Genomic and functional analyses of Polycomb group proteins in mouse preimplantation development

项目摘要 PcG蛋白在维持正常人群基因沉默的表观遗传记忆中起关键作用 发展和人类疾病。PCG蛋白在两种酶促多亚基复合体中发挥作用：Polycomb 抑制复合体1和2(Prc1和2)。Prc1在组蛋白H_2A(H_2Aub)上将单倍半乳糖沉积为赖氨酸119 而PrC2使组蛋白H3上赖氨酸27的所有状态甲基化(H3K27me1/2/3)。多梳域是怎样的 哺乳动物着床前发育过程中的重新编程在很大程度上仍不清楚。最近几年的研究进展 低投入的表观基因组图谱技术使研究哺乳动物染色质动力学成为可能 植入前胚胎。卵母细胞中非规范H3K27me3可介导生殖系DNA的发现 甲基化无关的基因组印迹在多梳结构域上提出了几个重要的问题 处于早期发展阶段的监管。例如，H2 Aub是否遵循类似的重新编程动态 H3K27me3，Prc1/2在调节3D染色质中起什么作用，以及Prc1/2是否形成正反馈 在植入前发育过程中相互加强的环。为了解决这些问题，我生成了 初步数据显示，与传统观点不同，H_2Aub和H_3K27me3在很大程度上是协同作用的 定位的H_2Aub和H_3K27me3在受精后经历了全基因组不同的重编程动态。在……里面 此外，在卵母细胞和植入前胚胎中，PRC1的H_2Aub沉积不依赖于PRC2， 这表明在这一发育窗口中，PRC1比PRC2具有更重要的作用。建立在意想不到的基础上 观察，我建议使用低投入的表观基因组学、生物信息学和快速蛋白质的组合 了解Prc1/2和染色质机制和功能的降解方法 它们分别在小鼠着床前发育过程中沉积修饰。在目标1(K99阶段)中，我将 确定受精后H_2Aub和H_3K27me3不同重编程动态的机制。 在目标2(K99期)中，I将快速降解受精卵中的Prc1，以评估其对受精卵基因组激活的影响， PRC2募集和3D染色质结构。我将接受波士顿儿童队张毅博士的培训 医院/哈佛医学院)、彼得·帕克(哈佛医学院)和宾古(密歇根州立大学) 建立低投入的表观基因组图谱分析工具、生化分析、计算管道和快速 蛋白质降解技术。在目标3(R00阶段)，我将利用技术和计算 在K99期建立管道来研究不同的PRC1亚复合体在植入前的作用 发展。NIH K99/R00独立之路奖，以及杰出的研究 BCH/HMS的环境将有助于我完成拟议的工作并过渡到独立的 调查员。总而言之，完成这些目标将揭示PcG招募的基本机制，定义 PcG介导的基因沉默在小鼠着床前发育中的作用和发现新的范式 哺乳动物配子到胚胎转变过程中染色质重编程的研究。

Project Summary Polycomb group (PcG) proteins play critical roles in maintaining epigenetic memory of gene silencing in normal development and human diseases. PcG proteins function in two enzymatic multi-subunit complexes: Polycomb repressive complex 1 and 2 (PRC1 and 2). PRC1 deposits monoubiquitin to lysine 119 on histone H2A (H2Aub) whereas PRC2 methylates all states of lysine 27 on histone H3 (H3K27me1/2/3). How Polycomb domains are reprogrammed during mammalian preimplantation development remains largely unknown. Recent advances on low input epigenomic profiling techniques make it feasible to investigate chromatin dynamics in mammalian preimplantation embryos. The discovery that non-canonical H3K27me3 in oocytes can mediate germline DNA methylation-independent genomic imprinting has raised several important questions on Polycomb domain regulation in early development. For example, whether H2Aub follows a similar reprogramming dynamic as H3K27me3, what’s the role of PRC1/2 in regulating 3D chromatin, and whether PRC1/2 form a positive feedback loop to reinforce each other during preimplantation development. To address these questions, I have generated preliminary data showing that, in contrast to conventional view that H2Aub and H3K27me3 are largely co- localized, H2Aub and H3K27me3 undergo genome-wide distinct reprogramming dynamics after fertilization. In addition, H2Aub deposition by PRC1 is independent of PRC2 in oocytes and preimplantation embryos, suggesting a more critical role of PRC1 than PRC2 during this developmental window. Built on the unexpected observations, I propose to use a combination of low input epigenomics, bioinformatics, and rapid protein degradation approach to understand mechanisms and functions of PRC1/2 as well as the chromatin modifications they respectively deposit in mouse preimplantation development. In Aim 1 (K99 phase), I will identify mechanisms underlying the distinct reprogramming dynamics of H2Aub and H3K27me3 after fertilization. In Aim 2 (K99 phase), I will rapidly degrade PRC1 in zygotes to assess its impact on zygotic genome activation, PRC2 recruitment, and 3D chromatin structures. I will be trained by Drs. Yi Zhang (Boston Children’s Hospital/Harvard Medical School), Peter Park (Harvard Medical School), and Bin Gu (Michigan State University) to establish low input epigenomic profiling tools, biochemical assays, computational pipelines, and the rapid protein degradation technique. In Aim 3 (R00 phase), I will take advantage of the techniques and computational pipelines established during the K99 phase to study the role of variant PRC1 subcomplexes in preimplantation development. The NIH K99/R00 Pathway to Independence Award, together with the outstanding research environment at BCH/HMS will facilitate my completion of the proposed work and transition to an independent investigator. Collectively, completion of these aims will reveal mechanisms underlying PcG recruitment, define the role of PcG-mediated gene silencing in mouse preimplantation development, and uncover new paradigms on chromatin reprogramming during mammalian gamete-to-embryo transition.