Regulation Of Cellular Proliferation And Diversity In Drosophila

果蝇细胞增殖和多样性的调节

基本信息

批准号：
7968545
负责人：
JAMES A. KENNISON
金额：
$ 73.77万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7968545
关键词：
Adult Binding Biological Assay Biological Models Cancer Etiology Cell Fate Control Cell Proliferation Cells Chromatin Remodeling Factor Chromosomal Rearrangement Chromosome Segregation Chromosomes Cis-Acting Sequence Comb animal structure Complex Cues DNA Development Developmental Gene Disease Drosophila genus Ectopic Expression Elements Embryo Gene Expression Regulation Gene Targeting Genes Genetic Genetic Enhancer Element Genetic Transcription Genomics Goals Homeobox Genes Homologous Gene Human Human Development Indium Lead Link Maintenance Microtubules Molecular Genetics Mutation Organism Pattern Play Proteins Regulation Regulator Genes Regulatory Element Regulatory Pathway Repression Role Screening procedure Specific qualifier value Staging Structure Tissues Trans-Activators Transcriptional Regulation Transgenes Von Hippel-Lindau Tumor Suppressor Protein Work brahma cis acting element cohesin derepression gamma Tubulin gene function gene replacement homeodomain man protein complex rhinoceros sex tool transcription factor

项目摘要

One goal of the Section on Drosophila Gene Regulation is to understand the regulation of homeotic gene function in Drosophila. The homeotic genes encode homeodomain-containing transcription factors that control cell fates by regulating the transcription of downstream target genes. The homeotic genes are expressed in precise spatial patterns that are crucial for the proper determination of segmental identities. Cis-acting transcriptional regulatory elements from the homeotic genes have been previously-identified by assays in transgenes in Drosophila. These assays have identified both tissue-specific enhancer elements, as well as cis-regulatory elements that are required for the maintenance of activation or repression throughout development. While these transgene assays have been important in defining the structure of the cis-regulatory elements and identifying trans-acting factors that bind them, their functions within the contexts of the endogenous genes is still not well understood. We have used a large number of existing chromosomal rearrangements in the Sex combs reduced homeotic gene to investigate the functions of the cis-acting elements within the endogenous gene. Characterization of the chromosomal rearrangements revealed that two genomic regions about 70 kb apart in the Sex combs reduced gene must be in cis to maintain proper repression. When not physically linked to each other, these genomic regions interact with regions on the homologous chromosome and cause derepression of its wild-type Sex combs reduced gene. Using a transgene assay, we have identified candidate fragments of DNA that may correspond to these regulatory genomic regions. Both regions appear to contain clusters of regulatory elements that can interact with elements on homologous chromosomes. We deleted two of these candidate elements from the endogenous Sex combs reduced gene by targetted gene replacement. Both deletions only partially disrupt silencing of the Sex combs reduced gene. This suggests that the elements in each cluster have partially redundant functions. Genetic studies identified the trithorax group of genes that are required for expression or function of the homeotic genes. One of the trithorax group genes that we first identified is verthandi. We have shown that verthandi encodes a subunit of cohesin, a protein complex already known to be required for proper segregation of chromosomes during mitotis. Both verthandi and Nipped-B (a cohesin-associated protein) are required for expression of homeotic genes, supporting a role for cohesin in transcriptional regulation. Screening for mutations that interact with the trithorax group gene brahma (a subunit of the BRM chromatin-remodeling complexes), we isolated a mutation in gamma-tubulin. Our results suggest that microtubule organizer complexes play a role in facilitating transcription. We have also isolated brahma-interacting mutations in the rhinoceros gene. Rhinoceros encodes the Drosophila homolog of a human transcription factor that physically interacts with the von Hippel Lindau tumor suppressor protein.

有关果蝇基因调节的部分的一个目标是了解果蝇中同源基因功能的调节。同源基因编码含同源域的转录因子，这些因子通过调节下游靶基因的转录来控制细胞的命运。同源基因以精确的空间模式表达，这对于适当确定分段身份至关重要。从果蝇中的转基因中的测定，来自同源基因的顺式转录调节元件已被先前鉴定出来。这些测定方法已经确定了组织特异性增强子元素以及在整个发育过程中维持激活或抑制所必需的顺式调节元件。尽管这些转基因测定对于定义顺式调节元件的结构并识别结合它们的跨性因子的结构很重要，但在内源基因的背景下它们的功能仍未得到很好的了解。我们已经在性梳中使用了大量现有的染色体重排可减少同源基因，以研究内源基因内顺式作用元件的功能。染色体重排的表征表明，在性梳子中，基因中约有70 kb的两个基因组区域必须在顺式中以保持适当的抑制作用。当没有物理上相互联系时，这些基因组区域与同源染色体上的区域相互作用，并导致其野生型性梳子的压抑减少基因。使用转基因测定法，我们已经确定了可能与这些调节基因组区域相对应的DNA的候选片段。这两个区域似乎都包含可以与同源染色体上元素相互作用的调节元素簇。我们从内源性梳中删除了其中两个候选元素，从而通过靶向基因置换降低了基因。两种删除仅部分破坏了性梳子的沉默减少基因。这表明每个集群中的元素具有部分冗余功能。遗传研究鉴定了综合基因表达或功能所需的Trithorax基因组。我们首先确定的Trithorax组基因之一是Verthandi。我们已经表明，verthandi编码粘蛋白的亚基，粘蛋白是一种已知在Mitotis期间染色体正确分离所需的蛋白质复合物。表达同型基因的表达需要Verthandi和Nipped-B（与粘着素相关的蛋白），这支持了粘蛋白在转录调控中的作用。筛选与Trithorax组基因梵天（BRM染色质复合物复合物的亚基）相互作用的突变，我们在γ-微管蛋白中分离了突变。我们的结果表明，微管组织者络合物在促进转录中起作用。我们还分离了犀牛基因中的梵天相互作用突变。 Rhinoceros编码人类转录因子的果蝇同源物，该果蝇与von Hippel Lindau抑制蛋白的物理相互作用。