Project 1: The biochemical, topological and functional impact of cancer associated Ctcfmutations and their contribution to cancer

摘要-项目1（SKOK） 众所周知，在发育过程中发生的细胞表型的变化是高度相关的。 这取决于基因组中表观遗传标记的模式和分布。事实上，少校 机制表观遗传学研究的焦点集中在组蛋白或DNA之间的相互作用 修饰蛋白质和转录因子。这些研究成果丰硕， 许多关于健康和疾病中基因调控的基本见解。然而， 基因组和需要大量的基因被调节和转录在一个协调的 方式对细胞提出了重大的能量和物理挑战。因此，毫不奇怪， 通过三维结构赋予了表观基因组控制的额外临界水平 染色体及其在细胞核内的组织。 尽管已知3D基因组组织在基因调控和癌症中起着至关重要的作用， 人们对这些联系的基本机制知之甚少。CTCF是核心，因为它 控制着基因组的组织并与癌症有关。事实上，CTCF的突变在 许多癌症，然而，它们扰乱3D染色体结构的程度， 导致恶性表型的原因尚不清楚。我们假设每个CTCF突变都会改变 根据其是否与（i）CTCF的完全丧失相关， 结合，（ii）结合亲和力的变化，（iii）结合基序偏好的改变或（iv）取向 的约束。此外，我们提出，突变，经常发生在癌症，没有 对结合或结合亲和力的明显影响将在破坏 CTCF分子或重要辅因子如粘附素的结合。为了测试这些模型， 使用三种创新的方法来检查突变对（i）结合亲和力和靶向的影响， 序列特异性，（ii）染色体结构和基因调控，和（iii）体内表型， 肿瘤开始/进展。我们将首先描述癌症相关CTCF的影响， 在它们的结合亲和力和结合基序上的突变。接下来，我们将分析它们对细胞的功能影响。 存活、基因表达和染色体组织。最后，小鼠模型将解决关键 关于癌症相关的CTCF突变单独是否可使恶性肿瘤易感的问题 转化或是否需要合作突变。我们建议，获得一个机械的 对CTCF癌症相关突变体影响的预测性理解对于理解 癌症基因组这可能会使一系列新的治疗方法，以抵消 突变结构蛋白的恶性效应。 ! 1

SUMMARY – PROJECT 1 (SKOK) It is well established that changes in cellular phenotypes that occur during development are highly dependent on the patterning and distribution of epigenetic marks in the genome. Indeed, the major focus of mechanistic epigenetic studies has centered around the interplay between histone or DNA modifying proteins and transcription factors. These studies have been immensely fruitful and yielded many fundamental insights into gene regulation in health and disease. Yet the enormous length of the genome and the requirement for large sets of genes to be regulated and transcribed in a coordinated manner poses significant energetic and physical challenges to cells. It is thus not surprising that an additional critical level of control of the epigenome is conferred through the three dimensional structure of chromosomes and their organization within the nucleus. Though it is known that 3D genome organization plays a crucial role in gene regulation and cancer, the underlying mechanisms connecting these are poorly understood. CTCF is central to these, as it governs genome organization and is implicated in cancer. In fact mutations in CTCF are detected in numerous cancers, however the extent to which they perturb 3D chromosomal architecture and contribute to the malignant phenotype is unknown. We hypothesize that each CTCF mutation will alter cellular function in a different manner depending on whether it is associated with (i) total loss of CTCF binding, (ii) a change in binding affinity, (iii) an alteration in binding motif preference or (iv) orientation of binding. Furthermore, we propose that mutations, which occur frequently in cancers that have no apparent effect on binding or binding affinity will have important functions in disrupting dimerization of CTCF molecules or binding of important cofactors such as cohesin. To test these models we aim to use three innovative approaches that examine the impact of mutations on (i) binding affinity and target sequence specificity, (ii) chromosome structure and gene regulation, and (iii) in vivo phenotype and tumor initiation/progression. We will start by characterizing the impact of cancer associated CTCF mutations on their binding affinity and binding motif. Next we will analyze their functional effect on cell survival, gene expression and chromosome organization. Finally, mouse models will address the key question of whether cancer associated CTCF mutations alone can predispose to malignant transformation or whether cooperating mutations are required. We propose that gaining a mechanistic predictive understanding of the impact of CTCF cancer associated mutants is essential to understand cancer genomes. This may enable a range of novel therapeutic approaches to counteract the malignancy effects of mutant architectural proteins. !1