Changes in chromatin states control gene expression during differentiation of stem cells and defects in intestinal stem cells (ISCs) underlie disorders such as colorectal cancer and inflammatory bowel diseases. In spite of recent advances in identification of ISCs, the role of epigenetic modifications in ISC gene expression, function and intestinal disease remains unknown. EZH2, which is part of the Polycomb repressive complex (PRC) 2, is overexpressed in many colorectal cancers. However, the current knowledge of the functions and mechanisms of most epigenetic enzymes (including PRC2) is derived from in vitro studies in cancer cell lines or ES cells. An in vivo validation and understanding of their working is imperative before the potential of such enzymes as drug targets can be fully realized. PRC2 is thought to repress gene expression in stem cells and work with other histone modifications to control stem cell differentiation. We propose to study the role of the PRC2-mediated histone mark H327me3 in Intestinal stem cell gene expression, function and differentiation. To achieve this, I will combine three experimental approaches: ChIP-seq for the H3K27me3 mark in various intestinal cells (Aim 1); genetic deletion of Eed to eliminate PRC2 activity (Aim 2); and RNA-seq for detailed transcriptome analysis in different intestinal cells with intact and disrupted PRC2 function (Aim 3). To understand the dynamic pattern of H3K27me3 modification during ISC differentiation, we have mapped H3K27me3-marked genes using ChIP assays in ISC and differentiated cells from intestinal villi. In order to understand how the H3K27me3 mark controls gene expression, we will perform detailed transcriptome analysis using RNA-seq in ISC and villus cells. By correlating the differential H3k27me3 mark and differential gene expression between stem and differentiated cells, we will obtain original and detailed information about the role of PRC2 in control of intestinal genes. To evaluate PRC2 requirements in ISC function and intestinal diseases, we have deleted the core PRC2 component gene Eed in the whole intestinal epithelium and also specifically in ISCs, using cell specific knock-out mouse models. Eed-null intestines show severe defects and in-depth analysis of this phenotype, together with gene expression analysis of wild type and Eed-null cells will allow us to identify specific genes, signaling pathways, and regulatory mechanisms involved in ISC maintenance and differentiation. Our studies hold promise to reveal new insights into gene regulation during differentiation and mechanistic details of PRC2-mediated chromatin modifications.
Using genetic and bioinformatics tools, we propose to understand how the fast growing intestinal stem cells control gene expression through epigenetic mechanisms. Our study will lead to a detailed understanding of the chromatin basis for rapid growth, function and regeneration of the intestinal mucosa. This knowledge is highly relevant to developing new diagnostic strategies and drugs for effective treatment of colorectal cancer and inflammatory bowel diseases faced by more than 1.5 million Americans every year.
|Saxena, Madhurima; Roman, Adrianna K San; O'Neill, Nicholas K et al. (2017) Transcription factor-dependent 'anti-repressive' mammalian enhancers exclude H3K27me3 from extended genomic domains. Genes Dev 31:2391-2404|
|Jadhav, Unmesh; Saxena, Madhurima; O'Neill, Nicholas K et al. (2017) Dynamic Reorganization of Chromatin Accessibility Signatures during Dedifferentiation of Secretory Precursors into Lgr5+ Intestinal Stem Cells. Cell Stem Cell 21:65-77.e5|
|Jadhav, Unmesh; Shivdasani, Ramesh A (2016) Natural Selection, Crypt Fitness, and Pol III Dependency in the Intestine. Cell Mol Gastroenterol Hepatol 2:714-715|
|Jadhav, Unmesh; Nalapareddy, Kodandaramireddy; Saxena, Madhurima et al. (2016) Acquired Tissue-Specific Promoter Bivalency Is a Basis for PRC2 Necessity in Adult Cells. Cell 165:1389-1400|
|Kim, Tae-Hee; Saadatpour, Assieh; Guo, Guoji et al. (2016) Single-Cell Transcript Profiles Reveal Multilineage Priming in Early Progenitors Derived from Lgr5(+) Intestinal Stem Cells. Cell Rep 16:2053-2060|