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：
8351123
负责人：
JAMES A. KENNISON
金额：
$ 91.2万
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/8351123
关键词：
Adult Binding Biological Assay Biological Models Cancer Etiology Cell Fate Control Cell Proliferation Cells Chromosomal Rearrangement Chromosomes Cis-Acting Sequence Comb animal structure Cues DNA Development Developmental Gene Disease Drosophila genome Drosophila genus Ectopic Expression Elements Embryo Fertility Gene Expression Regulation Gene Targeting Genes Genetic Genetic Enhancer Element Genetic Transcription Genomics Goals Homeobox Genes Human Human Development Indium Junk DNA Lead Link Maintenance Mitotic Molecular Genetics Mutation Organism Pattern Polycomb Proteins Regulation Regulator Genes Regulatory Element Regulatory Pathway Repression Specific qualifier value Staging Structure Tissues Trans-Activators Transgenes Work cis acting element derepression fly gene function gene replacement homeodomain human disease male man 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 Polycomb group of genes that are required for transcriptional silencing. To identify new Polycomb group genes, we have developed a transgene assay using the cis-regulatory elements within the homeotic genes that interact with the Polycomb group proteins. Recessive mutations that disrupt transgene silencing are recovered in mitotic clones in heterozygous flies. We have isolated mutations in most of the known Polycomb group genes on the second chromosome. We have also isolated mutations in several new Polycomb group genes. We have also characterized two regions of the Drosophila genome to identify all genes required for either zygotic viability or male fertility. Only about one-third of the genes in these regions are required for zygotic viability, with another 5% required only for male fertility. Almost two-thirds of the genes do not appear to be essential for either viability or fertility. Within one of these genomic regions, we identified a gene desert (a genomic region that is not required for either viability or fertility.

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。遗传学研究确定了转录沉默所需的基因多肉液。为了鉴定新的PolyComb基因基因，我们使用了与Polycomb基团蛋白相互作用的同源基因中的顺式调节元素开发了转基因测定。破坏转基因沉默的隐性突变在杂合蝇中有丝分裂克隆中回收。在第二个染色体上，我们在大多数已知多康布基因基因中都有分离的突变。我们还在几个新的PolyComb基因基因中分离了突变。我们还表征了果蝇基因组的两个区域，以识别合子生存力或男性生育所需的所有基因。这些区域中只有大约三分之一的基因才是合子生存能力，而男性生育只需要另外5％。几乎三分之二的基因似乎对于生存力或生育而言并不是必不可少的。在这些基因组区域之一中，我们确定了一个基因沙漠（一个基因组区域，这是生存力或生育能力所必需的。