Binding domain mutations provide insight into CTCF's relationship with chromatin and its contribution to gene regulation (chip-seq)

Although only a fraction of CTCF motifs are bound in any cell type, and approximately half of the occupied sites overlap cohesin, the mechanisms underlying cell-type specific attachment and ability to function as a chromatin organizer remain unknown. To investigate the relationship between CTCF and chromatin we applied a combination of imaging, structural and molecular approaches, using a series of brain and cancer associated CTCF mutations that act as CTCF perturbations. We demonstrate that binding and the functional impact of WT and mutant CTCF depend not only on the unique properties of each protein, but also on the genomic context of bound sites. Our studies also highlight the reciprocal relationship between CTCF and chromatin, demonstrating that the unique binding properties of WT and mutant proteins have a distinct impact on accessibility, TF binding, cohesin overlap, chromatin interactivity and gene expression programs, providing insight into their cancer and brain related effects. To study the impact of CTCF mutations, we established 10 cell lines wherein the mESC degron cell line was modified to express either a stable doxycycline-inducible control wild-type Ctcf or a mutant Ctcf (mCtcf) transgene in the absence of endogenous CTCF. We then performed chipmentation for cohesin (SMC3) and for transgene CTCF (FLAG). Cells expressing transgene CTCF mutant (indole acetic acid, IAA, an analogue of auxin + doxycycline, ID condition) were compared to cells expressing the transgene WT CTCF (WT, ID condition). To assess the rescue by the transgene WT CTCF, we performed chipmentation using CTCF antibody in WT untreated and WT ID conditions. Two replicates per condition were assessed.