Gene Activation by Remote Transcriptional Enhancers

描述(逐字摘自申请者摘要)：激活的增强剂 几千碱基之外的启动子对于正确表达 后生动物发育所需的几个基因。我们发现了 果蝇在基因筛查中夹住-B蛋白以寻找支持 被Cut基因中的一个远程增强子激活。NIPP-B也有助于激活 通过远程增强子获得Ubx基因，并参与姐妹 幼虫神经母细胞分裂过程中的染色单体凝聚。我们的目标是了解 NIPD-B如何参与基因激活。NIPP-B与人类同源 功能未知的蛋白质和酵母Scc2(S.cerevisiae)和Mis4(S. Pombe)是姐妹染色单体凝聚所必需的蛋白质。需要Scc2才能 粘附素蛋白复合体，将姐妹染色单体结合在一起 染色体。这些数据表明Scc2和Mis4完全与染色体结合 在细胞周期的各个阶段，并帮助在S阶段招募粘附素。这个 研究人员推测，NIPT-B通过以下方式发挥类似的作用来帮助基因激活 招募作用于染色体结构的蛋白质复合体以保持增强剂 推动者和推动者关系密切。粘附素含有两种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蛋白或粘附素复合体的其他成分的基因中 用来确定这些蛋白是否也参与了Cut或 UBX。作为一种更普遍的方法，与NIPED-B相互作用的蛋白质将是 通过生化分离，然后编码它们的基因突变将被 用于探索它们在体内对基因表达和染色单体凝聚力的作用。 NIPED-B的突变和构建的缺失突变体将被用来观察 NIPT-B蛋白的基因表达及染色单体凝聚功能 是可以分开的。免疫染色和染色质免疫沉淀实验 将用于确定NIPP-B蛋白是否直接作用于CUT和UBX，或者 在远离这些目标基因的位置。这些实验将有助于确定 NIPD-B在基因激活中的作用以及如何增加对其的理解 远程增强子调控转录。这将有助于阐明基本的 某些人类中发生的基因表达缺陷的潜在机制 疾病。

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.