GLI transcriptional repression regulates tissue-specific enhancer activity in response to Hedgehog signaling

GLI3 binding regions with called peaks with a false discovery rate (FDR) < 0.05 from two biological replicates of E10.5 (32–35S) forelimbs. Rank ordered coordinates, peak length, log2 fold change (log2FC) and FDR are listed for each peak.

H3K27ac called peaks with a FDR < 0.05 from two biological replicates from WT and Shh^-/- E10.5 forelimbs. For each peak, the assigned Peak ID, coordinates, peak type, fold change normalized to input for WT and Shh^-/- samples and fold change of WT over Shh^-/- are listed. Additional tabs include sorted datasets for sub-classifications. Tabs containing GBRs indicate intersections with GLI binding regions.

H3K4me1 called peaks with a false discovery rate (FDR) < 0.05 from two biological replicates of E10.5 WT forelimbs. Note that this is a reanalysis of a publicly available ENCODE dataset (see methods).

Table showing the top 20 motifs uncovered from de novo motif analysis on HH-responsive GBRs. The enrichment is relative to matched genomic controls. Note that ‘HH_resp_2’ is the only motif with an enrichment value of greater than two and corresponds with a known GLI binding motif.

H3K27me3 called peaks with a FDR < 0.05 from two replicates of Shh^-/- E10.5 forelimbs. For each peak, the assigned Peak ID, coordinates, log2 fold change normalized signal to input. Additional tab includes H3K27me3 peaks that overlap with GLI3 binding regions; the GBR sub-classifications are specified.

The first column indicates genes previously identified as differentially expressed between Shh^-/- and WT E10.5 limb buds (Lewandowski et al., 2015). The second column indicates the fold enrichment of H3K27me3 at the promoter compared to Input with the adjusted P-value indicated in the third column. The fourth column indicates whether the gene has a HH-dependent GBR (indicated by one and yellow shading) within the same presumptive TAD (Dixon et al., 2012). There are 22 HH-dependent target genes out of 80 HH-responsive genes.

H3K4me2 called peaks with a FDR < 0.05 from two replicates from WT and Shh^-/- E10.5 forelimbs. For each peak, the assigned Peak ID, coordinates, peak type, fold change normalized to input for WT and Shh^-/- samples and fold change of WT over. Shh^-/- are listed. Additional tabs include sorted files for each peak type. Under the ‘GLI3 binding’ column, ‘TRUE’ implies overlap with a GBR, while ‘FALSE’ indicates no overlap.

Coordinates for all ATAC peaks in the WT group that overlap with GBRs are listed. ‘Shh_ATAC_peak’ identifies the corresponding id# for that peak in the Shh^-/- data, and if a peak is not present in the Shh^-/- samples, it is marked as NA. A column for each GBR type identifies which GBR type a given ATAC peak overlaps with. The number indicates the peak ID. If a peak region does not overlap with the type of peak in that list, it will be marked as NA. The normalized log2 transformed signals are shown for each sample in addition to the ‘average’ signal across all samples. The ‘t’ statistic calculates the difference in signals between WT and Shh^-/- by taking into consideration fold-change and variance among samples. A positive t statistic values indicate a peak is more accessible in WT than Shh^-/- and a negative t statistic indicates higher accessibility in Shh^-/-. The ‘p.value’ is obtained from a moderated t-test using limma. The ‘p.value.adj’ is the adjusted p-value (FDR) using the Benjamini-Hochberg procedure.

H3K27ac called peaks with a FDR < 0.05 from two replicates of WT, Shh^-/- and Shh^-/-;Gli3^-/- E10.5 (33–34S) forelimbs. Separate tabs for each genotype include peak coordinates and log2 fold change over input. Additional tabs include a peak summary and differential analysis of WT vs. Shh^-/-;Gli3^-/-. Differential analysis tab lists peak coordinates, peak type, fold change normalized to input for WT and Shh^-/-;Gli3^-/- samples and fold change of WT over Shh^-/-;Gli3^-/-.

List of 31 HH-responsive GBRs with H3K27me3 enrichment in Shh^-/- limb buds and their enrichment of H3K27ac in Shh^-/-;Gli3^-/-. 20 of the 31 regions were detected in the H3K27ac MicroChIP analyses, those not detected are noted. For the 20 regions detected, they are classified as having H3K27ac in Shh^-/-;Gli3^-/- as ‘present/increased’ or ‘absent/decreased’ compared to WT controls.

HDAC1 binding regions with called peaks with a false discovery rate (FDR) < 0.05 from four biological replicates of E11.5 (40–44S) pooled forelimbs and hindlimbs. Rank ordered coordinates, peak length, log2 fold change (log2FC) and FDR are listed for each peak.

Tab 1. Columns indicate VISTA enhancer IDs, coordinates, number of tissues with limb enhancer activity as annotated by the VISTA database (Visel et al., 2007) and its corresponding GBR category. Tabs 2–5. Normalized signal and called peaks for H3K27ac, ATAC, H3K4me1 and H3K4me2 in WT, and Shh^-/- limbs if applicable, is listed for all HH-responsive GBRs (Tab 2- normalized signal; Tab 3-called peaks) and Stable GBRs (Tab 4- normalized signal, Tab 5-called peaks) that were shown to drive limb activity, as tested in the VISTA database. For called peaks in Tabs 3, 5, ‘NO’ denotes not a significantly called peak, ‘YES’ denotes a called peak.

H3K27ac called peaks with a FDR < 0.05 from two replicates of purmorphamine (‘pm’) treated or DMSO control NIH3T3 cells. For each peak, the assigned Peak ID, coordinates, peak type, fold change normalized to input for purmorphamine treated and control samples, and fold change of purmorphamine treated over control are listed. Additional tabs include sorted files for each peak type.