Nuclear Organization and Function

项目摘要。基因表达的适当调节对于细胞分化和分化是必不可少的。 体内平衡我们对控制转录过程的机制的理解大多来自于 一维基因组的研究，即10 nm染色质纤维。然而，基因组是折叠在 三维（3D）核空间，这种组织和基因表达之间的关系是 不太了解。使用果蝇作为模型系统，其中获得250 bp分辨率Hi-C是可行的 数据，我们已经发现，基因组被折叠成只有一种类型的域，我们称之为区室 域.这些结构域与其序列的转录状态精确相关。房室 结构域也存在于其它低等真核生物中。在此基础上，我们提出， 代表了基因组3D组织的进化保守原则。果蝇和低等真核生物 缺乏CTCF或这种蛋白质不能阻止粘附素挤出。然而，CTCF可以干扰 在脊椎动物中的粘着蛋白挤出的进展，这反过来又影响基因组中的其他类型的相互作用。 在这里，我们建议将从果蝇的3D组织分析中学到的概念扩展到哺乳动物 通过提出一个雄心勃勃的和实质性的多学科方法，结合遗传学，表观基因组学， 计算生物学和人类胚胎干细胞（hESC）分化为疾病相关组织。 这些实验的假设是基于这样一种观点，而不是普遍的观点， 哺乳动物的基因组是由保守的原则组成的， 分成相对较小的分区。粘连蛋白挤出作用于房室结构域的顶部 支架并影响其组织。为了验证这一新的假设，我们将消耗存在于 转录过程的各个方面所需的复合物。我们还将耗尽蛋白质复合物 负责H3 K27 me 3-和H3 K9 me 3-依赖性沉默。然后我们将使用Micro-C XL来获得非常 高分辨率的相互作用数据，并检查蛋白质消耗对自相互作用形成的影响。 域之间的相互作用。这些影响将在存在和 为了理解环挤出对增强子-启动子相互作用的贡献， 频率.我们将从一维表观遗传学角度研究3D基因组组织的可预测性。 信息使用机器学习计算工具。我们将研究CTCF循环形成的逻辑， 分析CTCF位点周围的局部染色质环境，所述CTCF位点能够或不能形成不同的环， 使用我们开发的一种新的计算工具。从这些实验中学到的原理将 通过分析3D组织的变化及其与基因表达的关系进行测试， hESC向胰腺细胞的分化。这项工作的结果将填补我们对以下问题的理解中的关键空白： 3D染色质组织和转录之间的关系，及其在人类疾病中的可能作用。

PROJECT SUMMARY. Proper regulation of gene expression is essential for cell differentiation and homeostasis. Most of our understanding of the mechanisms that control the transcription process comes from studies of the one-dimensional genome i.e. the 10 nm chromatin fiber. However, the genome is folded in the three-dimensional (3D) nuclear space, and the relationship between this organization and gene expression is poorly understood. Using Drosophila as a model system, where it is feasible to obtain 250 bp resolution Hi-C data, we have found that the genome is folded into only one type of domain, which we call compartmental domains. These domains precisely correlate with the transcriptional state of their sequences. Compartmental domains are also found in other lower eukaryotes. Based on this, we propose that compartmental domains represent an evolutionarily conserved principle of genome 3D organization. Drosophila and lower eukaryotes either lack CTCF or this protein is unable to stop cohesin extrusion. However, CTCF can interfere with the progression of cohesin extrusion in vertebrates, which in turn affects other types of interactions in the genome. Here we suggest extending concepts learned from the analysis of 3D organization in Drosophila to mammals by proposing an ambitious and substantive multi-disciplinary approach combining genetics, epigenomics, computational biology, and differentiation of human embryonic stem cells (hESC) into disease-relevant tissues. The hypothesis underlying the proposed experiments is based on the idea that, rather than the prevalent view of large compartments containing smaller TADs, the mammalian genome is organized by conserved principles into relatively small compartmental domains. Cohesin extrusion operates on top of the compartmental domain scaffold and affects its organization. To test this novel hypothesis, we will deplete specific proteins present in complexes required for various aspects of the transcription process. We will also deplete protein complexes responsible for H3K27me3- and H3K9me3-dependent silencing. We will then use Micro-C XL to obtain very high-resolution interaction data and examine effects of protein depletion on the formation of self-interacting domains and in the interactions between these domains. These effects will be examined in the presence and absence of cohesin in order to understand the contribution of loop extrusion to enhancer-promoter interaction frequency. We will examine the predictability of 3D genome organization from one-dimensional epigenetic information using machine learning computational tools. We will study the logic of CTCF loop formation by analyzing the local chromatin environment around CTCF sites able or unable to form loops of different strengths using a new computational tool we have developed. Principles learned from these experiments will be tested by analyzing changes in 3D organization and their relationship to gene expression during the differentiation of hESCs into pancreatic cells. Results from this work will fill critical gaps in our understanding of the relationship between 3D chromatin organization and transcription, and its possible role in human disease.