Genome binding/occupancy profiling by high throughput sequencing
Summary
CCCTC binding factor (CTCF) has been implicated in mediating chromatin insulation and long-distance interactions between enhancer and promoter. In this study, to study the roles of CTCF in somatic cell reprogramming, we performed RNA-seq, Hi-C, ChIP-seq and ATAC-seq at the specific stage of reprogramming. Through bioinformatics analysis, our data indicated that CTCF promotes enhancer-promoter interactions and chromatin accessibility to regulate the expression of key pluripotency genes; meanwhile, it serves as an insulator to suppress the expression of key somatic genes. Smarca5 loss inhibits CTCF-promoted reprogramming by reversing CTCF insulator activity on MEF-specific genes and leading to incorrect nucleosome positioning adjacent to CTCF binding sites. These findings reveal the dual functions of CTCF as both a structural regulator and insulator in conjunction with a key chromatin remodeler to drive cellular reprogramming towards pluripotency.
Overall design
Examination of the chromatin accessibility in reprogramming cells with OSKM plus control, Ctcf overexpression or Ctcf overexpression plus Smarca5 knockdown on day 10, sorted reprogramming intermediate cells in the TetOn lentivirus OSKM reprogramming system with both control shRNA and Ctcf knockdown by ATAC-seq.
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