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变化是非常重要的 取决于基因组中表观遗传标记的模式和分布。的确，少校 机械论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，成果丰硕 关于健康和疾病中的基因调控的许多基本见解。然而这本书的巨大篇幅 基因组和大量基因需要以协调的方式调节和转录 这种方式对细胞构成了巨大的能量和身体挑战。因此，不足为奇的是 表观基因组的额外临界控制水平是通过三维结构授予的 染色体及其在细胞核内的组织。 虽然已知3D基因组组织在基因调控和癌症中起着至关重要的作用， 人们对连接这些因素的潜在机制知之甚少。CTCF是这些项目的核心，因为它 管理基因组组织，并与癌症有关。事实上，CTCF中的突变是在 许多癌症，然而，他们扰乱3D染色体结构和 对恶性表型的贡献尚不清楚。我们假设每个CTCF突变都会改变 细胞功能的不同取决于它是否与(I)CTCF的完全丧失有关 结合，(二)结合亲和力的改变，(三)结合基序偏好的改变或(四)取向的改变 是有约束力的。此外，我们认为，在没有基因突变的癌症中频繁发生的突变 对结合或结合亲和力的明显影响将在破坏二聚化方面发挥重要作用 CTCF分子或重要辅因子(如粘附素)的结合。为了测试这些模型，我们的目标是 使用三种创新的方法来检查突变对(I)结合亲和力和靶标的影响 序列特异性，(Ii)染色体结构和基因调控，以及(Iii)体内表型和 肿瘤的发生/发展。我们将从表征与癌症相关的CTCF的影响开始 它们的结合亲和力和结合基序的突变。接下来，我们将分析它们对细胞的功能影响 生存、基因表达和染色体组织。最后，鼠标模型将解决关键问题 癌症相关的CTCF突变是否单独会导致恶性的问题 或者是否需要协同突变。我们建议获得一个机械论的 对CTCF癌症相关突变体的影响的预测性了解对于理解 癌症基因组。这可能使一系列新的治疗方法能够中和 突变建筑蛋白的恶性作用。 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