Auxin-inducible degron 2 system deciphers functions of CTCF domains in transcriptional regulation

Abstract

Background CTCF is a well-established chromatin architectural protein that also plays various roles in transcriptional regulation. While CTCF biology has been extensively studied, how the domains of CTCF function to regulate transcription remains unknown. Additionally, the original auxin-inducible degron 1 (AID1) system has limitations in investigating the function of CTCF. Results We employ an improved auxin-inducible degron technology, AID2, to facilitate the study of acute depletion of CTCF while overcoming the limitations of the previous AID system. As previously observed through the AID1 system and steady-state RNA analysis, the new AID2 system combined with SLAM-seq confirms that CTCF depletion leads to modest nascent and steady-state transcript changes. A CTCF domain sgRNA library screening identifies the zinc finger (ZF) domain as the region within CTCF with the most functional relevance, including ZFs 1 and 10. Removal of ZFs 1 and 10 reveals genomic regions that independently require these ZFs for DNA binding and transcriptional regulation. Notably, loci regulated by either ZF1 or ZF10 exhibit unique CTCF binding motifs specific to each ZF. Conclusions By extensively comparing the AID1 and AID2 systems for CTCF degradation in SEM cells, we confirm that AID2 degradation is superior for achieving miniAID-tagged protein degradation without the limitations of the AID1 system. The model we create that combines AID2 depletion of CTCF with exogenous overexpression of CTCF mutants allows us to demonstrate how peripheral ZFs intricately orchestrate transcriptional regulation in a cellular context for the first time.

Transposable Elements Shape Stemness in Normal and Leukemic Hematopoiesis

“…In HSPC populations, ZNF143, SMC3, CTCF cistromes, and those of their known partners SMC1A, RAD21 and CHD8 62,65,66 , enrich over LTR41B elements (q≤3.22e-34, OR≤9.36) (Extended Data Fig. 5D, Extended Data Table 10), in line with the role in 3D chromatin organization of LTR41B elements 67 . The accessibility of TE subfamilies serving as docking sites for known HSC factors and/or chromatin interaction factors parallels the enrichment of the GATA1 and CTCF DNA recognition sequences at some of these TEs (Extended Data Fig.…”

CTCF mutation at R567 causes developmental disorders via 3D genome rearrangement and abnormal neurodevelopment

“…To scrutinize the detailed binding properties affected by CTCF R567W , we performed motif enrichment analysis on our ChIP-seq data using a previously described method 41 . Using brain tissues as an example, we categorized CTCF sites into four groups based on alterations in CTCF binding strength following the CTCF R567W mutation.…”

An updated catalog of CTCF variants associated with neurodevelopmental disorder phenotypes

“…The effect of dysfunctional chromatin looping and gene expression during development is a growing area of research however the exact mechanisms of pathogenicity in CRD remain to be uncovered (Lupiáñez et al, 2015 ; Hanssen et al, 2017 ; Chakraborty et al, 2023 ). One puzzle that remains is that fast depletion of CTCF , using auxin-inducible degron systems in cell-based models, have not resulted in dramatic changes to enhancer-promoter interactions or transcription, highlighting a tolerance within cell assays to CTCF loss (Alharbi et al, 2021 ; Hsieh et al, 2022 ; Hyle et al, 2023 ). However, when CTCF is depleted in vivo , it does produce severe developmental phenotypes.…”

“…Hemizygous CTCF mice however, are viable and fertile, yet are predisposed to both spontaneous and induced tumor incidence, with global DNA methylation changes and deregulated gene expression patterns across tissues (Kemp et al, 2014 ; Alharbi et al, 2021 ). Depletion of CTCF in mammalian cell lines using the auxin-inducible degron system results in loss of chromatin looping and limited effects on gene transcription (Nora et al, 2017 ; Hyle et al, 2023 ). These studies highlight the necessity of correct CTCF gene dosage during development and throughout lifespan.…”