CTCF-RNA interactions orchestrate cell-specific chromatin loop organization [iCLIP2]

CCCTC-binding factor (CTCF) is essential for chromatin organization, but its role in dynamically shaping chromatin loops during cellular differentiation is not fully understood. We previously demonstrated that CTCF interacts with endogenous RNAs, and deletion of its ZF1 RNA-binding region disrupts chromatin loops in mouse embryonic stem cells (ESCs). Using an ESC-to-neural progenitor cell (NPC) differentiation model, we show that the ZF1 RNA-binding region of CTCF is crucial for maintaining cell-type specific chromatin loops. Expression of CTCF-?ZF1 leads to dysregulation of genes within these disrupted loops, particularly those involved in neuronal development and function. We identified NPC-specific, CTCF-interacting RNAs and chose Podxl and Grb10 for further study. We found that CRISPR-Cas9-mediated truncation of Podxl and Grb10 disrupts chromatin loops in cis, similar to the disruption seen in the NPC-?ZF1 mutant. Our study underscores the importance of CTCF-ZF1 RNA interactions in preserving cell-specific genome structure and cellular identity. Overall design: CTCF iCLIP2 analysis of CTCF-AID2 degron and CTCF rescue wild-type (WT) and ?ZF1 mutant in both embryonic stem cells (ESCs) and neural progenitor cells (NPCs).