Polycomb-Group Genes and Gene Regulation

描述（由申请人提供）：拟议研究的长期目标是了解果蝇Polycomb-group （PcG）蛋白维持靶基因转录沉默的分子机制。PcG蛋白最初被认为是天线虫和双胸虫复合物同源基因的负调控因子，在植物、蠕虫和哺乳动物中是保守的，在细胞决定和记忆细胞命运中起重要作用。PcG蛋白不启动靶基因的抑制。相反，在短寿命转录因子的初始抑制之后，PcG蛋白识别抑制状态并建立抑制染色质结构域，在许多细胞周期中遗传性地保持沉默。大多数PcG蛋白作为多聚体复合物的组分起作用。两种PcG复合物的多肽组成已被描述，其他复合物已被鉴定，但尚未纯化。在有丝分裂后，转录沉默的可遗传维持需要PcG复合物重复和忠实地募集到靶基因，在拟议的研究中，我们将研究序列特异性DNA结合PcG蛋白在增殖细胞中PcG复合物募集到靶位点中的作用。靶基因的pcg依赖性沉默涉及染色质结构的修饰。我们将使用RNA干扰（RNAi）和染色质免疫沉淀（X-ChIP）技术来检测PcG蛋白在靶位点的组装以及单个PcG蛋白和蛋白质复合物对染色质修饰的特定作用。利用这种方法，鉴定了一种新的PcG蛋白的活性。将进行反向遗传研究以检验其体内功能。以前未表征的PcG复合物将使用传统的色谱方法进行生物化学鉴定。其组成多肽的身份将用质谱法确定。从这些互补的实验方法中获得的信息将有助于更好地理解PcG蛋白维持靶基因转录沉默的分子机制。

DESCRIPTION (provided by applicant): The long-term goal of the proposed studies is to understand the molecular mechanisms by which the Drosophila Polycomb-group (PcG) proteins maintain the transcriptional silence of target genes. Originally identified as negative regulators of the homeotic genes of the Antennapedia and bithorax gone complexes, PcG proteins are conserved in plants, worms and mammals and play important roles in cellular determination and memorization of cell fate. PcG proteins do not initiate the repression of target genes. Rather, following initial repression by short-lived transcription factors, PcG proteins recognize the repressed state and establish repressive chromatin domains that heritably maintain silence through many cell cycles. Most PcG proteins function as components of multimeric complexes. The polypeptide compositions of two PcG complexes have been described, and other complexes have been identified, but not yet purified. Heritable maintenance of transcriptional silence requires repeated and faithful recruitment of PcG complexes to target genes following mitosis, in the proposed studies, we will examine the role of a sequence-specific DNA binding PcG protein in recruitment of PcG complexes to target sites in proliferating cells. PcG-dependent silencing of target genes involves modification of chromatin structure. We will use RNA interference (RNAi) and chromatin immunoprecipitation (X-ChIP) techniques to examine the assembly of PcG proteins at target sites and the specific effects of individual PcG proteins and protein complexes on chromatin modification. Using this approach, the activity of a novel PcG protein has been identified. Reverse genetic studies will be performed to examine its in vivo function. A previously uncharacterized PcG complex will be biochemically identified using traditional chromatographic methods. The identity of its constituent polypeptides will be determined using mass spectrometry. Information gained from these complimentary experimental approaches will lead to better understanding of the molecular mechanisms by which PcG proteins maintain the transcriptional silence of target genes.