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(逐字摘自申请者摘要)：的长期目标 本研究旨在了解基因调控的机制。 在人的一生中，适当地控制基因的表达是必不可少的 生物体；表达失调可导致发育异常 和疾病。异染色质蛋白1，hp1，是一种进化保守的 染色质蛋白质，是沉默染色质的一种成分。这项提议将 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对于沉默体内的基因是必不可少的 染色体的常染色质区。在小鼠身上，HP1已经被证明可以相互作用 与转录辅阻遏子相关联的启动子区域 很多基因。在人类中，HP1的减少与乳房转移有关 癌细胞，可能是由于失去了沉默所需的基因 转移表型。果蝇提供了细胞学和遗传学所不具备的 在哺乳动物系统中确定HP1在调节HP1中的作用 常色基因。提出的实验将检验以下假设：1) HP1使异色区域的形成成核，这些异色结构域沿着 染色体，使邻近基因沉默。剖析……的机制 异染色质的形成和扩散，HP1将被拴在256个串联 常染色质内的位置。遗传学、细胞学和生物化学实验 他将用来确定形成的沉默染色质的性质， 传播程度，以及招募到的其他因素的身份 区域。结果将与中心性异染色质的已知结果进行比较。 2)HP1是一种短程转录沉默因子。HP1将被拴在 8个串联位点战略性地位于 特征明确的基因。转录激活和转录激活之间的平衡 沉默将通过表达和染色质结构分析来探索。这个 结果将决定短程沉默的机制是否相似 那些负责异染色质介导的沉默的人。3)HP1为 对使常着色质基因沉默是必不可少的。HP1调控的常着色性基因 到目前为止还没有在任何生物体中发现。细胞学的结合， 染色质免疫沉淀实验，并将遗传学用于 确定HP1调控的常着色性基因。对这些基因的分析将 允许与由HP1人工沉默的基因进行比较，以及基因 在异染色质中被HP1沉默。