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.

Grant Number: 5R01GM074701-20
NIH Institute/Center: NIH
Principal Investigator: MICHAEL CAREY