The biochemical, topological and functional impact of cancer-associated Ctcfmutations and their contribution to cancer

New York University School Of Medicine, New York NY

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Abstract

PROJECT 1: SUMMARY CTCF is an eleven-zinc finger protein that is known to play a key role in organizing chromatin into self-interacting topologically associating domain (TAD) structures by promoting the formation of cohesin-mediated loops and insulated boundaries. A number of labs (including the Skok lab) have shown that disruption of insulated TAD boundaries by deletion of CTCF binding sites can alter gene regulation leading to defects in developmental processes and cancer initiation as a result of illegitimate enhancer promoter contacts. Physical contacts between cis regulatory elements (CREs) and the genes they control occurs via cohesin-mediated loop formation. Although contacts commonly form between a single CRE-promoter pair, we and others have found that gene regulation can also occur in ‘CRE hubs’, regions that encompass multiple genes and control elements that co-regulate each other through chromatin looping. CTCF dependent and independent interactions can be conserved, or cell-type specific, but while the role of transcription factors in promoting CTCF independent loops is known, the underlying mechanisms of cell-type specific CTCF mediated loops have not yet been elucidated. There are numerous CTCF motifs in the genome, but only a fraction of these are bound by CTCF in any cell type. Since CTCF’s binding profiles are known to be cell-type specific, chromatin accessibility is presumed to be important for CTCF binding. However, our preliminary data shows that CTCF can bind consensus motifs at both inaccessible and accessible sites. Nonetheless, CTCF has a stronger signal and is more competent at blocking cohesin and forming loops that orchestrate chromatin architecture when bound to accessible chromatin. But accessibility per se, does not predict CTCF binding because the majority of accessible motif containing sites are not bound by CTCF. Bioinformatic and RNA-seq analyses revealed that CTCF binding is linked to enrichment of motifs of expressed TFs abutting (within 20bp) these sites. Moreover, motifs are also enriched within 20bp of CTCF bound inaccessible sites but in contrast to bound accessible sites, the TFs are expressed at a lower level. This result indicates that a change in transcriptional program might be able to flip an inaccessible CTCF bound site to an accessible bound site in which newly bound TFs lead to a stronger CTCF signal capable of blocking cohesin. As part of this application, we aim to study the impact of transcriptional programming on CTCF binding and function at the global level and in CRE hubs across cell state transitions. Use of CTCF mutants (whose graded effects act as CTCF perturbations) will enable us to investigate direct and indirect CTCF-mediated effects on hub rewiring (Figure 1) at an unprecedented level to examine the underlying CTCF-dependent mechanisms of CRE hubs, using not only multi-way but also multi-omics and genome-wide single molecule approaches.