Interplay Between Chromatin and Co-Activator Complexes

Project Summary/Abstract Enhancer-promoter (E-P) communication enables gene activation in eukaryotes but despite advances in many other areas of the field, the nature of this communication is not well understood mechanistically, particularly within the native chromatin environment of a cell. We have developed novel systems in mouse embryonic stem cells (mESCs) to study E-P communication at both the physical level, E-P looping, and the functional level, Pol II Pre-Initiation Complex (PIC) assembly and gene activation. We will leverage a key transcription factor termed Estrogen Related Receptor Beta (ESRRB) that binds in abundance at many mESC enhancers. ESRRB also binds directly to Mediator, a major co-activator complex controlling enhancer and promoter function. ESRRB- responsive genes are typically found within looped domains termed Insulated Neighborhoods, bounded by strong peaks of Cohesin and CTCF. Depletion of ESRRB by siRNAs causes greatly diminished Mediator binding to ESRRB sites within enhancers and decreased target gene expression restricted to within Insulated Neighborhoods. In addition, we have developed a degron system that targets TAF12 and dismantles both TFIID and Pol II PICs, along with gene expression. We will use these systems, and build onto them, to address fundamental questions about the physical and functional mechanisms of E-P communication. In Aim 1, we will employ degrons of ESRRB, Cohesin and TAF12, along with a few select CRISPR deletions of enhancers, proximal promoters and core promoters. ChIP-seq of key factors, RNA-seq, and Promoter- Capture Hi-C will be used to quantitate the effects of each degron and genetic deletion on physical E-P looping and functional interactions including Mediator binding, PIC assembly, chromatin state, and gene expression. In Aim 2 we will approach the problem orthogonally. Paradoxically, some ESRRB regulons contain inactive genes expressed later in development. CRISPR-dCAS9 targeting strategies of activation domains (VPR), chromatin modification (p300), and remodeling machines (BRG1) will be used to ectopically activate these genes, and determine whether this process depends upon kidnapping the functional enhancer within the regulon. In sum, our proposal seeks to determine whether physical E-P looping enables or is independent from functional communication, and what determinants (i.e., PICs, chromatin state, activators, proximal promoters) control functional interactions. The results will provide important new information on E-P communication in a well-defined experimental model, highly relevant to development and differentiation.