Profiling and characterization of genome-wide 3D promoter-silencer interactions

Northwestern University At Chicago, Evanston IL

Investigators

Abstract

Project Summary/Abstract The spatiotemporal regulation of gene transcription is tightly orchestrated by cis-regulatory elements, including promoters, enhancers, insulators, and silencers. While transcriptional enhancers and promoter-enhancer interactions have been extensively investigated across various cell types, our understanding of distal silencers and how they regulate the target genes is still limited. Just like enhancers, they can regulate the target genes from distance and function independently of their positions and orientations. It has been shown that silencers also play a critical role in cell type-specific gene expression. Histone modifications have been frequently used to annotate human genome and predict potential regulatory elements. Among them, H3K27me3, which is catalyzed by the PRC2 complex, have been widely regarded as a potential marker to predict distal silencers. Recent large NIH consortia such as ENCODE have produced ChIP-Seq for H3K27me3 in many cell lines, but there have been limited effort to interrogate their roles in regulating gene expression and phenotypes through functional assays. More importantly, there have been limited effort to determine the target genes for silencers and how they control target genes from distance. To address these gaps, we propose the following aims: 1) To profile and characterize promoter-silencer interactions by HiChIP for H3K27me3 in a variety of cell lines representing different tissue origins. 2) To validate the function of distal silencers and promoter-silencer loops in gene regulation. We will perform genome-wide STARR-Seq experiment to test the predicted silencers in five widely used ENCODE cell lines. Then, we will also perform high-throughput CRISPRa screening (de-repressing) to investigate the silencer functions in cell phenotype (proliferation). 3) To investigate the mechanism underlying the formation of promoter- silencer loops. We will use AID system to knock out several potential factors involved in promoter-silencer interactions, including CTCF, RAD21, and EZH2 in the same five cell lines. To complement the protein degradation approach, we will also treat the cells with EZH2 inhibitor. We will perform RNA-Seq and HiChIP in cells before and after the protein degradation or inhibitor treatment to investigate their roles in chromatin loops and gene regulation.