Binding domain mutations provide insight into CTCF's relationship with chromatin and its contribution to gene regulation (Hi-C)

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, we established 10 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. We then performed Hi-C. Cells expressing transgene CTCF mutant (indole acetic acid, IAA, an analogue of auxin + doxycycline, ID condition) were compared to cells expressing the transgene WT CTCF (WT, ID condition). Two replicates per condition were assessed.

本研究使用一系列CCCTC结合因子（CTCF）突变体，探究CTCF与染色质的相互关系及其在基因调控中的功能贡献。CTCF的功能效应取决于其结合位点的基因组背景，以及野生型（Wild Type, WT）与突变型CTCF蛋白各自独特的结合特性。具体而言，CTCF的信号强度与染色质可及性的变化呈关联关系，而其阻断黏连蛋白（cohesin）的能力则与其结合稳定性密切相关。多元建模分析结果显示，CTCF与染色质可及性均能独立调控黏连蛋白结合与染色质绝缘过程，其中CTCF信号强度的调控作用更为显著。CTCF与染色质之间存在双向互作关系：在CTCF结合位点处，染色质可及性以黏连蛋白依赖性、突变体特异性的方式降低。此外，每种CTCF突变体均可通过间接方式改变转录因子（Transcription Factor, TF）结合与染色质可及性，此类变化对转录网络重构及细胞分化能力具有最为关键的调控作用。总体而言，这些突变体扰动实验为解析CTCF的位点特异性效应提供了宝贵的研究资源。为研究CTCF突变的功能影响，本研究构建了10株细胞系：以小鼠胚胎干细胞（mouse embryonic stem cell, mESC）降解标签细胞系为基础，在敲除内源性CTCF的背景下，分别诱导表达稳定的多西环素调控型野生型Ctcf转基因，或突变型Ctcf（mCtcf）转基因。随后开展高通量染色质构象捕获（Hi-C）实验。将表达CTCF突变体转基因的细胞（经吲哚乙酸（indole acetic acid, IAA，生长素类似物）与多西环素处理，即ID组）与表达野生型CTCF转基因的细胞（WT组，同ID条件）进行对照比较。每组均设置2个生物学重复进行检测分析。