De novo establishment of Polycomb-group-mediated repression

描述(由申请人提供)：聚梳组(PcG)蛋白组成一个表观遗传系统，维持目标基因的转录抑制。最初被鉴定为果蝇HOX基因的负调控因子，其功能和分子/生化活性从昆虫到哺乳动物都高度保守。哺乳动物的PcG同源基因需要维持干细胞的多能状态，而它们的错误表达有助于肿瘤的发生。PCG蛋白并不启动转录抑制，而是取代了基因特异性转录因子的抑制。一旦PcG介导的抑制被建立，它就能够在众多的细胞周期中稳定地和遗传地维持基因沉默。在PcG介导的抑制的维持阶段，DNA结合的PcG蛋白(例如多态同源蛋白)与多梳状反应元件(Pres)内的序列结合，并招募额外的PcG蛋白复合体，如PrC2，后者反过来在赖氨酸27(H3K27me3)甲基化组蛋白H3，促进Prc1的招募。对于PcG蛋白最初识别靶基因抑制状态的机制以及导致PcG介导的抑制建立的分子步骤，人们几乎一无所知。在很大程度上，这是由于在PcG介导的抑制建立的发育阶段，目的基因在胚胎中的异质性表达。在拟议的研究中，基因操作将被用来获得合体胚胎期和细胞胚胚期胚胎，其中PcG靶基因在合胞胚胚期普遍受到母体表达的转录因子的抑制，并且PcG需要维持其在细胞胚胚期的普遍抑制。提出了两个特定的目标：(1)定位PcG靶基因调控区域内的PreS的位置；(2)确定当PcG介导的抑制建立时，调控蛋白(包括PcG蛋白)在PcG靶基因上的物理和时间分布。这些研究的目的是深入了解从最初的转录因子介导的抑制到PcG介导的转录沉默的转变所涉及的分子和生化步骤。这些结果将与iPS细胞的产生和肿瘤的发生特别相关，这两者都涉及PcG介导的抑制的从头建立。 与公共健康相关：多梳状蛋白是高度保守的基因表达负调控因子，在从果蝇到人类的各种生物中，它维持着许多发育重要基因的沉默。多梳状蛋白的过度表达导致了一系列人类癌症，包括转移性前列腺癌和侵袭性乳腺癌以及血液病和上皮性癌。拟议的研究将为初步建立多梳基团介导的抑制的分子机制提供更多的洞察力，这可能导致开发抑制其活性、阻断其致癌作用的治疗方法。

DESCRIPTION (provided by applicant): Polycomb-group (PcG) proteins comprise an epigenetic system that maintains the transcriptional repression of target genes. Originally identified as negative regulators of Drosophila Hox genes, their functions and molecular/biochemical activities have been highly conserved from insects to mammals. Mammalian PcG orthologs are required to maintain the pluripotent state of stem cells and their misexpression contributes to oncogenesis. PcG proteins do not initiate transcriptional repression, but rather take over repression from gene-specific transcription factors. Once PcG- mediated repression is established, it is then capable of stably and heritably maintaining gene silence through numerous cell cycles. During the maintenance phase of PcG-mediated repression, DNA binding PcG proteins (e.g., Pleiohomeotic) bind to sequences within Polycomb Response Elements (PREs) and recruit additional PcG protein complexes, such as PRC2, which in turn methylates histone H3 at lysine 27 (H3K27me3), facilitating recruitment of PRC1. Virtually nothing is known about the mechanisms by which PcG proteins initially recognize the repressed state of target genes and the molecular steps that lead to establishment of PcG-mediated repression. In large part, this is due to the heterogeneous expression of target genes within embryos during the developmental stages when PcG-mediated repression is established. In the proposed studies, genetic manipulation will be used to obtain syncytial and cellular blastoderm stage embryos in which a PcG target gene is ubiquitously repressed at the syncytial blastoderm stage by a maternally expressed transcription factor, and in which the PcG is required to maintain its ubiquitous repression by the cellular blastoderm stage. Two specific aims are proposed: (1) Map the locations of PREs within the regulatory region of the PcG target gene; (2) Define the physical and temporal distributions of regulatory proteins, including PcG proteins, at the PcG target gene during the developmental window when PcG-mediated repression is established. The goal of these studies is to provide insight into the molecular and biochemical steps involved in the transition from initial transcription factor-mediated repression to PcG-mediated maintenance of transcriptional silence. These results will be particularly relevant to the generation of iPS cells and oncogenesis, both of which involve de novo establishment of PcG-mediated repression. PUBLIC HEALTH RELEVANCE: Polycomb-group proteins are highly conserved negative regulators of gene expression, which maintain the silence of numerous developmentally important genes in organisms ranging from fruit flies to humans. Overexpression of Polycomb-group proteins contributes to a range of human cancers, including metastatic prostate and aggressive breast cancers as well as hematologic and epithelial cancers. The proposed studies will provide greater insight into the molecular mechanisms by which Polycomb-group- mediated repression is initially established, which may lead to the development of therapies that inhibit their activities, blocking their oncogenic effects.