De novo establishment of Polycomb-group-mediated repression

描述（由申请人提供）：Polycomb-group （PcG）蛋白是进化上保守的表观遗传转录调控因子。在PcG蛋白最初被募集到靶位点后，它们能够在无限数量的细胞周期中维持转录抑制。然而，pcg介导的抑制是动态的，可以逆转以允许靶基因的表达。哺乳动物PcG蛋白的靶标包括胚胎干细胞中的分化特异性基因和干细胞诱导分化时的多能性基因。PcG蛋白的错误表达导致了多种人类癌症。多能基因的抑制和PcG蛋白的致癌作用都涉及先前活性基因的从头抑制。尽管我们对PcG蛋白在抑制维持阶段的活性了解很多，但对于PcG介导的抑制最初建立的机制，以及PcG蛋白最初如何区分转录抑制和活性靶标，我们几乎一无所知。果蝇PcG蛋白的募集和稳定维持需要被称为Polycomb Response Elements （PREs）的DNA序列的存在，该序列包含多个序列特异性蛋白的结合位点。在哺乳动物中，非编码rna和转录因子被认为可以招募PcG蛋白，尽管最近的证据表明PcG蛋白的基因组分布与未甲基化的CpG岛有更高的相关性。有人提出，哺乳动物未甲基化的CpG岛和果蝇的PREs可能提供类似的染色质环境，有利于PcG蛋白靶向。在拟议的研究中，我们将使用一个已建立的遗传系统来产生PcG靶基因普遍被抑制的胚胎。在建立pcg介导的抑制的发育窗口中，处于离散阶段的胚胎将进行染色质免疫沉淀，以确定蛋白质的时间募集和组蛋白修饰的沉积。提出了两个具体目标：(1)确定序列特异性DNA结合蛋白对靶基因PREs活性的各自贡献；(2)明确导致PcG介导的抑制建立的分子和生化事件，以及PcG蛋白区分被抑制和活性靶基因的机制。这些研究的结果将与胚胎和诱导多能干细胞的建立和操作以及PcG蛋白致癌活性的潜在机制有关。

DESCRIPTION (provided by applicant): Polycomb-group (PcG) proteins are evolutionarily conserved epigenetic transcriptional regulators. Following initial recruitment of PcG proteins to target loci, they are capable of maintaining transcriptional repression through an indefinite number of cell cycles. However, PcG-mediated repression is dynamic and can be reversed to allow the expression of target genes. Targets of mammalian PcG proteins include differentiation-specific genes in embryonic stem cells and pluripotency genes when stem cells are induced to differentiate. Mis-expression of PcG proteins contributes to a wide range of human cancers. Both repression of pluripotency genes and the oncogenic effects of PcG proteins involve de novo repression of previously active genes. Although much is known about the activities of PcG proteins during the maintenance phase of repression, virtually nothing is known about the mechanisms involved in the initial establishment of PcG-mediated repression or how PcG proteins initially distinguish between transcriptionally repressed and active targets. Recruitment and stable maintenance of Drosophila PcG proteins requires the presence of DNA sequences known as Polycomb Response Elements (PREs) that contain binding sites for multiple sequence-specific proteins. In mammals, noncoding RNAs and transcription factors have been proposed to recruit PcG proteins, although recent evidence suggests a higher correlation of the genomic distribution of PcG proteins with unmethylated CpG islands. It has been proposed that mammalian unmethylated CpG islands and Drosophila PREs may provide similar chromatin environments that are conducive to PcG protein targeting. In the proposed studies, we will use an established genetic system to produce embryos in which a PcG target gene is ubiquitously repressed. Embryos at discrete stages during the developmental window when PcG-mediated repression is established will be subjected to chromatin immunoprecipitation to identify the temporal recruitment of proteins and deposition of histone modifications. Two specific aims are proposed: (1) Determine the respective contributions of sequence-specific DNA binding proteins to the activities of a target gene's PREs; (2) Define the molecular and biochemical events that lead to establishment of PcG-mediated repression and the mechanism by which PcG proteins distinguish between repressed and active target genes. The results of these studies will be relevant to the establishment and manipulation of embryonic and induced pluripotent stem cells and to the mechanisms underlying the oncogenic activities of PcG proteins.