Nuclear organization and its role in gene regulation

摘要： 我的实验室的工作一直处于研究的前沿，表明核组织和远程染色质 相互作用在重组和基因调控中起着至关重要的作用。我们结合了分子和成像技术 (DNA/RNA FISH)方法与内部生成的计算管道，因此是为数不多的 在染色体折叠的实验和分析方面都有专业知识的实验室。在这 应用我们将把我们的工作扩展到两个主要的相互关联的重大生物学问题上 重要性：项目1：在有丝分裂染色质折叠的背景下理解书签。在……里面 有丝分裂(M)--M期活性基因的启动子--被“书签”标记，以保持某些 监管要素。这为基因子集的快速激活提供了一种机制，允许细胞 退出有丝分裂以保存前一个细胞周期的表观遗传程序的记忆。自.以来 与染色质相关的调节因子具有明显的亲和力，部分因子将保留在M期 染色质比其他的更好，而且因为结合是以动态方式发生的，所以 仍然受约束的因素在一群细胞中不会是一致的。书签的低效， 结合细胞间的可变性，赋予子细胞一定程度的表观遗传可塑性，使 以改变它们的表型以响应环境信号，这可能对 发育和生物过程。然而，人们对这种现象背后的机制知之甚少。 过程，特别是凝集素II在有丝分裂过程中介导的染色质折叠如何影响可及性。 项目2：静止细胞染色质组织和基因调控的潜在机制。 细胞可以在压力、毒性、营养或生长因子枯竭的条件下适应和生存/和 化学侮辱，通过退出细胞周期，进入一种可逆的休眠状态，称为静止(G0)。 以往的研究表明，CTCF/粘附素介导的TAD结构在进入G1后恢复，但 哺乳动物G0细胞的染色体结构还没有用现代分子仔细研究过 方法论。基因表达在G0细胞中被整体抑制，但我们现在知道静止的细胞 积极转录特定基因。值得注意的是，静止细胞中的染色质主要是致密的，就像在M 相位。因此，我们假设从M到G0的细胞部分地保持了 有丝分裂细胞通过保留凝聚素II介导的环。我们未来研究的目标是确定 (I)在M期功能开始时的凝集素II结合，以标记活性启动子的子集，(Ii)是否 在单个细胞之间被书签标记的位置存在变异性，以及(Iii)凝集素II- 介导的染色质环和书签与独特的基因组组织机械地联系在一起 和G0细胞的转录程序。鉴于我们在实验和计算方面的专业知识 在核组织领域，我们处于独特的地位，能够解决这些及时和相关的问题。

SUMMARY: My lab’s work has been at the forefront of studies showing that nuclear organization and long-range chromatin interactions play an essential role in recombination and gene regulation. We combine molecular and imaging (DNA/RNA FISH) approaches with in house generated computational pipelines, and are thus one of a handful of labs that has expertise in both the experimental and analytical aspects of chromosome folding. In this application we will extend our work to focus on two main interlinked problems of significant biological importance: Project 1: Understanding bookmarking in the context of mitotic chromatin folding. In mitosis (M), the promoters of M-phase active genes are “bookmarked” maintaining the accessibility of some regulatory elements. This provides a mechanism for the rapid activation of a subset of genes, allowing cells exiting from mitosis to preserve a memory of the epigenetic program of the previous cell cycle. Since regulatory factors associate with chromatin with distinct affinities, some factors will be retained on M-phase chromatin better than others, and furthermore, because binding occurs in a dynamic manner, sites at which factors remain bound will not be uniform across a population of cells. The inefficiency of bookmarking, combined with cell-to-cell variability, imparts daughter cells with a degree of epigenetic plasticity, enabling them to alter their phenotype in response to environmental signals, which can have a significant impact on developmental and biological processes. However, little is known about the mechanisms underlying this process, and in particular how condensin II-mediated chromatin folding during mitosis impacts accessibility. Project 2: Mechanisms underlying the chromatin organization and gene regulation of quiescent cells. Cells can adapt and survive under conditions of stress, toxicity, nutrient or growth factor depletion/and chemical insult, by exiting the cell cycle and entering a reversible dormant state known as quiescence (G0). Previous studies showed that CTCF/cohesin-mediated TAD structure is restored after entry into G1, but chromosome structure in mammalian G0 cells has not been studied carefully using modern molecular methodologies. Gene expression is globally repressed in G0 cells, but we now know that quiescent cells actively transcribe specific genes. Notably, chromatin in quiescent cells is predominantly compact, as in M phase. Accordingly, we hypothesize that cells exiting M into G0 partially preserve the compact organization of mitotic cells by retaining condensin II-mediated loops. The goal of our future studies is to determine whether (i) condensin II binding at the start of M-phase functions to bookmark a subset of active promoters, (ii) whether there is variability in the sites that are bookmarked between individual cells, and (iii) whether condensin II- mediated chromatin looping and bookmarking are mechanistically linked to the unique genome organization and transcriptional programs of G0 cells. Given our combined experimental and computational expertise in the field of nuclear organization, we are uniquely positioned to address these timely, relevant questions.