Background: Human cancers commonly contain mutations in transcription factors that lead to aberrant DNA binding or altered effector function at target sites. One such factor significantly mutated in cancer is the evolutionarily-conserved CCCTC-binding factor (CTCF), which has fundamental roles in maintaining chromatin architecture and transcriptional regulation. Numerous cancer genome sequencing and functional studies have revealed CTCF's role as a haploinsufficient tumour suppressor gene. However, to date, structure-function relationships of somatic CTCF mutations have not been examined. Methods: We collated somatic CTCF mutations from cancer genome portals and published studies to determine their nature, frequency, distribution and potential functional impact. We undertook an in-depth examination of 5 CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. We performed functional analyses including cell growth, colony-formation, chromatin immunoprecipitation and transcriptional reporter assays. Based on their homology, each ZF mutation was then modelled on CTCF's ZF domain crystal structure and its structural impact analysed using molecular dynamics simulations. Results: We observed an enrichment of somatic missense mutations occurring in the ZF region of CTCF, compared to the unstructured N- and C-termini. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. In silico mutagenesis revealed that L309P had the highest mutation energy and thus most severe impact on protein stability. Molecular docking and molecular dynamics simulations confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding (R339Q, R339W, R377H). Remarkably, R339Q and G420D were stabilised by the formation of new primary DNA bonds. All mutations exhibited some loss or gain of bonds at neighbouring residues, often in adjacent zinc fingers. Conclusions: Our data confirm the significant negative impact haploinsufficient CTCF ZF mutations have on its tumour suppressor function. A spectrum of loss-, change- and gain-of-function impacts in CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. We have established that diverse cellular phenotypes in CTCF are explained by examining structure-function relationships.

中文翻译：

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CTCF锌指中的体细胞突变产生细胞表型，由结构-功能关系解释

背景：人类癌症通常在转录因子中含有突变，导致目标部位的DNA结合异常或效应子功能改变。在癌症中显着突变的此类因子之一是进化保守的CCCTC结合因子（CTCF），其在维持染色质结构和转录调控中具有基本作用。众多的癌症基因组测序和功能研究表明，CTCF作为单倍型肿瘤抑制基因的作用。然而，迄今为止，尚未检查体细胞CTCF突变的结构-功能关系。方法：我们从癌症基因组门户网站中收集了体细胞CTCF突变，并发表了研究以确定其性质，频率，分布和潜在的功能影响。我们对ZF内部和内部ZF关键残基中发生的5个CTCF错义锌指（ZF）突变进行了深入研究。我们进行了功能分析，包括细胞生长，集落形成，染色质免疫沉淀和转录报告基因检测。基于它们的同源性，然后在CTCF的ZF域晶体结构上建模每个ZF突变，并使用分子动力学模拟分析其结构影响。结果：与未结构化的N和C末端相比，我们观察到CTCF的ZF区域中发生了丰富的体细胞错义突变。CTCF ZF突变的功能表征揭示了完全（L309P，R339W，R377H）或中间（R339Q）废除，以及CTCF的抗增殖作用的增强（G420D）。在特定位点的DNA结合被破坏，转录调节活性被取消。通过计算机诱变表明，L309P具有最高的突变能量，因此对蛋白质稳定性的影响最大。分子对接和分子动力学模拟证实，特异性接触DNA碱基或骨架的残基中的突变表现出DNA结合的丧失（R339Q，R339W，R377H）。值得注意的是，R339Q和G420D通过形成新的一级DNA键而得以稳定。所有突变通常在相邻的锌指中在相邻残基处表现出一些键的丢失或获得。结论：我们的数据证实，单倍体不足的CTCF ZF突变对其抑癌功能具有明显的负面影响。一系列损失 在细胞生长调节和基因调节活性中观察到CTCF锌指的功能改变和功能获得影响。我们已经建立了通过检查结构-功能关系来解释CTCF中不同的细胞表型。