Cis-regulatory circuitry underlying Hedgehog mediated limb development

描述(由申请人提供)：Hedgehog(HH)途径的缺陷是人类出生缺陷的广泛基础，包括无前脑畸形、腭裂和多指畸形。HH信号通过调节Gli转录因子的活性来调控转录。因此，彻底了解Gli蛋白如何识别和激活转录靶标，对于理解HH相关出生缺陷中被错误调控的基因和过程是必要的。然而，关于Gli蛋白如何识别目标基因以及如何与其他转录输入相互作用，几乎一无所知。这项建议的目的是定义活性的GLI增强剂，并确定它们在作为肢体发育基础的HH响应的顺式调控网络中的相互作用。在第一种方法中，我们将使用一种创新的方法来识别不同遗传背景的HH反应活性增强子。这些数据将被用来构建一个全球基因调控网络，定义一组具有集体潜力的重要基因外区，以介导HH途径的转录效应。在第二种以活体为中心的方法中，我们将确定在HH靶基因的转录调控中是否需要额外的共激活因子。最后，我们将确定HH和BMP通路是否共同调节靶基因，重点是调节发育重要的基因Gremlin。这项研究将通过识别肢体中的活性Gli增强子和靶基因，定义增强子域和确定辅助因素，来改变我们对肢体发育的理解。这些将为未来的研究提供宝贵的资源，并将为从系统层面理解肢体发育提供坚实的基础。这些增强子的鉴定也将为临床科学家提供重要的基因组资源，以寻求了解BMP和HH相关出生缺陷以及数百种临床上不同形式的人类多指畸形的潜在机制。 与公共健康相关：从这些实验中获得的结果将促进我们对刺猬信号如何导致受调控的转录反应的理解。它们还将增加我们对发育过程中转录过程的理解。综上所述，这些结果将有助于理解大量刺猬介导的出生缺陷背后的机制。

DESCRIPTION (provided by applicant): Defects in the Hedgehog (Hh) pathway underlie a vast spectrum of human birth defects, including holoprosencephaly, cleft palate, and polydactyly. Hh signaling controls transcription by regulating the activity of Gli transcription factors. A thorough understanding of how Gli proteins recognize and activate transcriptional targets is therefore necessary to understand the genes and processes that are misregulated in Hh-associated birth defects. However, virtually nothing is known about how Gli proteins recognize target genes and interact with other transcriptional inputs. The aim of this proposal is to define active Gli enhancers and to determine their interactions in a Hh- responsive cis regulatory network that underlies limb development. In the first approach, we will use an innovative approach to identify Hh-responsive, active enhancers different genetic backgrounds. This data will be used to construct a global gene regulatory network, defining an important set of extragenic regions with the collective potential to mediate the transcriptional effects of the Hh pathway. In a second in vivo-centered approach, we will determine the requirement for additional co-activating factors in the transcriptional regulation of Hh target genes. Finally, we will determine if the Hh and BMP pathways co-regulate target genes, focusing on the regulation the developmentally important gene, Gremlin. This study will transform our understanding of limb development by identifying active Gli enhancers and target genes in the limb, defining enhancer domains and determining co-factors. These will provide an invaluable resource for future studies and will provide a solid foundation for a systems-level understanding of limb development. The identification of these enhancers will also provide an important genomic resource for clinical scientists seeking to understand the mechanisms underlying the spectrum of BMP and Hh-associated birth defects and the hundreds of clinically distinct forms of human polydactyly. PUBLIC HEALTH RELEVANCE: The results obtained from these experiments will advance our understanding of how hedgehog signaling leads to a regulated transcriptional response. They will also increase our understanding of transcriptional processes during development. Together, these results will contribute to an understanding of the mechanisms underlying the large number of hedgehog-mediated birth defects.