Gene Activation by Remote Transcriptional Enhancers

远程转录增强子激活基因

• 批准号: 6344151
• 负责人: Dale L Dorsett
• 金额: $ 24.57万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6344151
• 关键词: Drosophilidae; cell cycle; chromosomes; developmental genetics; developmental neurobiology; gene deletion mutation; gene expression; gene mutation; genetic enhancer element; genetic mapping; immunoprecipitation; laboratory rabbit; larva; nerve stem cell; neurogenesis; neurogenetics; transcription factor

DESCRIPTION (Verbatim from the applicant's abstract): Enhancers that activate promoters several kilobases away are vital for the appropriate expression of several genes that are required for metazoan development. We discovered the Drosophila Nipped-B protein in a genetic screen for factors that support activation by a distant enhancer in the cut gene. Nipped-B also aids activation of the Ultrabithorax (Ubx) gene by remote enhancers, and participates in sister chromatid cohesion in dividing larval neuroblasts. The goal is to understand how Nipped-B participates in gene activation. Nipped-B is homologous to a human protein of unknown function and to the yeast Scc2 (S. cerevisiae) and Mis4 (S. pombe) proteins required for sister chromatid cohesion. Scc2 is needed for the Cohesin protein complex that holds sister chromatids together to bind to chromosomes. The data indicate that Scc2 and Mis4 bind to chromosomes at all stages of the cell cycle and help recruit Cohesin during S phase. The investigator postulates that Nipped-B acts similarly to aid gene activation by recruiting protein complexes that act on chromosome structure to hold enhancers and promoters close together. Cohesin contains two SMC proteins, which may directly affect DNA and chromosome structure. In the proposed work, mutations in genes encoding SMC proteins or other components of the Cohesin complex will be used to determine if these proteins also participate in activation of cut or Ubx. As a more general approach, proteins that interact with Nipped-B will be isolated biochemically, and then mutations in the genes encoding them will be used to explore their in vivo roles in gene expression and chromatid cohesion. Mutations in Nipped-B and constructed deletion mutants will be used to see if the gene expression and chromatid cohesion functions of the Nipped-B protein are separable. Immunostaining and chromatin immunoprecipitation experiments will be used to determine if Nipped-B protein acts directly at cut and Ubx, or at sites distant from these target genes. These experiments will help define the role of Nipped-B in gene activation and increase understanding of how remote enhancers regulate transcription. This will help illuminate the basic mechanisms underlying gene expression defects that occur in some human diseases.

描述（逐字研究申请人的摘要）：激活的增强剂 启动子几千碱基对于适当的表达至关重要 多唑开发所需的几种基因。我们发现了 果蝇在遗传筛选中的果蝇nipped-b蛋白支持支持的因素 切割基因中远处增强子的激活。 Nipped-B还有助于激活 远程增强剂的Ultrabithorax（UBX）基因，并参与姐妹 在分裂幼虫神经细胞中的染色单体凝聚力。目标是了解 Nipped-B如何参与基因激活。 Nipped-B对人是同源的 功能未知的蛋白质以及酵母SCC2（酿酒酵母）和MIS4的蛋白质（S. 姊妹染色单体内含度所需的POMBE）蛋白质。需要SCC2 将姐妹染色单体固定在一起以结合的粘着蛋白蛋白复合物 染色体。数据表明SCC2和MIS4完全结合染色体 细胞周期的阶段，并有助于在S期募集粘着素。这 研究人员假设Nipped-B的作用类似，以帮助通过 招募对染色体结构作用以保持增强剂的蛋白质复合物 和发起人在一起。粘着蛋白包含两个SMC蛋白，可能 直接影响DNA和染色体结构。在拟议的工作中，突变 在编码SMC蛋白或粘蛋白复合物的其他成分的基因中 用于确定这些蛋白质是否也参与切割的激活或 UBX。作为一种更通用的方法，与Nipped-B相互作用的蛋白质将是 从生化分离，然后在编码它们的基因中突变为 用于探索它们在基因表达和染色单体内聚会中的体内作用。 将使用Nipped-B和构造的缺失突变体中的突变来查看是否是否 Nipped-B蛋白的基因表达和染色单体凝聚功能 可以分离。免疫染色和染色质免疫沉淀实验 将用于确定Nipped-B蛋白是直接在切割和UBX上作用，还是 在远离这些靶基因的位点。这些实验将有助于定义 Nipped-B在基因激活中的作用并增加了对 远程增强器调节转录。这将有助于阐明基本 某些人类中发生的基因表达缺陷的机制 疾病。