Mechanisms of Hedgehog Target Gene Selection in Development and Cancer

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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.