Nuclear organization and its role in gene regulation

总结： 我的实验室的工作一直处于研究的前沿，表明核组织和长距离染色质 相互作用在重组和基因调控中起重要作用。我们将联合收割机和成像技术 (DNA/RNA FISH）方法与内部生成的计算管道，因此是少数之一， 拥有染色体折叠实验和分析方面的专业知识的实验室。在这 应用我们将扩大我们的工作，重点放在两个主要的相互关联的问题，重要的生物 重要性：项目1：理解有丝分裂染色质折叠背景下的书签。在 在有丝分裂（M）中，M期活性基因的启动子被“书签化”，维持一些启动子的可及性。 监管要素。这提供了一种快速激活基因子集的机制，使细胞 从有丝分裂中退出，以保留先前细胞周期的表观遗传程序的记忆。以来 调节因子与染色质有不同的亲和力，有些因子会保留在M期 染色质，而且，因为结合是以动态方式发生的， 保持结合的因子在整个细胞群中将不是均匀的。书签的低效率， 结合细胞间的变异性，赋予子细胞一定程度的表观遗传可塑性， 他们改变他们的表型响应环境信号，这可能会产生重大影响， 发育和生物过程。然而，人们对这一现象背后的机制知之甚少。 过程，特别是如何凝聚素II介导的染色质折叠在有丝分裂影响可及性。 项目2：静止细胞染色质组织和基因调控的机制。 细胞可以在应激、毒性、营养或生长因子耗尽的条件下适应和存活， 化学损伤，通过退出细胞周期并进入称为静止（G 0）的可逆休眠状态。 以前的研究表明，CTCF/cohesin介导的细胞膜结构在进入G1期后恢复， 哺乳动物G 0细胞的染色体结构尚未用现代分子生物学方法进行仔细研究。 方法论。基因表达在G 0细胞中被全面抑制，但我们现在知道， 活跃地转录特定的基因。值得注意的是，静止期细胞的染色质主要是致密的，如M 相位因此，我们假设从M进入G 0的细胞部分保留了细胞的紧凑组织。 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期开始时结合的缩合蛋白II功能标记活性启动子的子集，（ii）是否 在单个细胞之间标记的位点存在变异性，以及（iii）是否缩合蛋白II- 介导的染色质循环和书签机制与独特的基因组结构有关， 和G 0细胞的转录程序。鉴于我们结合实验和计算的专业知识， 在核组织领域，我们具有独特的地位来处理这些及时的相关问题。

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.