Epigenetics of replication stress in human cells

人类细胞复制应激的表观遗传学

• 批准号: 9270554
• 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
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-06 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9270554
• 关键词: Acetylation; Address; Adopted; Affect; Alpha Cell; Area; Base Sequence; Binding; Biological Assay; CRISPR/Cas technology; Cell Proliferation; Cell Survival; Cell division; Cells; Cellular biology; Cessation of life; Chromatin; Code; Complex; 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; Recruitment Activity; Research; Resistance; Resolution; Role; Signal Transduction; Site; Small Interfering RNA; Source; Stress; 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; synergism; tool

 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与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，因此具有更大的治疗操作潜力，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，其特征是复制叉的异常密度、分布和稳定性。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，它也与癌发生的早期步骤有关。最近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，复制应激是否会挑战或损害表观遗传遗传，从而为细胞退化或转化开辟另一条途径。我们将通过剖析一个具体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（在早衰维尔纳综合征中发生突变）中的作用。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，我们之前已经表明，WRN 缺失会损害细胞对复制应激的抵抗力，最近，组蛋白脱乙酰酶 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，我们将确定在正常和 WRN 缺陷细胞复制应激期间和之后发生的新生染色质成熟和表观遗传变化的背景下，WRN 和 HDAC 之间的合作机制，以及它们对细胞存活、增殖和寿命的影响。我们的 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相结合（分别为微流体辅助复制轨迹分析或 ma-RTA，以及新生 DNA 的免疫沉淀或 iPOND）。我们将使用 RNAi 和 CRISPR/Cas9 操作来灭活感兴趣基因的表达。我们还将使用有针对性和公正的方法来确定 WRN 和 HDAC1,2 之间功能相互作用所涉及的因素和过程。我们将查询特定候选蛋白质的参与情况，并对 WRN 或 HDAC1,2 缺陷和正常细胞中与停滞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流程对 HDAC、WRN 依赖性复制应激表型的表观遗传修饰物进行 siRNA 筛选。