Polycomb-Group Genes and Gene Regulation

描述（由申请方提供）：拟定研究的长期目标是了解果蝇多梳组（PcG）蛋白维持靶基因转录沉默的分子机制。PcG蛋白最初被鉴定为双胸虫和双足虫复合体的同源异型基因的负调控因子，在植物、蠕虫和哺乳动物中是保守的，并且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阻遏。相反，在短暂的转录因子的初始抑制后，PcG蛋白识别被抑制的状态并建立抑制性染色质结构域，其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。已经描述了两种PcG复合物的多肽组成，并且已经鉴定了其他复合物，但尚未纯化。转录沉默的遗传性维持需要重复和忠实的招募PcG复合物的靶基因后，有丝分裂，在拟议的研究中，我们将检查的序列特异性DNA结合PcG蛋白在招募PcG复合物的增殖细胞中的靶位点的作用。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）技术来研究PcG蛋白在靶位点的组装以及单个PcG蛋白和蛋白复合物对染色质修饰的特定影响。使用这种方法，一种新的PcG蛋白的活性已被确定。将进行反向遗传研究以检查其体内功能。一个以前未表征的PcG复合物将使用传统的色谱方法进行生化鉴定。将使用质谱法确定其组成多肽的身份。从这些互补的实验方法获得的信息将导致更好地了解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.