Single base-pair resolution analysis of binding motif with diffMotif uncovers the oncogenic impact of CTCF mutations in breast cancer [RNA-Seq]

Most cancer associated CTCF mutations are involved in the recognition of CTCF core binding motif. Interestingly, mutations of the currently uncharacterized ZF1 of CTCF are practically exclusive to breast cancer, where they are associated with increased metastatic ability and therapeutic resistance. Although unnecessary to bind the core-binding motif, it is still unknown if the ZF1 of CTCF promotes binding to an altered CTCF motif. Further, classical motif analysis tools are powerless to identify minute changes in core binding motif. Therefore, the biological and epigenetic mechanism of action of CTCF ZF1 mutations is still unexplored. In order to explain the impact of CTCF ZF1M in breast models, we developed a new tool, diffMotif, that allows the identification, at the single base-pair resolution, of extension or alteration of core binding motif. Using diffMotif, we identified an extension of CTCF core binding motif, necessitating a functional ZF1 to bind appropriately. Using a combination of ChIP-Seq and RNA-Seq, we discover that the inability to bind the extended motif led to altered gene expression driving increased invasive ability and drug metabolism, mimicking the harmful oncogenic phenotypes observed clinically. Our study demonstrates the oncologic relevance of the characterisation of mutation induced altered DNA binding ability, by diffMotif. Examination of mRNA expression and CTCF binding changes in the context of CTCF ZF1 mutations

绝大多数与癌症相关的CCCTC结合因子（CTCF）突变均参与CTCF核心结合基序的识别过程。值得注意的是，目前尚未被功能表征的CTCF锌指1（ZF1）突变几乎仅见于乳腺癌，此类突变与肿瘤侵袭转移能力增强及治疗耐药性提升密切相关。尽管ZF1并非CTCF结合核心结合基序所必需，但CTCF的ZF1是否会促进其与变异型CTCF结合基序的结合，目前仍未明确。此外，传统的基序分析工具无法识别核心结合基序的细微变化。因此，CTCF ZF1突变发挥作用的生物学及表观遗传机制仍有待探索。为阐明CTCF ZF1突变在乳腺癌模型中的影响，我们开发了一款全新工具diffMotif，该工具可实现单碱基分辨率下核心结合基序延伸或变异的精准识别。借助diffMotif工具，我们发现了CTCF核心结合基序的一种延伸形式，该形式需要具备功能活性的ZF1才能实现正常结合。通过联合使用染色质免疫共沉淀测序（ChIP-Seq）与转录组测序（RNA-Seq）技术，我们发现无法结合该延伸基序会导致基因表达异常，进而驱动侵袭能力增强与药物代谢改变，模拟了临床中观察到的有害致癌表型。本研究通过diffMotif工具证实了突变诱导的DNA结合能力改变的肿瘤学研究价值。针对CTCF ZF1突变背景下的mRNA表达及CTCF结合变化的研究