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Epigenetics of replication stress in human cells

人类细胞复制应激的表观遗传学

基本信息

批准号：
9900812
负责人：
JULIA SIDOROVA
金额：
$ 32.45万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-06 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9900812
关键词：
Acetylation Address Adopted Affect Area Base Sequence Binding Biological Assay CRISPR/Cas technology Cell Proliferation Cell Survival Cell division Cells Cellular biology Cessation of life Chromatin Chromatin Remodeling Factor Code DNA DNA Damage DNA Sequence Alteration DNA replication fork Data Deacetylation Environment Epigenetic Process Event Fiber Gene Expression Genes Genome Genome Stability Genomic Instability Genomics Goals HDAC1 gene HDAC2 gene Hand Health Histone Deacetylase Histone Deacetylation Histone H4 Histones Homeostasis Human Human Genome Human body Immunoprecipitation Impairment Individual Left Longevity Maintenance Malignant Neoplasms Measures Mediating Microfluidics Modeling Monitor Multiprotein Complexes Mutate Normal Cell Nucleotides Organism Outcome Pathway interactions Phenotype Post-Translational Protein Processing Predisposition Premature aging syndrome Process Protein Analysis Proteins RNA Interference Research Resistance Resolution Role Signal Transduction Site Small Interfering RNA Source Stretching Techniques Testing Text Therapeutic Time Translating WRN gene Werner Syndrome Work Writing base carcinogenesis cell growth chemotherapy chromatin modification chromatin remodeling density follow-up genetic information helicase in vivo interest protein function public health relevance recruit replication stress synergism tool

项目摘要

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.

描述（由申请人提供）：我们的长期目标是确定人类细胞中复制应激和表观遗传状态和过程之间的联系。表观遗传学变化预计比基因突变或缺失更容易逆转，因此它们在治疗操作方面具有更大的潜力，这一概念强调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。化疗可以引发复制应激。作为基因组不稳定性的来源，它也涉及癌发生的早期步骤。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细胞对复制应激的抵抗力，相反，复制应激是否可以挑战或损害表观遗传，从而为细胞变性或转化开辟另一条途径。我们将通过解剖一个具体的问题来解决这些问题：围绕移动和停滞复制叉的染色质修饰和重塑及其在RECQ解旋酶WRN缺陷的背景下的作用，在过早衰老的沃纳综合征中突变。一方面，最近的研究表明WRN与染色质维持和表观遗传稳定性有关。另一方面，我们以前已经表明，WRN的缺乏损害细胞的复制压力，最近，组蛋白去乙酰化酶HDAC 1和2与WRN合作，抵消复制叉失活过程中所造成的复制压力的核苷酸池耗尽。为了更好地了解复制改变，染色质改变和WRN缺陷的细胞生物学之间的联系，我们将确定WRN和HDAC之间的合作机制，在新生染色质成熟和表观遗传变化的背景下，在正常和WRN缺陷细胞复制应激期间和之后发生，以及它们对细胞存活，增殖和寿命的影响。我们该方法将标准细胞生物学测定与DNA和蛋白质水平的体内基因组复制的高分辨率功能分析（分别为微流体辅助复制跟踪分析或ma-RTA，以及新生DNA的免疫沉淀或iPOND）相结合。我们将使用RNAi和CRISPR/Cas9操作来抑制感兴趣基因的表达。我们还将使用有针对性和公正的方法来确定WRN和HDAC 1和2之间的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的参与，以及执行与WRN或HDAC 1，2缺陷和正常细胞中的停滞复制叉相关的蛋白质的质谱分析。我们还将使用之前验证的管道来进行SiRNA筛选，以寻找HDAC、WRN依赖性复制应激表型的表观遗传修饰剂。