Binding domain mutations provide insight into CTCF's relationship with chromatin and its contribution to gene regulation (RNA-seq, exit from pluripotency)

Here we used a series of CTCF mutations to explore CTCF’s relationship with chromatin and its contribution to gene regulation. CTCF’s impact depends on the genomic context of bound sites and the unique binding properties of WT and mutant CTCF proteins. Specifically, CTCF’s signal strength is linked to changes in accessibility, and the ability to block cohesin is linked to its binding stability. Multivariate modelling reveals that both CTCF and accessibility contribute independently to cohesin binding and insulation, however CTCF signal strength has a stronger effect. CTCF and chromatin have a bidirectional relationship such that at CTCF sites, accessibility is reduced in a cohesin-dependent, mutant specific fashion. In addition, each mutant alters TF binding and accessibility in an indirect manner, changes which impart the most influence on rewiring transcriptional networks and the cell’s ability to differentiate. Collectively, the mutant perturbations provide a rich resource for determining CTCF’s site-specific effects. To study the impact of CTCF mutations on lineage commitment, we cultured 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 in cell media with or without LIF (leukemia inhibitory factor). We then performed gene expression profiling using data generated from total RNA-seq. Pluripotent mESCs expressing transgene CTCF WT or mutant (indole acetic acid, IAA, an analogue of auxin + doxycycline, ID condition) cultured in LIF-containing media were compared to mESCs undergoing exit from pluripoitency cultured in no LIF media for 3 days. Two replicates per condition were assessed.