The Involvement of E-box binding domains in the activation of Sonic Hedgehog target genes

Sonic Hedgehog (Hh) signaling pathway is involved in development of tissues such as the cerebellum, and also the development of medulloblastomas in which there is over-activation of the pathway and differential Hh target gene expression. Hh pathway activation leads to activation of Gli1 transcription factors responsible for expression of Hh target genes. Findings from the Scott lab showed Gli1 often binds DNA near E-box binding motifs. These E-boxes are enriched in Gli-ChIP experiments in medulloblastomas versus Gli-ChIP in granule neuron precursors – normal cells thought to give rise to tumorous medulloblastomas. My goal is to investigate how E-boxes affect the expression of Hh target genes (Hes5, Fez1, Cdk6, Rab34, PTCH, Tle1 and Tspan11). These genes were chosen because they bind Gli1 in ChIP experiments, were responsive to increased Hh signaling in cell culture, and contained a variety of E-box motifs. I have cloned 9 enhancers of these genes upstream of a luciferase reporter. I will use luciferase transcription to quantify these enhancers’ activity under the following conditions: 1)in 3T3 cells which normally have little Hh activity, 2) 3T3 cells treated with Hh agonist, SAG, 3) Med1 cells - a medulloblastoma-derived cell line with naturally high Hh pathway activity, and 4)in Med1 cells treated with Hh antagonist, SANT1. E-boxes will then be mutated in these enhancers and quantify the transcriptional effect and its relation to Hh activity to determine if E-box binding domains affect Gli1 controlled gene expression. Future experiments may include using siRNA to identify the culpable E-box binding protein(s).