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: 10456597
• 负责人: Jonathan Snedeker
• 金额: $ 2.04万
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10456597
• 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，其分裂以产生自我更新干细胞和分化干细胞。 在多细胞生物体中，子细胞在维持组织稳态中起着至关重要的作用。很好理解的 大多数细胞类型的表观遗传机制调节基因表达，从而控制细胞的命运。然而，它在很大程度上仍然是一个 在成体干细胞的不对称分裂过程中产生的两个子细胞如何继续获得不同的 表观基因组该实验室先前发现，组蛋白是表观遗传信息的关键载体， 在雄性果蝇生殖系干细胞（GSC）的不对称分裂过程中，在这个过程中，老 组蛋白保留在GSC中，而分化细胞继承了新合成的组蛋白，这表明 通路可以维持GSC中的表观遗传信息，同时引发分化的子细胞获得新的 分化过程中的表观遗传信息。最近，我们发现这一过程的机制是由两个- 在DNA复制过程中，组蛋白首先不对称地沉积在姐妹染色单体上， 在有丝分裂期间被区别识别和分离。在复制过程中，这种不对称性主要通过以下方式实现： 在前导链上掺入旧的组蛋白，而滞后链随后掺入新的组蛋白。的发现 不对称组蛋白遗传是由DNA复制驱动的，这开启了DNA复制发挥作用的令人兴奋的可能性。 未被重视的作用。然而，组蛋白在细胞中的精确分子机制 姐妹染色单体上的不对称掺入仍不清楚。 在这里，我提出，在GSC复制过程中，组蛋白掺入的不对称性是由增强固有的 DNA复制的不对称我发现了初步证据表明RPA和滞后链的蛋白质水平 聚合酶DNA Polα和DNA Polδ可能驱动该过程中的不对称性。我建议进一步测试 使用一系列方法来改变这些蛋白质的水平，利用遗传和生物化学来改变这些蛋白质的水平 接近。我计划使用染色质纤维的超分辨率成像来读出这些操作的效果， 复制偶联核小体组装的单分子分辨率。此外，我提出了一个新的假设， 这些蛋白质水平的改变可能通过将滞后链合成与组蛋白遗传不对称性分离而驱动组蛋白遗传不对称。 复制叉进展。为了明确地测试这个模型，我计划将染色质纤维与生物化学物质结合起来， 操纵细胞周期进程，以直接测量前叉进程后的复制时间， 落后股最后，使用基于测序的方法，我将分析组蛋白掺入是否也显示局部的 基因组中的差异。这项研究的结果不仅大大提高了我们对 DNA复制和表观遗传学，但也可以提供一个全新的框架来思考干细胞生物学和细胞 命运规格