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，发育过程中发生的细胞表型变化是高度变化的。 取决于基因组中表观遗传标记的模式和分布。确实，主要 表观遗传学机制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 关于健康和疾病基因调控的许多基本见解。然而，其巨大的长度 基因组以及对大量基因进行协调调节和转录的要求 方式对细胞提出了重大的能量和身体挑战。因此，毫不奇怪， 通过三维结构赋予表观基因组额外的关键控制水平 染色体及其在细胞核内的组织。 尽管众所周知 3D 基因组组织在基因调控和癌症中发挥着至关重要的作用， 连接这些的基本机制人们知之甚少。 CTCF 是其中的核心，因为它 控制基因组组织并与癌症有关。事实上，CTCF 的突变是在 许多癌症，但是它们扰乱 3D 染色体结构的程度和 导致恶性表型的因素尚不清楚。我们假设每个 CTCF 突变都会改变 细胞功能以不同的方式取决于它是否与 (i) CTCF 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) 方向 的绑定。此外，我们提出，在没有基因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具有重要功能 CTCF 分子或重要辅助因子（如粘连蛋白）的结合。为了测试这些模型，我们的目标是 使用三种创新方法来检查突变对 (i) 结合亲和力和目标的影响 序列特异性，(ii) 染色体结构和基因调控，以及 (iii) 体内表型和 肿瘤发生/进展。我们将首先描述癌症相关 CTCF 的影响 其结合亲和力和结合基序发生突变。接下来我们来分析一下它们对细胞的功能作用 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 突变是否单独会诱发恶性的问题 转化或是否需要协同突变。我们建议获得机械 预测性了解 CTCF 癌症相关突变体的影响对于理解 CTCF 癌症相关突变体的影响至关重要 癌症基因组。这可能使一系列新的治疗方法能够抵消 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