The Nucleosome Remodelling & Deacetylase complex: Genome folding & transcriptional regulation

Abstract

Genome function is highly dependent on the three dimensional organisation of chromatin within a cell’s nucleus. Approximately 2 metres of DNA is folded to fit within a nucleus which is on the order of a few micrometers. In addition to simply fitting within the space available, this folding directly influences gene regulation. At the very largest scales, chromosomes are known to occupy distinct chromosome territories, intermingling only at the borders. At the very smallest scale, chromatin is known to be organised into a beads on a string like structure of nucleosomes. Until the advent of Chromosome Conformation Capture (3C), and related experiments, relatively little was known at scales between these two. In particular, the Hi-C experiment provides genome-wide pairwise information about the frequency with which two genomic loci come into contact and has been extensively used in the last decade to establish key structural features such as chromatin compartments, topologically associating domains (TADs) and chromatin loops mediated by key architectural proteins such as CTCF and Cohesin. It has further been used to establish physical models of gene regulation such as loop extrusion. Chromatin remodeling protein complexes - complexes that can dynamically modify chromatin architecture to control access of key transcriptional machinery to genomic sites - play essential roles in development and control of gene expression. The Nucleosome Remodeling and Deacetylase (NuRD) complex is one of four major ATP-dependent chromatin remodeling complexes and has been shown to be crucial for early embryonic development, to maintain transcriptional heterogeneity and to modulate the physical dynamics of regulatory genomic elements. In this thesis I will investigate the role of 3D chromatin organisation in NuRD-mediated transcriptional regulation in mouse embryonic stem cells. Specifically, I will make use of experimental data generated from a knock-out of Mbd3, a core component of NuRD linking together it's chromatin remodelling and histone deacetylase subcomplexes. Comparing steady state wild-type (WT) and Mbd3- knock out (KO) conditions as well as a timecourse of reintroduction of Mbd3 to Mbd3-KO cells I will firstly identify features of chromatin folding associated with NuRD reassembly. I will also construct a temporal model of NuRD-mediated regulation and compare this with established theories of transcriptional regulation via chromatin structure. The main findings are as follows: (i) coarse scale features of chromatin folding (i.e. chromatin compartments & TADs) strongly differ between steady state wild-type (WT) and Mbd3-KO conditions but differences occur downstream of significant NuRD-mediated regulation, (ii) Upon reassembly of NuRD in Mbd3-KO cells, regulatory interactions between enhancers and promoters undergo rapid changes which are then associated with downstream transcriptional regulation, (iii) changes to regulatory interactions upon NuRD reassembly are strongly associated with rapid NuRD-mediated control of chromatin accessibility at key enhancer sites, and (iv) NuRD-mediated changes to the regulatory landscape occur independently of (or at least prior to) significant changes in CTCF/Cohesin mediated chromatin looping.