Mechanisms of Gene Silencing

基因沉默的机制

• 批准号: 6636474
• 负责人: Lori L Wallrath
• 金额: $ 24.4万
• 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/6636474
• 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（申请人摘要的逐字记录）：长期目标 这项研究旨在了解基因调控所涉及的机制。 在生命周期中，适当控制基因表达至关重要 生物;表达失调可能导致发育异常 和疾病。异染色质蛋白一 HP1 是一种进化保守蛋白 染色质蛋白是沉默染色质的组成部分。该提案将 重点研究 HP1 介导的常染色质沉默的分子机制 地点。最近的数据表明 HP1 对于沉默基因中的基因至关重要 染色体的常染色质区域。在小鼠中，HP1 已被证明可以相互作用 与与启动子区域相关的转录辅阻遏物 许多基因。在人类中，HP1 的减少与乳腺转移相关 癌细胞，可能是由于失去了沉默所需的基因 转移表型。果蝇提供了无法获得的细胞学和遗传学 在哺乳动物系统中确定 HP1 在调节中的作用 常染色基因。提出的实验将检验以下假设：1) HP1 使异染色质域的形成成核，这些异染色质域沿着 染色体，沉默邻近基因。剖析其机制 异染色质形成和扩散，HP1 将被拴在 256 串联上 常染色质内的位点。遗传、细胞学和生化实验 他将用于确定形成的沉默染色质的特性， 传播的程度，以及招募到的其他因素的身份 地区。结果将与已知的中心异染色质结果进行比较。 2) HP1充当短程转录沉默子。 HP1 将被拴在 8个串联位点战略性地位于启动子区域内 特征明确的基因。转录激活与转录激活之间的平衡 将通过表达和染色质结构分析来探索沉默。这 结果将确定短程沉默机制是否相似 那些负责异染色质介导的沉默的人。 3) HP1 为 对于沉默常染色质基因至关重要。 HP1 调控的常染色质基因 迄今为止尚未在任何生物体中发现。结合细胞学， 染色质免疫沉淀实验和遗传学将用于 鉴定受 HP1 调节的常染色质基因。对这些基因的分析将 允许与 HP1 人工沉默的基因和基因进行比较 异染色质中的 HP1 沉默。