Mechanisms of Hedgehog Target Gene Selection in Development and Cancer

描述（由申请人提供）：Hedgehog （Hh）信号是控制分化和生长的基础。在小脑发育过程中，浦肯野神经元释放出Sonic hedgehog (Shh)，这是邻近颗粒神经元前体（GNPs）的一种有效的丝裂原。GNPs通过改变激活或抑制靶基因的Gli转录因子的加工、位置和修饰来响应Shh。人类或小鼠编码Shh受体的patched1的突变，促进GNP细胞向髓母细胞瘤（MBs）的转化，这是最常见的儿童恶性脑肿瘤。我们使用染色质免疫沉淀（ChIP）和高通量测序技术确定了Gli1在小鼠GNPs和MB细胞染色质中的结合位置。这让我们找到了glii反应性转录增强子。结合ChIP数据和基因表达数据，我们确定了可能由Shh直接调控的靶基因。当小脑前体细胞变成癌细胞时，Gli1的靶向性和靶基因表达发生了巨大的变化。我们将探讨靶基因选择的机制，Hh靶基因与细胞周期的联系，以及靶基因在正常发育和肿瘤发生中的作用。特异性目的1：研究Gli转录因子如何协调调节小脑发育和肿瘤发生过程中的基因表达。我们的ChIP数据导致了许多新的靶基因，以及诸如Ptch1、Gli1和N-myc等已建立的靶标。132个基因在正常和肿瘤细胞类型中是一致的靶标。值得注意的是，尽管GNPs和MB细胞之间关系密切，但许多假定的靶基因对一种或另一种细胞类型是特异性的。我们将确定在两种细胞类型中选择性识别增强子元件的机制。特异性目标2：确定染色质修饰剂如何影响gli调控的基因表达。我们已经确定了与GNPs和MBs中特定靶基因的调控变化相关的组蛋白修饰。我们将研究Hh靶基因规范中这些变化的机制重要性。特异性目的3：研究Gli蛋白与其他转录因子的相互作用。对Gli1结合的DNA区域的计算分析揭示了两种转录因子的证据，即E盒结合蛋白和NFI蛋白，它们可能与Gli1蛋白并行或协同工作。我们将探讨它们在靶基因控制中的作用。具体目标4：确定Gli目标对国民生产总值发展和肿瘤发生的贡献。我们将研究在GNPs和肿瘤的背景下，介导Hh信号和细胞周期之间联系的选定靶基因。这些计划中的研究对于理解Hedgehog通路在许多组织和器官中的发育和致瘤作用具有直接的重要性。当信号转导错误导致出生缺陷或癌症时，发现直接受Hh信号调控的基因将带来新的干预方法。

DESCRIPTION (provided by applicant): Hedgehog (Hh) signaling is fundamental to the control of differentiation and growth. During development of the cerebellum, Purkinje neurons emit Sonic hedgehog (Shh), a potent mitogen for adjacent granule neuron precursors (GNPs). GNPs respond to Shh by altering the processing, location, and modification of Gli transcription factors that activate or repress target genes. Mutations in human or mouse patched1, which encodes the Shh receptor, promote transformation of GNP cells into medulloblastomas (MBs), the most common childhood malignant brain tumor. We used chromatin immunoprecipitation (ChIP) and high-throughput sequencing to identify locations of Gli1 binding in the chromatin of murine GNPs and MB cells. This led us to Gli-responsive transcriptional enhancers. Combining ChIP data with gene expression data we identified putative target genes that are directly regulated by Shh. Dramatic changes in targeting of Gli1, and target gene expression, occur when cerebellum precursor cells become cancer cells. We will investigate the mechanisms of target gene selection, the connections between Hh target genes and the cell cycle, and the roles of target genes in normal development and tumorigenesis. Specific Aim 1: Investigate how Gli transcription factors coordinate to regulate gene expression in cerebellum development and tumorigenesis. Our ChIP data led to many novel target genes, and well-established targets like Ptch1, Gli1, and N-myc. 132 genes are consistent targets in normal and tumor cell types. Remarkably, despite the close relation between GNPs and MB cells, many putative target genes are specific to one cell type or the other. We will determine the mechanism of selective recognition of enhancer elements in the two cell types. Specific Aim 2: Determine how chromatin modifiers influence Gli-regulated gene expression. We have identified histone modifications that correlate with the regulatory changes for specific target genes in GNPs vs. MBs. We will investigate the mechanistic importance of these changes in Hh target gene specification. Specific Aim 3: Investigate interactions of Gli proteins with other transcription factors. Computational analyses of the DNA regions bound by Gli1 revealed evidence for two types of transcription factors, E box-binding proteins and NFI proteins, that may work in parallel, or collaborate, with Gli1 protein. We will investigate their roles in target gene control. Specific Aim 4: Define contributions of Gli targets to GNP development and tumorigenesis. We will investigate selected target genes that mediate the connection between Hh signaling and the cell cycle, in the context of GNPs and tumors. The planned studies have direct importance for understanding developmental and tumorigenic roles of the Hedgehog pathway in many tissues and organs. Discovering genes that are directly regulated by Hh signaling will lead to new ways to intervene when errors in signal transduction lead to birth defects or cancer.