Linking Chromatin remodelers and CTCF in mammalian Topological Domain structure and function – 3D-reMODEL

Members of the ATP-dependent chromatin remodeling complexes (remodelers), including the BAF complex, are recurrently mutated in human cancers. We recently reported a genome-wide investigation of remodeler function in embryonic stem (ES) cells (De Dieuleveult et al., Nature 2016). We detected extensive binding of remodelers to promoters and enhancer elements, and unexpectedly, to CTCF-occupied elements. CTCF is a DNA-binding protein that divides the genome into separated chromatin domains called ‘Regulatory Neighborhoods’. This 3D organization is thought to minimize illegitimate contacts between (onco-)genes and nearby unrelated enhancers, with recent data confirming that mutations in CTCF-sites are recurrent in human cancers. By combining the expertise of the three partners in the project, we will investigate the function of chromatin remodelers at CTCF-sites and determine how they contribute to organize 3D genome structure.
Our 3D-reMODEL project has four objectives that will systematically elucidate the functional link between chromatin remodelers and CTCF in the structuring of mammalian 3D DNA domains and how perturbed collaboration between remodelers and CTCF may contribute to the cancerous phenotype:
1. We will describe how CTCF co-localizes with nine different remodelers in an orientation dependent manner at different types of DNA domains (TADs and various types of sub-TADs) in mouse ES cells.
2. Using ChIP-seq, we will dissect how the nine remodelers contribute to CTCF binding and Cohesin stabilization or vice-versa.
3. We will unravel the nature of CTCF-remodeler co-occupancy by testing for (direct) interactions between CTCF and remodelers. We will also characterize protein-protein interaction (PPI) networks of CTCF/Cohesin using co-immunoprecipitation and mass spectrometry approaches.
4. Using a combination of high resolution Hi-C and HiChIP approaches, we will analyze how loss-of-function of each remodeler affects the formation of different types of 3D DNA domains. In particular, we will monitor the consequence of Brg1 loss of function to determine if its function in regulating 3D DNA domains can predict part of its tumor suppressor function.
We have performed preliminary experiments in which we mapped the ChIP-seq distribution of CTCF in ES cells depleted of either Chd4, Brg1 or Ep400 remodeler. We found that loss of each these remodeler alters CTCF binding with a specific pattern, suggesting a major function for this family of factors in the regulation of 3D chromatin domains. It will now be important to test how the other remodelers affect CTCF binding, if CTCF itself plays a role on the recruitment of remodelers and how changes in CTCF occupancy may cause changes in the 3D organization of the ES cell genome.