Control Of Gene Expression During Development

发育过程中基因表达的控制

• 批准号: 6813874
• 负责人: JUDITH A KASSIS
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 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/6813874
• 关键词: Drosophilidae; arthropod genetics; cell differentiation; developmental genetics; gene induction /repression; genetic regulation; genetic regulatory element; growth /development; immunoprecipitation; intermolecular interaction; nucleic acid sequence; tissue /cell culture; transcription factor

During development and differentiation, genes become competent to be expressed or are stably silenced in an epigenetically heritable manner. This selective activation/repression of genes leads to the differentiation of tissue types. Our group is interested in the molecular mechanisms that lead to the heritable transmission of the silenced state. To address this problem, we are studying the mechanism of gene silencing by the Polycomb group genes (PcG) in Drosophila. The PcG genes encode a diverse group of proteins known to be important for silencing of homeotic and segmentation genes during development. Some PcG genes encode histone-modifying proteins that silence transcription by transforming chromatin to an inactive state, but how do these non-DNA binding PcG proteins get to the DNA? PcG proteins act through poorly defined cis-acting DNA sequences called Polycomb group Response Elements (PREs). We have been studying two proteins that bind to PREs, Pho and Phol. Pho and Phol are zinc-finger proteins closely related to the mammalian transcription factor YY1. Pho and Phol are 80% identical in their zinc-finger regions and bind to the same DNA sequence in vitro. The phenotype of phol, pho double mutants is much more severe than the phenotype of either single mutant suggesting that phol and pho encode redundant activities. It has been proposed that Pho and Phol recruit and/or anchor non-DNA binding Polycomb proteins to the DNA. We have recently completed experiments to begin to address this hypothesis. Chromatin-immunoprecipitation experiments have shown that Pho and Phol are bound to a PRE in the Ubx gene in tissue culture cells and in wing disks from Drosophila larvae. The non-DNA binding Polycomb group proteins Pc and E(z) are also closely associated with this PRE. Treatment of tissue culture cells with Pho-RNAi to deplete Pho protein causes a loss of E(z) and Pc binding to the PRE, suggesting that Pho anchors E(z) and Pc to the DNA. In wing disks from pho mutants which lack Pho protein, E(z) and Pc are still bound to the DNA. However in pho, phol double mutant wing disks, E(z) and Pc are lost from the DNA, suggesting that Phol and Pho act redundantly to anchor these proteins to the DNA in wing disks. Surprisingly, although Phol and Pho are bound to many of the same DNA fragments in vivo, their distribution is not identical. Thus, although these two proteins have overlapping activities, they may interact with some DNA sequences and proteins differently. Regulatory sequences in eukaryotic genes can be located many tens of kilobases away from the promoter. How do these distant regulatory sequences activate or repress the promoter? One model is that proteins bound to a distant enhancer or silencer interact with proteins bound near the promoter causing a loop of the intervening DNA. One of the fragments of DNA from the Drosophila engrailed gene that has PRE activity can also mediate interactions between distant DNA fragments. We are currently trying to understand the function of this DNA in vivo by deleting it from the endogenous engrailed gene. Our results suggest that this fragment of DNA acts to facilitate interactions between a distant enhancer involved in positively regulating engrailed expression in the wing and the engrailed promoter. Thus, this fragment of DNA acts as a PRE (a negative regulatory element) in some transgenes, but as a positive regulatory element in its natural context. These results are consistent with the idea that the role of this element is to facilitate the interactions between enhancers or silencers with distant promoter elements.

在开发和差异化过程中，基因能够以表观遗传遗传的方式表达或稳定地沉默。这种基因的选择性激活/抑制导致组织类型的分化。我们的小组对导致沉默状态的传播传播的分子机制感兴趣。为了解决这一问题，我们正在研究果蝇中多康姆基基因（PCG）基因沉默的机理。 PCG基因编码了一组多样的蛋白质，对于在发育过程中的同源和分割基因沉默很重要。一些PCG基因编码组蛋白修饰蛋白，通过将染色质转化为无活性态，但是这些非DNA结合PCG蛋白如何转化为DNA？ PCG蛋白通过称为PolyComb组响应元件（PRES）的定义定义的顺式作用DNA序列起作用。我们一直在研究与PRES，PHO和PHOL结合的两种蛋白质。 PHO和PHOL是与哺乳动物转录因子YY1密切相关的锌指蛋白。 PHO和PHOL在其锌指区域的80％相同，并在体外与相同的DNA序列结合。 PHOL，PHO双突变体的表型比任何一个单个突变体的表型都要严重得多，这表明PHOL和PHO编码冗余活动。已经提出，PHO和PHOL募集和/或锚定非-DNA结合多孔蛋白与DNA。我们最近完成了实验，以开始解决这一假设。染色质 - 免疫沉淀实验表明，PHO和PHOL与组织培养细胞和果蝇幼虫的翼盘中的UBX基因中的PR结合。非DNA结合聚蛋白蛋白PC和E（Z）也与该PRES密切相关。用pho-RNAI处理组织培养细胞以耗尽pHO蛋白会导致E（Z）和PC与PRE的结合，这表明PHO将E（Z）和PC锚定与DNA。在缺乏Pho蛋白的Pho突变体的机翼磁盘中，E（Z）和PC仍与DNA结合。但是，在Pho中，Phol双重突变翼磁盘，E（Z）和PC从DNA中丢失，这表明PHOL和PHO作用是将这些蛋白固定在机翼磁盘中的DNA上。令人惊讶的是，尽管Phol和Pho与体内许多相同的DNA片段结合，但它们的分布并不相同。因此，尽管这两种蛋白质具有重叠活性，但它们可能会与某些DNA序列和蛋白质相互作用。 真核基因中的调节序列可以位于距启动子范围的许多千倍酶。这些遥远的调节序列如何激活或抑制启动子？一种模型是与远处增强子或消音器结合的蛋白质与启动子附近结合的蛋白质相互作用，从而导致中间DNA的环。具有果蝇植入的基因的DNA的片段之一，具有前活性的基因也可以介导遥远的DNA片段之间的相互作用。我们目前正在尝试通过从内源性一个处依恋基因中删除该DNA在体内的功能。我们的结果表明，这种DNA的片段起着促进参与正面调节机翼和狭窄启动子的正面表达的遥远增强子之间的相互作用。因此，这种DNA的碎片在某些转基因中充当了Pre（负调节元件），但在其自然背景下是一个积极的调节元件。这些结果与该元素的作用是促进具有远距离启动子元素的增强子或消音器之间的相互作用的想法一致。