Identification of Gli-Act targets in the mouse ventral neural tube by ChIP

In studies investigating Sonic hedgehog (Shh) mediated patterning of the ventral neural tube, a process where Shh acts as a morphogen, we have investigated the transcriptional network underlying neural tube specification. Adopting an ES-cell based Shh neuronal specification assay (embryoid body; EBs) and a FLAG-tagged Gli protein (a transcriptional effector of the Shh pathway), we identified a number of direct targets of Gli action using Chromatin Immunoprecipitation (ChIP). These results will provide a first survey of the genome level response to this critical signaling input in vertebrate patterning Keywords: ChIP-chip, Gli transcription factors, Gli1, neural specification, mouse Custom genomic tiling arrays based on expression data from a variety of Hh mutant backgrounds were constructed. These include 132 genes tiled with 60-mer oligo probes at a density of one probe per 125 bases spanning between 50-125kb of DNA. This region spans the TSS. Therefore there is very substantial upstream coverage and more variable downstream coverage (depending on gene size). There are three biological replicates, each with 4 technical replicates. The 4 technical replicates contain dye-swaps such that 1 and 2 are identical and reps 3 and 4 are identical. Within each hybridization, the Input sample is a small fraction of sheared chromatin that was not precipitated with the M2 Flag antibody as was the experimental ChIP sample, but was otherwise treated equivalently.