Nuclear Organization and Function

项目总结。适当的基因表达调控对于细胞分化和 动态平衡。我们对转录过程控制机制的大部分了解来自于 一维基因组即10纳米染色质纤维的研究。然而，基因组被折叠在 三维(3D)核空间，这种组织和基因表达之间的关系是 人们对此知之甚少。以果蝇为模型系统，获得250个BP的Hi-C分辨率是可行的 数据，我们发现基因组只折叠成一种类型的结构域，我们称之为区间结构域 域名。这些结构域与其序列的转录状态精确相关。隔间的 在其他低等真核生物中也发现了结构域。在此基础上，我们提出了划分域 代表了基因组3D组织在进化上保守的原则。果蝇和低等真核生物 要么是缺乏CTCF，要么是这种蛋白质无法阻止粘附素的分泌。但是，CTCF可能会干扰 脊椎动物粘附素挤出的进展，进而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3D组织分析中学到的概念推广到哺乳动物 通过提出一种雄心勃勃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，以及人类胚胎干细胞(HESC)向疾病相关组织的分化。 提出的实验的假设是基于这样的想法，而不是流行的观点 在含有较小TADS的大间隔中，哺乳动物的基因组是按照保守的原则组织的 形成相对较小的隔区。粘附素挤出作用于间隔区的顶部 脚手架，并影响其组织。为了验证这一新的假设，我们将耗尽存在于 转录过程的各个方面所需的复合体。我们还将耗尽蛋白质复合体 负责H3K27me3和H3K9me3依赖的沉默。然后我们将使用Micro-C XL来获得非常 高分辨率相互作用数据和蛋白质耗竭对自相互作用形成的影响 以及这些域之间的相互作用。这些影响将在现场和 缺乏粘附素以了解环挤出对增强启动子-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考察3D基因组组织的可预测性 使用机器学习计算工具获取信息。我们将通过以下方式研究CTCF循环形成的逻辑 分析CTCF位点周围能够或不能形成不同环的局部染色质环境 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则将 通过分析3D组织的变化及其与基因表达的关系来进行测试 人胚胎干细胞向胰腺细胞的分化。这项工作的结果将填补我们对 3D染色质组织和转录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.