Mechanisms of Gene Silencing

基因沉默的机制

• 批准号: 6323802
• 负责人: Lori L Wallrath
• 金额: $ 26.75万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6323802
• 关键词: Drosophilidae; alternatives to animals in research; biochemistry; chromatin; cytology; gene induction /repression; genetic promoter element; genetic regulation; genetic transcription; heterochromatin; immunofluorescence technique; immunoprecipitation; in situ hybridization; molecular genetics; molecular site; northern blottings; transfection; western blottings

DESCRIPTION (Verbatim from the applicant's abstract): The long-term goal of this research is to understand the mechanisms involved in gene regulation. Proper control of gene expression is essential during the life span of an organism; misregulation of expression can lead to developmental abnormalities and disease. Heterochromatin protein one, HP1, is an evolutionary conserved chromatin protein that is a component of silent chromatin. This proposal will focus on the molecular mechanisms of HP1 -mediated silencing at euchromatic locations. Recent data suggest that HP1 is essential for silencing genes within euchromatic regions of chromosomes. In mice, HP1 has been shown to interact with transcriptional corepressors that associate with the promoter regions of many genes. In humans, a reduction in HP1 correlates with metastasis of breast cancer cells, possibly due to the loss of silencing of genes required for the metastatic phenotype. Drosophila offers the cytology and genetics not available in mammalian systems to determine the role of HP1 in the regulation of euchromatic genes. Experiments proposed will test the following hypotheses: 1) HP1 nucleates the formation of heterochromatic domains that spread along the chromosome, silencing neighboring genes. To dissect the mechanisms of heterochromatin formation and spreading, HP1 will be tethered to 256 tandem sites within euchromatin. Genetic, cytological, and biochemical experiments will he used to determine the properties of the silent chromatin formed, the extent of spreading, and the identity of additional factors recruited to the region. Results will be compared with those known for centric heterochromatin. 2) HP1 acts as a short-range transcriptional silencer. HP1 will be tethered to 8 tandem sites strategically located within promoter regions of well-characterized genes. The balance between transcriptional activation and silencing will be explored by expression and chromatin structural analyses. The results will determine whether mechanisms of short-range silencing are similar to those responsible for heterochromatin mediated silencing. 3) HP1 is essential for silencing euchromatic genes. Euchromatic genes regulated by HP1 have not been identified in any organism to date. A combination of cytology, chromatin immunoprecipitation experiments, and genetics will be used to identify euchromatic genes regulated by HP1. An analysis of these genes will allow for comparisons with genes artificially silenced by HP1, and genes silenced by HP1 in heterochromatin.

描述（逐字研究申请人的摘要）： 这项研究是了解基因调节所涉及的机制。 适当控制基因表达是必不可少的 生物;表达的正调可能导致发育异常 和疾病。异染色质蛋白ONE HP1是一种进化保守的 染色质蛋白是静音染色质的组成部分。该提议将 专注于HP1介导的沉默的分子机制 位置。最近的数据表明，HP1对于沉默基因至关重要 染色体的圣灵区域。在小鼠中，HP1已被证明是相互作用的 带有与启动子区域相关的转录核心器 许多基因。在人类中，HP1的降低与乳房转移相关 癌细胞，可能是由于基因所需的基因的沉默丧失所致 转移表型。果蝇提供的细胞学和遗传学不可用 在哺乳动物系统中，确定HP1在调节中的作用 正式基因。提出的实验将检验以下假设：1） HP1核定沿着 染色体，沉默的相邻基因。剖析机制 异染色质形成和扩散，HP1将被束缚至256串联 菌膜中的位置。遗传，细胞学和生化实验 他将用来确定形成静音染色质的特性， 扩散程度以及招募到其他因素的身份 地区。结果将与中心异染色质已知的结果进行比较。 2）HP1充当短距离转录消音器。 HP1将束缚在 8个串联站点在战略上位于发起人区域内 特征良好的基因。转录激活与 沉默将通过表达和染色质结构分析来探索。这 结果将确定短程沉默的机制是否相似 给负责异染色质介导的沉默的人。 3）HP1是 对沉默的圣体基因必不可少的。由HP1调节的光子基因 迄今为止尚未在任何生物体中鉴定出来。细胞学的结合， 染色质免疫沉淀实验和遗传学将用于 识别由HP1调节的正式基因。对这些基因的分析将 允许与人工沉默的基因和基因进行比较 在异染色质中被HP1沉默。