Figure 3

Vertebrate GAGA factor ( Th-POK ) is associated with intergenic HFRs. Sequence analysis of HFRs from Hox clusters reveals the presence of multiple GAF binding sites. (A) Schematic map (drawn to scale) depicting the location of all the GAGA motifs (vertical red lines). A few motifs appearing close together are merged. Blue boxes denote the Hox genes with transcriptional orientation (the large intronic regions of Hoxa3 and Hoxd3 genes have been omitted for clarity); green boxes indicate the HFRs. (B) Binding of Th-POK (vertebrate GAF) and CTCF across the Hox clusters was assessed by real time ChIP-qPCR assays using a panel of primers designed at the HFRs as well as some genic regions enriched for histone H3. The known Th-POK binding region at the Evx2-Hoxd13 boundary [22] was used as a reference (control). ChIP with Th-POK antibody (left) showed robust binding measured as percentage of input at the control and intergenic HFRs (blue bars) enriched above the background level of the nonspecific immunoglobulin G (IgG, red bars). Genic regions did not show such a high enrichment profile (middle). ChIP with the CTCF antibody (right) did not show any enrichment over the background at the HFRs. Results represent mean ± standard error from three independent experiments with each qPCR assay performed in triplicate. Inset depicts the motif discovered from the HFRs (left) and matched to the GAGA binding site (right) by MEME analysis.