Chromatin Topology and Transcription in Myogenesis

High-throughput sequencing and the resulting development of biochemical "-Seq" experiments such as ChIP-Seq, DNase-Seq, and Methyl-Seq over the past decade has given rise to a wealth of predicted enhancers and other cis-regulatory regions (CRMs). These new assays provide a new opportunity to compare the number, location, and possible nature of CRMs that are predicted by various new biochemical techniques to instances of known CRMs, which until recently have primarily been located—for reasons of technological limitations—at a few tens of highly expressed, mostly developmentally-specific genes and the several kilobases (kb) upstream of their promoters. For example, an early surprise in the first ChIP-Seq experiments was that the number of predicted tissue-specific transcription factor-occupied sites outnumbered the number of tissue-specific genes by at least a factor of 10, and that many of these occupied sites were nowhere near developmentally relevant genes. In this thesis, I use the ChIA-PET technique, which preserves factor-containing physical interactions between loci in the genome that are far from each other (10kb-2Mb), where the factors used in this thesis are RNA Polymerase II (pol2) to capture active genes, and separately the developmental transcription factor Myogenin to additionally capture CRMs not at promoters. Overall, I report that (1) the closer together two occupied regions are, the more likely they are to be connected, and (2) that a gene’s activity level is highly correlated with its likelihood of being physically engaged with a distant occupied locus. These lead to the discoveries that occupied regions tend to engage with the active genes nearest to them regardless of the developmental profile of the genes, that many genes engage with multiple individual loci, and that many occupied regions interact with multiple genes, including genes that are not at all related in terms of their expression patterns. Individual elements that have multiple connections likely represent sequential rather than simultaneous interactions, and developmental genes may require more engaged enhancers than genes that are expressed in all cell types. Most excitingly, it is possible that many genes with unchanging expression patterns, including so-called "housekeeping genes," use CRMs; very few such genes have ever been assayed with respect to gene regulation, and they are the vast majority of genes in the genome

A comparative encyclopedia of DNA elements in the mouse genome

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases

An encyclopedia of mouse DNA elements (Mouse ENCODE)

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome

Extensive Promoter-Centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation

Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIAPET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells

An encyclopedia of mouse DNA elements (Mouse ENCODE)

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome

A Comparative Encyclopedia of DNA Elements in the Mouse Genome

Summary As the premier model organism in biomedical research, the laboratory mouse shares the majority of protein-coding genes with humans, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications, and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases

Perspectives on ENCODE

The Encylopedia of DNA Elements (ENCODE) Project launched in 2003 with the long-term goal of developing a comprehensive map of functional elements in the human genome. These included genes, biochemical regions associated with gene regulation (for example, transcription factor binding sites, open chromatin, and histone marks) and transcript isoforms. The marks serve as sites for candidate cis-regulatory elements (cCREs) that may serve functional roles in regulating gene expression1. The project has been extended to model organisms, particularly the mouse. In the third phase of ENCODE, nearly a million and more than 300,000 cCRE annotations have been generated for human and mouse, respectively, and these have provided a valuable resource for the scientific community.11Nsciescopu