Polycomb-Group Genes and Gene Regulation

描述(申请人提供)：拟议研究的长期目标是了解果蝇多梳组(PcG)蛋白维持目标基因转录沉默的分子机制。PcG蛋白最初被认为是触角线虫和双胸复合体同源异型基因的负调控因子，在植物、蠕虫和哺乳动物中保守，在细胞决定和记忆细胞命运方面发挥重要作用。PCG蛋白不会启动对靶基因的抑制。相反，在被短暂的转录因子最初抑制后，PcG蛋白识别抑制状态并建立抑制染色质结构域，该结构域在许多细胞周期中保持沉默。大多数PcG蛋白作为多聚体复合体的组成部分发挥作用。已经描述了两个PcG络合物的多肽组成，还鉴定了其他络合物，但尚未纯化。在有丝分裂后，可遗传地维持转录沉默需要重复和忠实地招募PcG复合体到靶基因，在拟议的研究中，我们将研究序列特异性DNA结合PcG蛋白在PcG复合体募集到增殖细胞靶点中的作用。依赖于PCG的靶基因沉默涉及染色质结构的改变。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蛋白在目标位置的组装，以及单个PcG蛋白和蛋白复合体对染色质修饰的特异性影响。利用这种方法，已经鉴定了一种新的PcG蛋白的活性。将进行反向遗传学研究，以检查其在体内的功能。将使用传统的层析方法对以前未表征的PcG络合物进行生化鉴定。它的组成多肽的鉴定将使用质谱学。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.