Investigating the mechanisms and generality of asymmetric histone inheritance in multiple stem cell systems

研究多个干细胞系统中不对称组蛋白遗传的机制和普遍性

• 批准号: 9319549
• 负责人: Matthew Wooten
• 金额: $ 4.4万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9319549
• 关键词: Address; Alpha Cell; Anatomy; Biological Assay; Biological Models; Cell Lineage; Cell Maintenance; Cell division; Cell physiology; Cells; Color; Complement; Coupled; DNA; DNA Sequence; DNA biosynthesis; DNA replication fork; Daughter; Deposition; Development; Diabetes Mellitus; Disease Progression; Drosophila genus; Ensure; Epigenetic Process; Exhibits; Female; Future; Gene Expression; Genome; Germ Cells; Heritability; Histone H4; Histones; Image; Infertility; Inheritance Patterns; Inherited; Label; Laboratories; Ligation; Malignant Neoplasms; Methods; Molecular; Morphology; Muscular Dystrophies; Mutate; Mutation; Neurodegenerative Disorders; Ovary; Pattern; Play; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; RNA interference screen; Regenerative Medicine; Reproductive Biology; Research; Role; Sister Chromatid; Site-Directed Mutagenesis; Specific qualifier value; Stem cells; System; Testing; Testis; Therapeutic; To specify; Work; base; cell behavior; cell type; daughter cell; epigenetic memory; fly; gene function; germline stem cells; human disease; improved; in vivo; innovation; insight; leukemia; male; novel; prevent; public health relevance; segregation; self-renewal; stem cell biology; stem cell division; stem cell fate

 DESCRIPTION (provided by applicant): Epigenetic phenomena create heritable changes in gene expression or function without altering primary DNA sequences. Epigenetic information directs cells with the same genome to take on distinct morphologies and different functions necessary for proper development. Many types of stem cells undergo asymmetric cell divisions to give rise to daughter cells with distinct cell fates: one that retains stem cell identity and another that differentiates. A long-standing question has been how the epigenetic information of a stem cell is transferred to the daughter cells. Using the Drosophila male germline stem cell (GSC) lineage, recent studies in our laboratory have shown that epigenetic information is inherited asymmetrically during asymmetric stem cell division (ACD). My research addresses two fundamental questions raised by this finding. (1) What are the molecular mechanisms used to ensure proper passage of epigenetic information during asymmetric cell division? (2) What other stem cell systems demonstrate asymmetric inheritance of epigenetic information? Using an innovative dual-color histone labelling method, our laboratory has been able to visualize the segregation patterns of the H3 histones during GSC ACD. By selectively labeling preexisting vs. newly synthesized H3, we have been able to demonstrate that preexisting histones preferentially segregate to the daughter cell fated to become the GSC, whereas newly synthesized H3 preferentially segregate to the daughter fated to become the gonialblast (GB). Histone H3.3, which is incorporated in a replication manner, shows a symmetric mode of inheritance. This finding, coupled with the fact that a majority of canonical histones are incorporated during DNA replication, suggests that the process of DNA replication may play a novel role in helping to establish asymmetric histone distribution observed in H3. We hypothesize that certain aspects of replication machinery are involved in retention and deposition of preexisting histones during DNA replication. Using an image-based protein-protein proximity ligation assay (PLA), we will test potential interactions between H3 (or modified H3) and DNA replication machinery components. We will test the functional role of key molecules identified in the PLA using an RNAi screen. Using the dual-color histone labelling method, I will explore the mode of inheritance exhibited by histone proteins H1, H2A, H2B and H4 to better understand what components of the chromatosome may play a part in establishing the epigenetic identity of a cell following ACD. I will also adapt the histone labelling method to labe histone proteins in the GSC of Drosophila ovary to determine if asymmetric histone distribution is present in multiple stem cell systems Improving our understanding of the molecular mechanisms governing epigenetic inheritance in stems cells will have significant implications on the fields of stem cell biology and regenerative medicine.

 描述（由申请人提供）：表观遗传现象在不改变初级DNA序列的情况下产生基因表达或功能的可遗传变化。表观遗传信息指导具有相同基因组的细胞呈现不同的形态和正常发育所需的不同功能。许多类型的干细胞经历不对称的细胞分裂，产生具有不同细胞命运的子细胞：一个保留干细胞身份，另一个分化。一个长期存在的问题是干细胞的表观遗传信息如何转移到子细胞。利用果蝇雄性生殖系干细胞（GSC）谱系，我们实验室最近的研究表明，表观遗传信息在不对称干细胞分裂（ACD）期间是不对称遗传的。我的研究解决了这一发现提出的两个基本问题。(1)在不对称细胞分裂过程中，有哪些分子机制可以确保表观遗传信息的正确传递？(2)还有哪些干细胞系统表现出表观遗传信息的不对称遗传？使用创新的双色组蛋白标记方法，我们的实验室已经能够可视化的分离模式的H3组蛋白在GSC ACD。通过选择性标记预先存在的与新合成的H3，我们已经能够证明预先存在的组蛋白优先分离到注定成为GSC的子细胞，而新合成的H3优先分离到注定成为成角细胞（GB）的子细胞。组蛋白H3.3以复制方式掺入，显示出对称的遗传模式。这一发现，再加上事实上，大多数典型的组蛋白在DNA复制过程中纳入，表明DNA复制的过程可能发挥了新的作用，帮助建立不对称的组蛋白分布观察H3。我们假设复制机制的某些方面参与了DNA复制过程中预先存在的组蛋白的保留和沉积。使用基于图像的蛋白质-蛋白质邻近连接分析（PLA），我们将测试H3（或修饰的H3）和DNA复制机制组件之间的潜在相互作用。我们将使用RNAi筛选来测试在PLA中鉴定的关键分子的功能作用。使用双色组蛋白标记方法，我将探索组蛋白H1，H2 A，H2 B和H4所表现出的遗传模式，以更好地了解哪些成分的染色体可能发挥作用，在建立ACD后细胞的表观遗传特性。我也将适应组蛋白标记方法来标记组蛋白蛋白在GSC的果蝇卵巢，以确定是否不对称的组蛋白分布存在于多个干细胞系统提高我们的理解的分子机制，在干细胞表观遗传将有显着的影响，在干细胞生物学和再生医学领域。