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Huber, Jonas; Tanasie, Nicoleta-Loredana; Zernia, Sarah; Stigler, Johannes (2024): Single-molecule imaging reveals a direct role of CTCF’s zinc fingers in SA interaction and cluster-dependent RNA recruitment. Nucleic Acids Research. ISSN 0305-1048

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Huber_et_al._-_2024_-_Single-molecule_imaging_reveals_a_direct_role_of_CTCF’s_zinc_fingers_in_SA_interaction_and_cluster-d.pdf

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Abstract

CTCF is a zinc finger protein associated with transcription regulation that also acts as a barrier factor for topologically associated domains (TADs) generated by cohesin via loop extrusion. These processes require different properties of CTCF-DNA interaction, and it is still unclear how CTCF’s structural features may modulate its diverse roles. Here, we employ single-molecule imaging to study both full-length CTCF and truncation mutants. We show that CTCF enriches at CTCF binding sites (CBSs), displaying a longer lifetime than observed previously. We demonstrate that the zinc finger domains mediate CTCF clustering and that clustering enables RNA recruitment, possibly creating a scaffold for interaction with RNA-binding proteins like cohesin's subunit SA. We further reveal a direct recruitment and an increase of SA residence time by CTCF bound at CBSs, suggesting that CTCF-SA interactions are crucial for cohesin stability on chromatin at TAD borders. Furthermore, we establish a single-molecule T7 transcription assay and show that although a transcribing polymerase can remove CTCF from CBSs, transcription is impaired. Our study shows that context-dependent nucleic acid binding determines the multifaceted CTCF roles in genome organization and transcription regulation.

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