Control Of Gene Expression During Development

发育过程中基因表达的控制

• 批准号: 7208942
• 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/7208942
• 关键词: DNA binding protein; Drosophilidae; arthropod genetics; binding sites; chromatin; developmental genetics; enzyme induction /repression; epigenetics; gene expression; genetic promoter element; genetic regulation; homeobox genes; protein binding; protein protein interaction; protein structure function; 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 genes during development in organisms as diverse as Drosophila and man. Many PcG genes encode chromatin-associated proteins that are thought to silence transcription through modification of histones and formation of inactive chromatin. PcG proteins act through cis-acting DNA sequences called Polycomb group Response Elements (PREs). PREs contain binding sites for multiple proteins that act together to recruit PcG protein complexes to the DNA. Our lab is working to identify all the DNA binding-proteins necessary to recruit PcG protein complexes. Interestingly, PREs also mediate interactions between distant DNA elements suggesting they may bring together distant silencers and enhancers with promoters. Our lab is investigating this possibility at the Drosophila engrailed gene. Many of the PcG proteins are associated in two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181-bp PRE from the Drosophila engrailed gene. At least 8 binding sites are present within this 181-bp PRE. Two of the binding sites are for the Polycomb-group proteins Pleiohomeotic (Pho) and Pleiohomeotic-like (Phol). The proteins GAGA factor and Pipsqueak bind to another two sites, but the identity of the proteins that bind to the other four sites was not known. We have recently found that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc-finger proteins. This family of proteins encodes transcription factors and has been extensively studied in mammals. There are 20 Sp1/KLF family members in mammals. In Drosophila there are 10 Sp1/KLF family members, and nine of them bind to Site 2. We derived a consensus-binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila. Our laboratory is currently studying these factors to elucidate their role in PRE function. 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基因编码了多种蛋白质，对于在果蝇和人类等多样性的生物体中发育过程中对同源基因的沉默很重要。许多PCG基因编码与染色质相关的蛋白质，这些蛋白质被认为是通过修饰组蛋白和非活性染色质的形成来沉默的转录。 PCG蛋白通过称为PolyComb组响应元件（PRES）的顺式作用DNA序列作用。 PRES包含多种蛋白质的结合位点，这些蛋白质共同起作用以募集PCG蛋白复合物到DNA。我们的实验室正在努力确定募集PCG蛋白复合物所需的所有DNA结合蛋白。有趣的是，PRES还介导遥远的DNA元素之间的相互作用，这表明它们可能会将遥远的消音器和增强子与启动子汇集在一起​​。我们的实验室正在研究果蝇所植入的基因的这种可能性。 许多PCG蛋白与通过修饰染色质抑制基因表达的两种蛋白质复合物相关。但是，这两种蛋白质复合物都与体内PRES专门缔合，但是，尚不清楚它们是如何招募或保存在Pre的。 PRES是复杂的元素，由许多蛋白质的结合位点组成。我们的实验室一直在努力定义从果蝇构成的基因的181 bp活动所需的所有序列和DNA结合蛋白。在这个181 bp的pre中至少存在8个结合位点。其中两个结合位点用于多肉瘤蛋白pleiohomeotic（Pho）和肾瘤状（PHOL）。蛋白质Gaga因子和Pipsqueak结合了另外两个位点，但是与其他四个位点结合的蛋白质的身份尚不清楚。我们最近发现，预活活动2（站点2）的一个位点可以由锌指蛋白的SP1/KLF家族的成员约束。这个蛋白质家族编码转录因子，并在哺乳动物中进行了广泛的研究。哺乳动物中有20个SP1/KLF家庭成员。在果蝇中，有10个SP1/KLF家族成员，其中9个与站点2结合。我们得出了SP1/KLF果蝇家庭成员的共识结合位点，并表明大多数分子表征的PRES都存在这种共识序列。这些数据表明，一个或多个SP1/KLF家庭成员在果蝇的Pre功能中起作用。我们的实验室目前正在研究这些因素，以阐明其在Pre功能中的作用。 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。