation, RNA- seq, and other high-throughput sequencing applications to comprehensively map RNA inheritance and transcriptional activity of seven invariant cell divisions in early C. elegans development, (2) to test the biological significance of asymmetric RNA segregation in development, and (3) to define the cis and trans-acting mechanisms required for asymmetric RNA segregation. To achieve these goals, we will also apply new approaches and technologies to the study of asymmetric segregation of cellular components. Our research plan is likely to identify cell-fate determinants and mechanisms that are generalizable to other systems, including humans.