Epigenetics of replication stress in human cells

 描述(由申请人提供)：我们的长期目标是确定复制压力和人类细胞的表观遗传状态和过程之间的联系。表观遗传变化预计比基因突变或缺失更容易逆转，因此它们可以拥有更大的治疗操作潜力，这一概念突显了这一研究领域对人类健康的重要性。复制应激是基因组复制的一种状态，其特征是复制叉的密度、分布和稳定性异常。化疗可能会引发复制压力。作为基因组不稳定性的一个来源，它也与癌症发生的早期步骤有关。从最近的研究中出现的重要问题是，表观遗传因素是否调节了细胞对复制应激的抵抗，反过来，复制应激是否可以挑战或损害表观遗传，从而为细胞退化或转化开辟了另一条途径。我们将通过剖析一个特定的问题来解决这些问题：染色质修改和围绕移动和停滞复制叉子的重塑，以及它在RecQ解旋酶WRN缺陷的背景下所起的作用，RRN在过早衰老的Werner综合征中突变。一方面，最近的工作表明WRN与染色质的维持和表观遗传的稳定性有关。另一方面，我们以前已经证明，WRN的缺失会损害细胞对复制应激的抵抗力，最近，组蛋白去乙酰基酶HDAC1和2与WRN合作，在核苷酸池耗尽引起的复制应激期间抵消复制叉失活。为了更好地了解WRN缺陷的细胞生物学与复制改变、染色质改变之间的联系，我们将从正常和WRN缺陷细胞在复制应激过程中和复制应激后发生的表观遗传学变化的背景下，确定WRN和HDAC在新生染色质成熟和表观遗传变化方面的合作机制，以及它们对细胞存活、增殖和寿命的影响。我们的 方法将标准细胞生物学分析与DNA和蛋白质水平上对体内基因组复制的高分辨率功能分析(分别是微流控辅助复制跟踪分析(MA-RTA)和新生DNA免疫沉淀(IPOND))相结合。我们将使用RNAi和CRISPR/Cas9操作来灭活目的基因的表达。我们还将使用有针对性和无偏见的方法来确定WRN与HDAC1和2之间功能相互作用所涉及的因素和过程。我们将查询特定的候选蛋白质的参与，并对WRN或HDAC1，2缺陷和正常细胞中与停滞的复制叉相关的蛋白质进行质谱分析。我们还将使用先前验证的管道来进行siRNA筛选，以寻找依赖于HDAC和WRN的复制应激表型的表观遗传修饰物。

 DESCRIPTION (provided by applicant): Our long-term goal is to define connections between replication stress and epigenetic states and processes in human cells. The importance of this research domain to human health is underscored by the notion that epigenetic changes are expected to be more easily reversible than a genetic mutation or deletion, can thus they can hold greater potential for therapeutic manipulation. Replication stress is a state of genomic replication characterized by abnormal density, distribution, and stability of replication forks. Replication stress can be triggered by chemotherapy. As a source of genomic instability it is also implicated in early steps of carcinogenesis. The important questions that emerge from recent studies are whether epigenetic factors modulate cellular resistance to replication stress, and conversely, whether replication stress can challenge or compromise epigenetic inheritance thus opening another avenue to cellular degeneration or transformation. We will address these questions by dissecting a specific problem: chromatin modification and remodeling around moving and stalling replication forks and its roles in the context of a deficiency in the RECQ helicase WRN, mutated in the Werner syndrome of premature aging. On the one hand, recent work now implicates WRN in chromatin maintenance and epigenetic stability. On the other hand, we have previously shown that WRN absence compromises cellular resistance to replication stress and, more recently, that histone deacetylases HDAC1 and 2 cooperate with WRN in counteracting replication fork inactivation during the replication stress caused by nucleotide pool depletion. With the aim of a greater understanding of the connections between altered replication, altered chromatin, and the cellular biology of WRN deficiency, we will determine the mechanism of cooperation between WRN and HDACs in the context of nascent chromatin maturation and epigenetic changes occurring during and after replication stress in normal and WRN-deficient cells, and their effect on cell survival, proliferation, and lifespan. Our approach integrates standard cellular biology assays with high-resolution, functional analyses of genomic replication in vivo at DNA and protein levels (respectively, microfluidic-assisted replication track analysis or ma-RTA, and immunoprecipitation of nascent DNA, or iPOND). We will use RNAi and CRISPR/Cas9 manipulation to inactivate expression of the genes of interest. We will also use both targeted and unbiased approaches to identify the factors and processes involved in the functional interaction between WRN and HDAC1 and 2. We will query for specific protein candidates' involvement as well as perform mass spectrometric analyses of proteins associated with stalled replication forks in WRN or HDAC1,2-deficient and normal cells. We will also use a previously validated pipeline to perform a siRNA screen for epigenetic modifiers of HDAC, WRN- dependent replication stress phenotypes.