Repression via Facultative Heterochromatin

通过兼性异染色质抑制

• 批准号: 10297973
• 负责人: DANNY REINBERG
• 金额: $ 40.26万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10297973
• 关键词: Acute Myelocytic Leukemia; Adult Acute Myeloblastic Leukemia; Affect; Auxins; Base Pairing; Binding; Biological; Biological Assay; Cell Cycle; Cell Cycle Stage; Cell division; Cells; Chromatin; Chromatin Structure; Complex; Cytoplasm; DNA; DNA Methylation; DNA biosynthesis; DNA replication fork; Daughter; Deposition; Disease; Epigenetic Process; Essential Genes; Event; Exhibits; Exons; Family; Foundations; Funding; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Heterochromatin; Histone H3; Histones; Homeobox Genes; Human; In Vitro; Individual; Inherited; Investigation; Karyotype; Label; Lysine; Maintenance; Mammalian Cell; Mediating; Methods; Microscopy; Modification; Molecular; Molecular Chaperones; Monitor; Mus; Mutate; Mutation; Myeloproliferative disease; NPM1 gene; Nucleosomes; Oncogenes; Optics; Polycomb; Post-Translational Protein Processing; Protein Isoforms; Proteins; Proteomics; Reporting; Repression; Role; S Phase; Series; Transcript; Ursidae Family; Writing; acute myeloid leukemia cell; embryonic stem cell; epigenetic regulation; epigenome; gene repression; grasp; in vivo; knock-down; leukemia; leukemogenesis; metaplastic cell transformation; microscopic imaging; mouse model; mutant; nucleophosmin; nucleoplasmin; preservation; protein complex; reconstruction; segregation; single molecule; tissue culture; tumorigenic

Project Summary A critical question in the field of Epigenetics/Mammalian Gene Regulation is how a cellular identity is inherited by progeny cells during cell division. This fundamental aspect of epigenetic regulation was recently clarified in our lab: repressed, but not active, chromatin domains are inherited. Repressed chromatin domains in facultative heterochromatin are maintained by the multi-subunit complex, Polycomb Repressive Complex 2 (PRC2), that catalyzes the histone post-translational modification, H3K27me3. PRC2 exhibits a notable “read and write” feature whereby its recognition of H3K27me3 results in its allosteric activation. Thus, PRC2 can fully restore repressive chromatin domains upon inheritance of H3K27me3-nucleosomes. Remarkably, our findings point to a previously reported histone chaperone, NPM1, as facilitating this inheritance of repressed chromatin: NPM1 is exclusively localized to chromatin in late S-phase when repressed chromatin is replicated, and interacts directly with PRC2. Our latest findings demonstrated specific de-repression of PRC2-regulated genes upon auxin- mediated depletion of NPM1 during S-phase of the cell cycle. We will expand our mechanistic studies of epigenetic inheritance by investigating the role of NPM1 as an S-phase-specific histone chaperone and its interplay with PRC2 in a series of histone chaperone assays performed in vitro with distinct candidate oligonucleosomal templates. We will investigate the role of NPM1 in epigenetic inheritance by adapting our in vivo assay for chromatin domain inheritance as a function of the presence of NPM1 and pertinent NPM1 mutants. The interactive dynamics of NPM1 and PRC2 in the context of a replication fork is critical information towards understanding the transfer of parental nucleosomes to daughter DNA strands. Thus, single-molecule localization microscopy as well as stochastic optical reconstruction microscopy (STORM) are expected to bear directly on the role of NPM1 and the significance of its interaction with the epigenetic regulator, PRC2. Importantly, mutant NPM1c associated with ~35% of all Acute Myelogenous Leukemia (AML) is mis-localized to the cytoplasm. We propose that NPM1c hampers normal PRC2 function. Indeed, similar to our findings above upon NPM1 depletion, known HOX gene targets of PRC2 are aberrantly expressed in NPM1c AML, participating in the establishment of the leukemic state. Deposition of H3K27me3 by PRC2 and DNA methylation by DNMT3A result in repressed chromatin, but are usually mutually exclusive. Yet, NPM1 and DNMT3A mutations synergize in leukemogenesis. Thus, we further propose that DNMT3A partially compensates for our proposed NPM1c- mediated thwarting of PRC2⏤which is lost upon DNMT3A mutation. Through temporal expression of NPM1c as a function of the presence of mutant DNMT3A, we will track the repercussions to gene expression, features of repressed chromatin domains, PRC2 chromatin occupancy and DNA methylation in both tissue culture and a mouse model of AML to fully grasp the sequence of aberrant epigenetic events as they occur in leukemogenesis.

项目摘要 表观遗传学/哺乳动物基因调控领域的一个关键问题是细胞身份是如何遗传的 在细胞分裂过程中由子代细胞。表观遗传调控的这一基本方面最近在 我们的实验室：受抑制但不活跃的染色质结构域是遗传的。兼性抑制染色质结构域 异染色质由多亚单位复合体，多梳抑制复合体2(PRC2)维持 催化组蛋白翻译后修饰，H3K27me3。PRC2展示了值得注意的“读写” 它对H3K27me3的识别导致了它的变构激活。因此，PRC2可以完全恢复 H3K27me3-核小体遗传的抑制染色质结构域。值得注意的是，我们的发现指向 先前报道的一种组蛋白伴侣NPM1促进了这种被抑制的染色质的遗传：NPM1是 当抑制的染色质被复制时，仅定位于S晚期的染色质，并直接相互作用 与PRC2合作。我们的最新发现证明了PRC2调节的基因对生长素的特异性抑制。 S期细胞周期中NPM1的介导耗竭。我们将扩展我们的机制研究 通过研究NPM1作为S相特异性的组蛋白伴侣及其作用的表观遗传 在体外进行的一系列组蛋白伴侣蛋白检测中与PrC2的相互作用 寡核小体模板。我们将通过调整我们的基因来研究NPM1在表观遗传中的作用 NPM1和相关NPM1突变体的存在对染色质区域遗传的活体检测。 在复制分叉的上下文中，NPM1和PRC2的交互动态是关键信息 了解亲代核小体向子代DNA链的转移。因此，单分子定位 显微镜以及随机光学重建显微镜(STORM)预计将直接作用于 NPM1的作用及其与表观遗传调控因子PrC2相互作用的意义。重要的是，变种人 约35%的急性髓系白血病(AML)患者中，NPM1c基因错误定位于细胞质。我们 提出NPM1c阻碍了正常的PRC2功能。事实上，与我们上面关于NPM1的发现类似 耗竭，已知的PRC2的HOX基因靶点在NPM1c AML中异常表达，参与 白血病状态的确立。PRC2沉积H3K27me3和DNMT3A结果DNA甲基化 在被抑制的染色质中，但通常是相互排斥的。然而，NPM1和DNMT3A突变在 白血病的发生。因此，我们进一步建议DNMT3A部分补偿我们提议的NPM1c- 介导抑制因DNMT3A突变而丢失的PrC2DNMT3A⏤。通过NPM1c AS的时间表达 突变DNMT3A的存在的函数，我们将跟踪对基因表达的影响，特征 组织培养和组织培养中的抑制染色质结构域、PRC2染色质占有率和DNA甲基化 建立小鼠急性髓系白血病模型，以充分掌握白血病发生过程中发生的异常表观遗传事件的序列。