Hedgehog (Hh) signaling is essential for the development and maintenance of many tissues. Damage to Hh signaling causes birth defects and cancer. In the developing cerebellum Sonic Hedgehog (Shh) secreted from Purkinje neurons modulates the relative activator or repressor function of Gli transcription factors to driv the proliferation of granule neuron precursors (GNP). Chronic Hh pathway activation in GNPs leads to medulloblastoma (MB), the most common childhood malignant brain tumor. To better understand Hh-dependent gene networks our lab evaluated genome-wide Gli1 binding in GNPs using chromatin immunoprecipitation and high throughput sequencing (ChIP-seq). Gli1-occupied regions were found to be enriched for consensus binding sites for Nuclear Factor One (Nfi) transcription factors. Several lines of evidence suggest Nfi and Gli proteins may function together: Nfi expression overlaps with Hh responsive regions in the developing cerebellum and lung, Nfi and Gli act synergistically on a representative enhancer, and different Nfi family members uniquely promote or antagonize Gli1-mediated reporter activity. Our data indicate Gli and Nfi proteins interact to modulate transcription, but the mechanism of this interaction and how it affects target genes remains unknown.
Aim 1 : Investigate the mechanism of interaction between Nfi and Gli Proteins. ChIP-seq studies combined with bioinformatic analysis identified many Gli1-bound regions that were enriched for Nfi binding motifs. I will evaluate whole-genome occupancy of Nfi proteins, allowing us to determine which enhancers are regulated by both Nfi and Gli proteins. I will determine whether Gli and Nfi function through a direct physical interaction, an interaction mediated by a third molecule, or act as parallel regulators that suppor each other.
Aim 2 : Investigate chromatin remodeling at Hh target genes. To determine the role of chromatin remodeling in the differentiation of Hh-dependent GNPs we will characterize the patterns of target gene occupancy by Gli and Nfi proteins in Hh-responsive GNPs and granule neurons that no longer respond to Hh. I will determine whether ATP-dependent chromatin remodeling complexes are necessary for Nfi-Gli-mediated transcriptional activation. I will determine whether Nfi proteins function to modulate nucleosome occupancy at Hh target genes in the cerebellum.
Aim 3 : Investigate the requirement for Nfi proteins in Hh target gene expression. I will determine whether synergy seen between Nfi and Gli proteins reflects increased duration of transcription or enhanced sensitivity to the concentration of Hh ligands. I will test for a genetic interaction between Hh and Nfi in MB progression.
These aims will allow me to do pioneering work on the first and best candidates for Gli cofactors, and to investigate a fundamental regulatory pathway in relation to brain development and cancer.
The Hedgehog (Hh) signaling pathway controls the relative activator and repressor function of Gli transcription factors to guide patterning, proliferation an differentiation in many organs and tissues. Using genomic methods, we have identified Nuclear Factor One (Nfi) transcription factors as potential Gli cofactors, a novel mechanism for Hh target gene selection. We will investigate the role of Nfi genes as Gli cofactors and determine the molecular mechanisms by which Hh target genes are chosen, and in doing so, gain a better understanding of a signaling pathway frequently associated with birth defects and cancers.