CHIP_QC, COMPUTATIONAL PLATFORM FOR MULTIVARIATE EPIGENETIC STUDIES AND ITS APPLICATION IN UNCOVERING ROLE OF POLYCOMB DEPENDENT METHYLATIONS STATES

ABSTRACT During my PhD tenure, I have been involved in developing a user-friendly cross-platform system capable of analyzing epigenomic data and further use it in understanding the role of the Polycomb Repressive Complex 2 (PRC2) in genome regulation. From current trending in epigenetics research, we can sense increasing ease of high throughput sequencing and greater interest towards genome wide epigenomic studies. As a result of which we experience an exponential flooding of epigenetic related data such as Chromatin immunoprecipitation followed by sequencing (ChIP-seq), and RNA sequencing (RNA-seq) in public domain. This creates an opportunity for crowd sourcing and exploring data outside the boundaries of specific query centered studies. Such data has to undergo standard primary analysis, which with the aid of multiple programs has been stabilized courtesy to the scientific community. Further downstream, out of many, genome wide comparative, correlative and quantitative studies have proven to be critical and helpful in deciphering key biological features. For such studies we lack platforms, which can be capable of handling, analyzing and linking multiple interdisciplinary (ChIP-seq/RNA-seq) datasets with efficient analytical methods. With this aim we developed ChIP_QC, a user-friendly standalone computational program with an ability to support numerous datasets with high/moderate sequencing depth for performing genome wide analysis. First, using ENCODE project (Consortium, 2012) data, we illustrated few applications of the program by posing different biological scenarios and showed the comfort with which some known observations can be verified and also how it can be helpful in deducing some other novel observations. Second, we were interested in understanding the functionality of the products generated through catalytic activity of PRC2. It is known that Lysine 27 of histone H3 (H3K27) undergoes posttranslational modification (PTM) and methylation is one such dominant PTM. Methylation on H3K27 can be either mono/di/tri-methylation form. Out of all three forms, it is very well demonstrated that trimethylation of H3K27 (H3K27me3) is PRC2 dependent and at the same time its role in gene repression is well characterized, but functional roles of other forms of methylation on H3K27 are still poorly characterized. For understanding this, we used mouse embryonic stem cells (mESC) as model system of our study and we were able to provide an extensive characterization of other forms of methylation, highlighting their differential deposition along the genome, their fundamental role in transcriptional regulation, and their indispensability during differentiation program. Using ChIP_QC and with other computational methods along with experimental evidences, our data demonstrated that the monomethylation of Lys27 (H3K27me1) is required for correct transcription of genes and positively correlates with trimethylated Lys36 (H3K36me3); on the other hand dimethylated Lys27 (H3K27me2), that we identified to be the principal activity of PRC2, prevents firing of non cell type specific enhancers

The H3K36me2 Methyltransferase Nsd1 Demarcates PRC2-Mediated H3K27me2 and H3K27me3 Domains in Embryonic Stem Cells

The Polycomb repressor complex 2 (PRC2) is composed of the core subunits Ezh1/2, Suz12, and Eed, and it mediates all di- and tri-methylation of histone H3 at lysine 27 in higher eukaryotes. However, little is known about how the catalytic activity of PRC2 is regulated to demarcate H3K27me2 and H3K27me3 domains across the genome. To address this, we mapped the endogenous interactomes of Ezh2 and Suz12 in embryonic stem cells (ESCs), and we combined this with a functional screen for H3K27 methylation marks. We found that Nsd1-mediated H3K36me2 co-locates with H3K27me2, and its loss leads to genome-wide expansion of H3K27me3. These increases in H3K27me3 occurred at PRC2/PRC1 target genes and as de novo accumulation within what were previously broad H3K27me2 domains. Our data support a model in which Nsd1 is a key modulator of PRC2 function required for regulating the demarcation of genome-wide H3K27me2 and H3K27me3 domains in ESCs. The Polycomb repressor complex 2 (PRC2) deposits H3K27me2 and H3K27me3 repressive histone modifications in spatially defined chromatin domains to maintain cellular identity. Streubel et al. identify the H3K36me2 methyltransferase Nsd1 as a key modulator of PRC2 to restrict H3K27me3 deposition and, thereby, to demarcate H3K27me3 from H3K27me2 domains in ESCs