Dissecting the roles of Polycomb repressive complex 2 accessory subunits during early developmental transitions

剖析 Polycomb 抑制复合物 2 辅助亚基在早期发育转变过程中的作用

• 批准号: 10441538
• 负责人: Ana Petracovici
• 金额: $ 1.81万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-12-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10441538
• 关键词: Acute; Affect; Auxins; Biochemical; CRISPR/Cas technology; Cell Lineage; Cells; Cellular Morphology; ChIP-seq; Characteristics; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Complex; Data; Defect; Development; Drosophila genus; ES Cell Line; EZH2 gene; Embryo; Embryonic Development; Embryonic Lethal Mutation; Engineering; Epiblast; Epigenetic Process; Excision; Exhibits; Gene Activation; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Transcription; Genomic Segment; Hematologic Neoplasms; Heterochromatin; Histone H3; Histones; Hour; Immunofluorescence Immunologic; Kinetics; Knock-out; Light; Link; Lysine; Malignant Neoplasms; Measures; Mediating; Molecular; Monitor; Mus; Pattern; Phenotype; Play; Polycomb; Process; Proteins; Refractory; Repression; Resolution; Role; Scheme; Structure; Testing; Time; Weaver Syndrome; base; cell type; developmental disease; differentiation protocol; embryonic stem cell; experimental study; gene repression; genetic approach; insight; nerve stem cell; pluripotency; prevent; stem cell differentiation; transcriptome sequencing

Project Summary The objective of this proposal is to determine the distinct molecular roles and temporal requirements of the two subtypes of the Polycomb Repressive Complex 2 (PRC2), PRC2.1 and PRC2.2, during differentiation. PRC2 is an essential chromatin modifier that trimethylates lysine 27 on histone H3 (H3K27me3), which leads to the formation of facultative heterochromatin, a structure that is refractory to transcription and prevents ectopic gene activation. The epigenetic function of PRC2 is essential for embryogenesis, and defects in this complex cause Weaver Syndrome, a congenital multi-system developmental disorder. Despite its ubiquitous expression during development, PRC2 is capable of targeting distinct sets of genes for silencing in different cell lineages, resulting in cell-type specific gene repression. However, the mechanisms regulating the targeting of PRC2 are poorly understood. PRC2 interacts with a variety of accessory subunits, resulting in the formation of two biochemically distinct subcomplexes—PRC2.1 and PRC2.2. Genetic knockouts of subunits comprising PRC2.1 and PRC2.2 in mice result in embryonic lethality at different times and with different phenotypes, demonstrating that the two PRC2 subcomplexes have non-overlapping essential functions in development. Unlike PRC2, the accessory subunits comprising PRC2.1 and PRC2.2 exhibit dynamic expression patterns during development, suggesting they may regulate distinct developmental stages. However, the molecular roles that PRC2.1 and PRC2.2 may play at different stages of development, as well as whether they target different genomic regions for silencing, remain unknown. I will investigate these questions using auxin- inducible degradation (AID) to acutely degrade subunits specific to PRC2.1 and PRC2.2 during the differentiation of mouse embryonic stem cells (mESCs). Because of its rapid kinetics, this approach will allow me to define the immediate molecular consequences of removing the selected subunit, avoiding confounding secondary and compensatory effects that have plagued previous genetic approaches. In Aim 1 I will combine AID with nascent RNA sequencing to determine the functions of PRC2.1 and PRC2.2 in regulating the transcriptional state of pluripotent mESCs. In Aim 2 I will use AID to selectively degrade PRC2.1 and PRC2.2 subunits at different times during the differentiation of mESCs to epiblast-like cells (EpiLCs) and neuronal progenitors (NPCs). I will measure resulting changes in gene expression, chromatin structure, and cell morphology to determine the functions of and temporal requirements for each PRC2 subcomplex at different developmental stages. Collectively, these Aims will shed mechanistic light on the distinct molecular roles of PRC2.1 and PRC2.2 in regulating pluripotency and differentiation.

项目摘要 该提议的目的是确定两者的不同分子角色和临时要求 分化过程中Polycomb抑制复合物2（PRC2），PRC2.1和PRC2.2的亚型。 Prc2是 一种基本的染色质修饰剂，在组蛋白H3（H3K27me3）上对赖氨酸27进行三甲基甲基化，这导致 兼性异染色质的形成，这种结构是对转录难治性并防止生态的结构 基因激活。 PRC2的表观遗传功能对于胚胎发生至关重要，并且在该复合物中的缺陷 导致韦弗综合征，一种先天性多系统发育障碍。尽管无处不在 在开发过程中，PRC2能够靶向不同的基因集以在不同的细胞系中沉默， 导致细胞型特异性基因表达。但是，调查PRC2靶向的机制是 理解不佳。 PRC2与各种附件亚基相互作用，导致两个形成 生化不同的子复合物-prc2.1和prc2.2。完成PRC2.1的亚基的遗传敲除 小鼠中的Prc2.2在不同时间和不同的表型中导致胚胎致死性， 证明这两个PRC2子复合物在开发中具有非重叠的基本功能。 与PRC2不同，配件亚基完成PRC2.1和PRC2.2暴露的动态表达模式 在开发过程中，表明它们可能会调节不同的发展阶段。但是，分子 PRC2.1和PRC2.2的角色可能在开发的不同阶段扮演，以及它们是否针对 沉默的不同基因组区域仍然未知。我将使用生长素调查这些问题 - 在Prc2.1和Prc2.2的急性降解（AID）中诱导降解（AID） 小鼠胚胎干细胞（MESC）的分化。由于其快速动力学，这种方法将允许 我定义去除选定亚基的直接分子后果，避免混淆 困扰先前的遗传方法的次要和补偿作用。在AIM 1中，我将结合 帮助新生的RNA测序确定PRC2.1和PRC2.2在调节中的功能 多能MESC的转录状态。在AIM 2中，我将使用援助有选择地降级Prc2.1和Prc2.2 在MESC分化为层状细胞（EPILC）和神经元的不同时间的亚基 祖先（NPC）。我将测量产生的基因表达，染色质结构和细胞的变化 在不同 发展阶段。总的来说，这些目标将使机械灯阐明 PRC2.1和PRC2.2在调节的丰富性和分化中。