Cis-regulatory circuitry underlying Hedgehog mediated limb development

描述（由申请人提供）：Hedgehog（Hh）通路缺陷是人类出生缺陷的基础，包括前脑无裂、腭裂和多指（趾）畸形。Hh信号通过调节Gli转录因子的活性来控制转录。因此，深入了解Gli蛋白如何识别和激活转录靶点对于了解Hh相关出生缺陷中调控不当的基因和过程是必要的。然而，几乎没有人知道Gli蛋白如何识别靶基因并与其他转录输入相互作用。该建议的目的是定义活性Gli增强子并确定它们在构成肢体发育基础的Hh-响应顺式调节网络中的相互作用。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响应性活性增强子。这些数据将被用来构建一个全球性的基因调控网络，定义一组重要的基因外区域的集体潜力，介导的Hh途径的转录效应。在第二个以体内为中心的方法中，我们将确定在Hh靶基因的转录调控中对额外的共激活因子的需求。最后，我们将确定Hh和BMP通路是否共同调节靶基因，重点是对发育重要基因Gremlin的调节。这项研究将通过识别肢体中的活性Gli增强子和靶基因，定义增强子结构域和确定辅助因子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，并为系统水平的肢体发育的理解提供坚实的基础。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，以了解BMP和Hh相关出生缺陷谱的机制以及数百种临床上不同形式的人类多指（趾）畸形。

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.