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

项目摘要 Polycomb group（PcG）蛋白在维持正常人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物中起作用：Polycomb 抑制复合物1和2（PRC 1和2）。PRC1将monoubiquitin沉积到组蛋白H2A（H2Aub）上的赖氨酸119上 而PRC2甲基化组蛋白H3上赖氨酸27的所有状态（H3K27me1/2/3）。Polycomb domain是什么 在哺乳动物植入前发育过程中的重编程在很大程度上仍然未知。研究进展 低输入的表观基因组分析技术使研究哺乳动物的染色质动力学成为可能 植入前胚胎发现卵母细胞中的非典型H3K27me3可介导生殖系DNA 非甲基化依赖的基因组印迹引起了关于Polycomb结构域的几个重要问题 在早期发展中的监管。例如，H2Aub是否遵循类似的重编程动态， H3K27me3，PRC1/2在3D染色质调控中的作用，PRC1/2是否形成正反馈 在着床前发育过程中相互加强。为了解决这些问题，我已经生成了 初步数据显示，与H2Aub和H3K27me3主要是共的传统观点相反， H2Aub和H3K27me3在受精后经历全基因组不同的重编程动态。在 此外，在卵母细胞和植入前胚胎中，PRC1的H2Aub沉积不依赖于PRC2， 这表明在该发育窗口期间PRC1比PRC2起更关键的作用。建立在意想不到的 观察，我建议使用低投入表观基因组学，生物信息学和快速蛋白质 降解的方法来了解PRC1/2以及染色质的机制和功能 修饰，它们分别存款在小鼠植入前发育。在目标1（K99阶段），我将 确定受精后H2Aub和H3K27me3不同重编程动力学的机制。 在Aim 2（K99期），我将快速降解合子中的PRC1，以评估其对合子基因组激活的影响， PRC2募集和3D染色质结构。我将接受张毅博士（波士顿儿童医院）的培训 医院/哈佛医学院）、Peter Park（哈佛医学院）和Bin Gu（密歇根州立大学） 建立低投入的表观基因组分析工具、生化分析、计算管道和快速的 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阶段建立的管道，以研究变体PRC1亚复合物在植入前的作用 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，并过渡到一个独立的 调查员总的来说，完成这些目标将揭示PcG招募的机制， PcG介导基因沉默在小鼠着床前发育中的作用，并揭示了新的范例 在哺乳动物配子到胚胎的转变过程中染色质重编程。

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.