The Role of Asymmetric Histone Inheritance in Establishing Distinct Cell Identities

项目摘要 细胞需要分裂和组织成组织。组织动态平衡的一种方法是利用成体 干细胞能够通过经历不对称细胞分裂(ACD)来取代细胞，以产生更新的干细胞和 分化的细胞。这种分裂的中断可能会导致癌症或组织退化。尽管ACD是一个至关重要的部分 在这个过程中，指定两个独特的细胞身份所涉及的潜在机制是 未知。之前研究ACD在果蝇雄性种系中的表观遗传机制的工作发现，通过 不同地标记新旧组蛋白，旧的H3组蛋白保留在更新的干细胞中，而新的H3组蛋白保留在更新的干细胞中 优先分离到分化细胞。 为了研究组蛋白不对称遗传是否是ACD的重要机制，我将研究组蛋白 果蝇肠道干细胞的遗传。ISCs经历ACD以产生两个分化细胞 类型：肠细胞和肠内分泌细胞。ISCs还能够经历对称细胞分裂(SCD)以产生 两个ISC。ISC谱系是研究组蛋白遗传的理想方法，因为它是高通量的，因为在一个谱系中有许多ISCs 肠道，ISC是谱系中唯一经历有丝分裂的细胞，转基因只能在祖细胞中表达。 细胞。我将利用双色转基因系统来区分标记新旧组蛋白，并跟踪它们的 ISC分部期间的隔离。我的初步数据表明，H3组蛋白在 ISCs的ACD，而H3组蛋白在SCD过程中对称分离。相反，H_2A组蛋白是分离的 ACD和SCD均为对称性。这表明H3对于建立不同的细胞身份可能是重要的。 为了研究组蛋白遗传的细胞和分子机制，我将首先研究旧的和新的组蛋白。 在最近复制的姐妹染色单体中沉积，然后研究有丝分裂过程中姐妹染色单体的分离。我 将使用染色质纤维技术来研究组蛋白在ISCs中DNA复制后的掺入。那我会的 分析识别和分离这些表观遗传上不同的姐妹染色单体的有丝分裂机制的组件 在细胞分裂过程中。此外，我将调查H3组蛋白尾部的突变，此前已发现 在果蝇生殖系中引起异常的组蛋白遗传和细胞缺陷，如肿瘤和组织退化。 在描述了细胞分裂过程中H3遗传的机制之后，我将研究H3遗传是如何 会影响细胞的身份。为了研究这一点，我将研究对细胞特性很重要的基因座上的染色质背景。 以确定是否通过细胞分裂建立和遗传了不同的染色质环境。我也会调查基因 在野生型和突变型H3背景中表达，以确定H3的错误遗传是否导致基因表达异常。 这将有助于理解组蛋白如何影响细胞特性，以及它们在癌症等疾病中可能扮演的角色。 在约翰霍普金斯大学和陈实验室的出色研究和培训将使我能够 完成建议的研究并培养在学术界发展事业所需的技能。

Project Summary Cells are required to divide and organize into tissues. One method of tissue homeostasis is the utilization of adult stem cells that are able to replace cells by undergoing asymmetric cell division (ACD) to produce a renewed stem cell and a differentiating cell. Disruptions to this division can lead to cancers or tissue degeneration. Although ACD is a vital part of tissue homeostasis, the underlying mechanisms involved in specifying two unique cell identities during this process are unknown. Previous work studying the epigenetic mechanisms of ACD in the Drosophila male germline found that, by labeling old and new histones distinctively, old H3 histones are retained in the renewed stem cell, whereas new H3 histones are preferentially segregated to the differentiating cell. To investigate whether asymmetric histone inheritance is an important mechanism of ACD, I will study histone inheritance in the intestinal stem cells (ISCs) of Drosophila. The ISCs undergo ACD to produce two differentiated cell types, enterocyte and enteroendocrine cells. The ISCs are also able to undergo symmetric cell division (SCD) to produce two ISCs. The ISC lineage is ideal to study histone inheritance as it is high throughput as there are many ISCs in one intestine, the ISC is the only cell in the lineage to undergo mitosis, and transgenes can be expressed in only the progenitor cells. I will take advantage of a two-color transgenic system to label old and new histones differentially and track their segregation during ISC division. My preliminary data suggest that H3 histones are segregated asymmetrically during the ACD of ISCs, while H3 histones are segregated symmetrically during SCD. In contrast, H2A histones are segregated symmetrically in both ACD and SCD. This suggests that H3 may be important for establishing different cell identities. To investigate the cellular and molecular mechanisms of histone inheritance, I will first look at old and new histone deposition in recently replicated sister chromatids and then study the segregation of the sister chromatids during mitosis. I will use a chromatin fiber technique to study the incorporation of histones following DNA replication in ISCs. I will then analyze components of the mitotic machinery that recognizes and segregates these epigenetically distinct sister chromatids during cell division. Further, I will investigate a mutation in the tail of the H3 histone, which has been previously found to cause aberrant histone inheritance and cellular defects such as tumors and tissue degeneration in the Drosophila germline. After characterizing the mechanism of H3 inheritance during cell division, I will investigate how H3 inheritance influences cell identity. To study this, I will examine the chromatin context at gene loci that are important for cell identity to determine if different chromatin contexts are established and inherited through cell division. I will also investigate gene expression in a wildtype and mutant H3 background to determine if mis-inheritance of H3 causes aberrant gene expression. This will provide understanding of how histones affect cell identity and the roles they may play in diseases such as cancer. The excellent research and training at Johns Hopkins University and in the Chen laboratory will allow me to both complete the proposed research and develop the skills necessary to pursue a career in academia.