Molecular dissection of Drosophila Hox gene cis-regulatory modules

描述(由申请人提供)： 摘要：我们DNA中的关键基因是如何调控的？在胚胎发育过程中，它们是如何在适当的空间和时间内协调表达的？这些问题对我们理解动物和人类的发育计划有着巨大的影响。尽管如此，我们目前对基因表达调控在分子水平上的理解仍然有些肤浅。已有研究表明，关键发育基因的转录受邻近非编码基因组DNA序列的特定区域以多种方式控制。这些不同的顺式调控模块(CRM)，包括转录增强子、绝缘体和消音器，已经被发现在所有已研究的真核模型系统中调节基因的表达。在某些情况下，已发现特定的转录因子与CRM中的序列结合并调节其活性。然而，决定转录因子在大多数CRM序列中结合的位置以及这种结合如何调节CRM功能的分子机制仍不清楚。果蝇双胸复合体的HOX基因是决定胚胎发育过程中动物身体计划的关键基因。在发育中的胚胎中，HOX基因在时间和空间上的表达受CRM调控。我们的初步研究表明，果蝇双胸复合体CRM上的序列在果蝇属内进化迅速。这与HOX基因形成鲜明对比，HOX基因在所有双边动物中都是保守的。果蝇双胸复合体中已知的大量增强子CRM，以及这些CRM上序列在不同果蝇物种中的快速进化，为研究CRM上的序列、转录因子结合和顺式调节功能之间的关系提供了一个令人兴奋的机会。这一建议的最终目的是在序列水平上阐明控制增强子CRM功能活性的分子机制。我们提出的研究还将为不同物种之间顺式调节功能的进化提供洞察力。首席研究员的长期科学目标是全面调查分子机制，通过这些分子机制，调节区实现对整个双胸复合体基因表达的协调控制。这项建议的主要重点将是对果蝇双胸复合体中已知的CRM中特定转录因子结合位点的生物信息学鉴定和功能表征。进一步的实验将测试已知CRM的保守亚区的功能活性，并研究远缘果蝇CRM亚区的功能兼容性，这将有助于揭示CRM体系结构和功能活性的进化。 公共卫生相关性： 叙述：基因调控问题是我们理解发育和疾病的核心。关键发育基因的精心时间和空间控制的表达，其中许多是调节下游基因表达的转录因子，是正常发育的关键基础。胚胎中HOX基因表达的早期模式在分子水平上启动了一系列调控事件，对后来的发育具有深远的影响。目前，我们对HOX基因顺式调控机制的认识还很肤浅。我们的初步研究使我们研究了顺式调节模块的功能，以及转录因子如何在调节序列水平上控制果蝇双胸复合体中HOX基因的表达。在所有动物中保存许多这些关键的发育基因和基因调控机制，这些研究将为果蝇以外的生物体的研究提供信息。

DESCRIPTION (provided by applicant): SUMMARY: How are key genes in our DNA regulated? How is their expression coordinated in the proper space and time during embryonic development? These questions have enormous implications in our comprehension of the developmental program in animals and humans. Despite this fact, our current understanding of regulation of gene expression at the molecular level remains somewhat superficial. It has been shown that the transcription of critical developmental genes is controlled in a variety of ways by specific regions of neighboring non-coding genomic DNA sequences. These distinct cis-regulatory modules (CRMs), which include transcriptional enhancers, insulators, and silencers, have been found to regulate gene expression in all the model eukaryotic systems studied so far. In some cases, specific transcription factors have been found to bind at sequences in the CRMs and to regulate their activity. However, the molecular mechanisms that determine where transcription factors bind within most CRM sequences and how this binding regulates CRM function remain unclear. The Hox genes of the Drosophila bithorax complex are critical genes that determine the animal body plan during embryonic development. The expression of Hox genes spatially and temporally in the developing embryo is controlled by CRMs. Our initial studies show that sequences at the CRMs of the Drosophila bithorax complex are rapidly evolving within the Drosophila genus. This is in sharp contrast to the Hox genes, which are conserved across all bilaterian animals. The large set of known enhancer CRMs at the Drosophila bithorax complex, and the rapid evolution of the sequences at these CRMs across different Drosophila species, provide an exciting opportunity to study the relationship between sequences at the CRMs, transcription factor binding and cis-regulatory function. The ultimate goal of this proposal is to elucidate the molecular mechanisms which control functional activity of the enhancer CRMs at the sequence level. Our proposed studies will also provide insight into the evolution of cis-regulatory function across different species. The long term scientific goal of the Principal Investigator is to fully investigate the molecular mechanisms by which the regulatory regions achieve coordinated control of gene expression across the entire bithorax complex. The major focus of this proposal will be on the bioinformatic identification and functional characterization of specific transcription factor binding sites within known CRMs of the Drosophila bithorax complex. Further experiments to test conserved sub-regions of known CRMs for functional activity and to investigate the functional compatibility of CRM sub-regions from distantly related Drosophila species will shed light on the evolution of CRM architecture and functional activity. PUBLIC HEALTH RELEVANCE: NARRATIVE: The issue of gene regulation is at the heart of our understanding of development and disease. Carefully timed and spatially controlled expression of key developmental genes, many of which are transcription factors that regulate the expression of downstream genes, serves as the critical basis for normal development. The early patterns of Hox gene expression in the embryo initiate a cascade of regulatory events at the molecular level that has profound consequences for later development. Currently our understanding of the mechanisms of cis-regulation for the Hox genes is still very superficial. Our preliminary studies have led us to investigate the function of cis-regulatory modules and how transcription factors act at the regulatory sequence level to control the expression of Hox genes at the Drosophila bithorax complex. The conservation of many of these key developmental genes and mechanisms of gene regulation across all animals promises that these studies will be informative for studies in organisms beyond Drosophila.