Genome wide maps of chromatin modification in response to Hh signaling in mouse embryonic forelimb buds

Purpose: The response to Hedgehog signaling in the limb is driven by GLI bound enhancers and the majority of Hh targets in the developing limb bud are regulated solely by the activity of GLI-repressor. Currently we do not have a comprehensive understanding of how GLI bound enhancers respond Hedgehog signaling. The goal of this study is to identify how GLI bound enhancers are regulated by Hedgehog signaling and specifically by GLI-repressor. Methods: Histone modification ChIP-sequencing was done in Embryonic day 10.5 limb buds from control and Shh null embryos to identify histone modifications that change in response to Hedgehog signaling. We also did a comparative analysis of histone modificaiton response to Hedgehog stimulation in NIH3T3 cells to assess tissue specificity of GLI bound enhancer response to Hedgehog signaling. Results: We found that Hedgehog signaling specifically regulates the acetylation status of Histone 3 lysine 27 at a subset of GLI binding regions. These regions correlate with both known Hedgehog target genes in the limb bud and with characterized enhancers in the limb. This set of Hedgehog responsive GLI bound enhancers demonstrate enhanced tissue specificity. ChIP-sequencing of 3 different histone modifications (H3K27ac, H3K4me2, and H3K27me3) in Shh-/- and WT control E10.5 forelimb buds. Additional ChIP-seq of H3K27ac in NIH3T3 cells with and without Hedgehog pathway stimulation. Two biological replicates were done for each ChIP experiment. Enrichment values were determined using input control samples from one or both replicates for each ChIP experiment.