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

项目概要 多梳族（PcG）蛋白在维持正常基因沉默的表观遗传记忆中发挥着关键作用 发展与人类疾病。 PcG 蛋白在两种酶促多亚基复合物中发挥作用：Polycomb 抑制复合体 1 和 2（PRC1 和 2）。 PRC1 将单泛素沉积到组蛋白 H2A (H2Aub) 上的赖氨酸 119 上 而 PRC2 则甲基化组蛋白 H3 (H3K27me1/2/3) 上赖氨酸 27 的所有状态。 Polycomb 域是怎样的 在哺乳动物植入前发育过程中重新编程的情况仍然很大程度上未知。最新进展 低输入表观基因组分析技术使得研究哺乳动物染色质动态成为可能 植入前胚胎。发现卵母细胞中的非典型 H3K27me3 可以介导种系 DNA 不依赖甲基化的基因组印记引发了有关 Polycomb 结构域的几个重要问题 早期发展的监管。例如，H2Aub 是否遵循与 H3K27me3，PRC1/2在调节3D染色质中的作用是什么，PRC1/2是否形成正反馈 环在植入前发育过程中相互加强。为了解决这些问题，我生成了 初步数据表明，与传统观点相反，H2Aub 和 H3K27me3 在很大程度上是共同的 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 沉积的 H2Aub 独立于 PRC2， 表明在此发育窗口期间 PRC1 比 PRC2 发挥更重要的作用。建立在意想不到的基础上 观察，我建议结合使用低输入表观基因组学、生物信息学和快速蛋白质 降解方法来了解 PRC1/2 以及染色质的机制和功能 它们分别在小鼠植入前发育中沉积的修饰。在目标1（K99阶段）中，我将 确定受精后 H2Aub 和 H3K27me3 不同重编程动态的潜在机制。 在目标 2（K99 阶段）中，我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 Drs 的培训。张毅（波士顿儿童医院 医院/哈佛医学院）、Peter Park（哈佛医学院）和 Bin Gu（密歇根州立大学） 建立低输入表观基因组分析工具、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）中，我将利用技术和计算 K99 阶段建立的管道，用于研究变异 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独立 调查员。总的来说，完成这些目标将揭示 PcG 招募的机制，定义 PcG 介导的基因沉默在小鼠植入前发育中的作用，并揭示新的范例 关于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.