Investigating the molecular mechanisms of asymmetric histone incorporation during DNA replication

研究 DNA 复制过程中不对称组蛋白掺入的分子机制

• 批准号: 10314925
• 负责人: Jonathan Snedeker
• 金额: $ 4.6万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314925
• 关键词: Affect; Biochemical; Biological Assay; Biology; Cell Cycle Progression; Cell Differentiation process; Cell Lineage; Cell division; Cells; Chromatin; Chromatin Fiber; Clustered Regularly Interspaced Short Palindromic Repeats; Coupled; DNA; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Deposition; Development; Drosophila genus; Epigenetic Process; Event; Gene Expression; Genetic; Genome; Genomic Segment; Genomic approach; Germ Cells; Histones; Homeostasis; Image; Inheritance Patterns; Inherited; Knock-in; Level of Evidence; Light; Measures; Mediating; Methods; Mitosis; Mitotic; Modeling; Molecular; Nucleosomes; Organism; Pathway interactions; Pattern; Play; Polymerase; Positioning Attribute; Process; Proteins; Recycling; Replication-Associated Process; Reporting; Resolution; Role; Series; Sister; Sister Chromatid; Specific qualifier value; Stains; Testing; Time; Tissues; adult stem cell; base; cell fate specification; cell type; daughter cell; epigenome; experimental study; germline stem cells; histone modification; improved; male; novel; nucleoside analog; overexpression; segregation; self-renewal; single molecule; small molecule; spatiotemporal; stem cell biology; stem cells

Asymmetrically dividing adult stem cells, which divide to create both a self-renewing stem cell and a differentiating daughter cell, play a crucial role in maintaining tissue homeostasis in multicellular organisms. It is well understood in most cell types that epigenetic mechanisms regulate gene expression and thereby govern cell fate. Yet, it largely remains a mystery how the two daughter cells generated during the asymmetric division of adult stem cells go on to acquire different epigenomes. The lab previously discovered that histones, key carriers of epigenetic information, are segregated asymmetrically during the asymmetric division of male Drosophila Germline Stem Cells (GSC). In this process old histones are retained in the GSC while the differentiating cell inherits newly synthesized histones, suggesting that this pathway could maintain epigenetic information in the GSC while priming the differentiating daughter cell to acquire new epigenetic information during differentiation. Recently, we found that this process is mechanistically underlied by a two- step process in which histones are first asymmetrically deposited on sister chromatids during DNA replication before being differentially recognized and segregated during mitosis. During replication this asymmetry is primarily achieved by incorporating old histones on the leading strand while the lagging strand later incorporates new histones. The finding that asymmetric histone inheritance is driven by DNA replication opens the exciting possibility that DNA replication plays unappreciated roles in patterning cell fate. However, the precise molecular mechanism by which histones are asymmetrically incorporated on sister chromatids remains unclear. Here I propose that the asymmetry in histone incorporation during replication in GSCs is driven by enhancing the inherent asymmetry of DNA replication. I have found preliminary evidence that protein levels of RPA and lagging strand polymerases DNA Polα and DNA Polδ may drive the asymmetry in this process. I propose testing further manipulating the levels of these proteins using a series of approaches to alter the levels of these proteins using genetic and biochemical approaches. I plan to read out the effects of these manipulations using superresolution imaging of chromatin fibers to gain single molecule resolution of replication coupled nucleosome assembly. Further, I propose the novel hypothesis that the altered levels of these proteins may drive the histone inheritance asymmetry by decoupling lagging strand synthesis from replication fork progression. To explicitly test this model I plan to couple chromatin fibers with biochemical manipulations of cell cycle progression to directly measure replication timing after fork progression for the leading and lagging strand. Finally, using a sequencing-based approach I will assay whether histone incorporation also displays local differences throughout the genome. The results of this study stand to dramatically improve our understanding not only of DNA replication and epigenetics but could also provide a whole new framework to think about stem cell biology and cell fate specification.

不对称分裂的成体干细胞，分裂产生自我更新的干细胞和分化的干细胞