Cross-regulation between loop extrusion, chromatin fiber structure and chromatin-associated RNAs

黏连蛋白复合物是驱动哺乳动物基因组在数十倍尺度上的三维组织的主要因素 到千分之一到百万分之一最近的单分子实验表明，它可以挤出DNA环， 这是基因组结构的组织原则。CTCF是一种DNA结合蛋白， 粘附素的易位和定义环边界，以及促进加载的粘附素的几个调节因子， 如NIPBL，或从染色质释放，如WAPL，粘着蛋白在染色体中定义相互作用域 在发育过程中影响基因表达模式，并可能导致发育疾病或癌症 当中断。尽管已经研究了裸DNA上的环挤出，但细胞中的粘着蛋白必须在细胞中导航， 核小体包装的染色质纤维，限制进入DNA上的结合位点，自组织成 类似表观遗传状态的区室独立于环结构域并与环结构域竞争，可能 调节DNA超螺旋，并被染色质相关RNA修饰。cohesin和CTCF如何相互作用 与细胞中染色质的关系是理解三维基因组结构的下一个前沿。这一竞技场的进展将 需要多尺度的方法，探测核小体尺度和兆碱基尺度的特征。 我提出实验来探讨（1）如何环挤压的凝聚扰动局部结构的染色质 （2）染色质纤维的局部结构如何通过连接组蛋白的消耗和 核小体的不稳定，调节粘附素加载和挤出环的能力;（3）如何改变核小体的稳定性， 由于过度的粘着蛋白成环导致的超螺旋的平衡影响局部核小体-核小体相互作用;以及（4） CTCF的染色质相关RNA相互作用组位于RNA依赖和RNA非依赖环边界。 为了分析粘附素、其调节因子和染色质纤维的特殊作用，我们将结合使用 在人和小鼠细胞系中稳定的蛋白质消耗、急性降解和药理学抑制。我们 将使用RICC-seq读取染色质纤维结构和染色质相关RNA的变化， 最近开发的用于在完整细胞中以亚核小体分辨率测量DNA-DNA接触，并使用 新的技术开发，以探测特定蛋白质的染色质相关RNA相互作用组。这些 方法将与更成熟的表观基因组和转录分析工具相结合，并与粗- 颗粒模拟，以开发和测试多尺度模型的相互作用，环挤压机械与 染色质纤维我预计这些实验的结果将揭示新的光如何循环挤出 和染色质的自我联合在特定的环境中相互作用，这是粘蛋白与DNA结合的模型 与环挤出，超螺旋如何在染色体上传播，以及局部 分子环境定义了环边界。这些知识可能会揭示新的补偿策略 由于粘着蛋白或其调节因子的突变导致的转录失调， 染色质纤维，粘着蛋白的天然底物。

The cohesin complex is a major factor driving the 3-D organization of mammalian genomes at the scale of tens to kilobases to megabases. Recent single-molecule experiments have shown that it can extrude loops of DNA, which are an organizing principle of genome architecture. Together with CTCF, a DNA-binding protein that stalls cohesin’s translocation and defines loop boundaries, and several regulators of cohesin that promote loading, such as NIPBL, or release from chromatin, such as WAPL, cohesin defines interaction domains in chromosomes that affect patterns of gene expression during development and can lead to developmental diseases or cancer when disrupted. Although loop extrusion on naked DNA has been studied, cohesin in cells must navigate nucleosome-packed chromatin fibers that restrict access to binding sites on DNA, self-organize into compartments of similar epigenetic state that are independent of and compete with loop domains, potentially regulate DNA supercoiling, and are decorated with chromatin-associated RNAs. How cohesin and CTCF interact with chromatin in cells is the next frontier in understanding 3-D genome organization. Progress in this arena will require a multi-scale approach, with methods that probe both nucleosome-scale and megabase-scale features. I propose experiments to probe (1) how loop extrusion by cohesin perturbs the local structure of the chromatin fiber; (2) how the local structure of the chromatin fiber, modulated by depletion of linker histones and destabilization of nucleosomes, regulates cohesin’s ability to load and extrude loops; (3) how changes in the balance of supercoiling due to excess cohesin looping affect local nucleosome-nucleosome interactions; and (4) the chromatin-associated RNA interactome of CTCF at RNA-dependent and RNA-independent loop boundaries. To dissect the specific effects of cohesin, its regulators and the chromatin fiber, we will use a combination of stable protein depletion, acute degradation, and pharmacological inhibition in human and mouse cell lines. We will read out changes in chromatin fiber structure and chromatin-associated RNAs using RICC-seq, a method I recently developed for measuring DNA-DNA contacts at sub-nucleosome resolution in intact cells, and using novel technology development to probe the chromatin-associated RNA interactome of specific proteins. These methods will be combined with more established epigenome and transcription profiling tools and with coarse- grained simulations to develop and test multi-scale models for the interaction of loop extrusion machinery with the chromatin fiber. I anticipate that the results of these experiments will shed new light on how loop extrusion and chromatin’s self-association interact in specific contexts, which models for cohesin’s engagement with DNA are relevant to loop extrusion, how supercoiling is disseminated across chromosomes, and how the local molecular context defines loop boundaries. This knowledge may reveal new strategies for compensating transcriptional dysregulation due to mutations in cohesin or its regulators using targetable factors that regulate the chromatin fiber, cohesin’s native substrate.