Liu Lian, Tang Yuanxiao, Zhang Yan, Wu Qiang
Center for Comparative Biomedicine, Ministry of Education Key Laboratory of Systems Biomedicine, State Key Laboratory of Medical Genomics, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China.
iScience. 2024 Nov 22;27(12):111452. doi: 10.1016/j.isci.2024.111452. eCollection 2024 Dec 20.
As an essential regulator of higher-order chromatin structures, CCCTC-binding factor (CTCF) is a highly conserved protein with a central DNA-binding domain of 11 tandem zinc fingers (ZFs), which are flanked by amino (N-) and carboxy (C-) terminal domains of intrinsically disordered regions. Here we report that CRISPR deletion of the entire C-terminal domain of alternating charge blocks decreases CTCF DNA binding but deletion of the C-terminal fragment of 116 amino acids results in increased CTCF DNA binding and aberrant gene regulation. Through a series of genetic targeting experiments, in conjunction with electrophoretic mobility shift assay (EMSA), circularized chromosome conformation capture (4C), qPCR, chromatin immunoprecipitation with sequencing (ChIP-seq), and assay for transposase-accessible chromatin with sequencing (ATAC-seq), we uncovered a negatively charged region (NCR) responsible for weakening CTCF DNA binding and chromatin accessibility. AlphaFold prediction suggests an autoinhibitory mechanism of CTCF via NCR as a flexible DNA mimic domain, possibly competing with DNA binding for the positively charged ZF surface area. Thus, the unstructured C-terminal domain plays an intricate role in maintaining proper CTCF-DNA interactions and 3D genome organization.
作为高阶染色质结构的重要调节因子，CCCTC结合因子（CTCF）是一种高度保守的蛋白质，具有由11个串联锌指（ZF）组成的中央DNA结合结构域，其两侧是内在无序区域的氨基（N-）和羧基（C-）末端结构域。在这里，我们报告称，通过CRISPR删除交替电荷块的整个C末端结构域会降低CTCF与DNA的结合，但删除116个氨基酸的C末端片段会导致CTCF与DNA的结合增加以及异常的基因调控。通过一系列基因靶向实验，结合电泳迁移率变动分析（EMSA）、环状染色体构象捕获（4C）、qPCR、染色质免疫沉淀测序（ChIP-seq）以及转座酶可及染色质测序分析（ATAC-seq），我们发现了一个带负电荷的区域（NCR），该区域负责削弱CTCF与DNA的结合以及染色质可及性。AlphaFold预测表明，CTCF通过NCR存在一种自抑制机制，NCR作为一个灵活的DNA模拟结构域，可能与DNA竞争带正电荷的ZF表面积的结合。因此，无结构的C末端结构域在维持适当的CTCF-DNA相互作用和三维基因组组织中发挥着复杂的作用。
