Figure 7. Alterations in the chromatin landscape around the BU-1 TSS as a function of the position of the +3.5 kb G4 motif.

A–D H3K4me3 (A), H3K9/14ac (B), H3K9me3 (C) and H3K36me3 (D) around the TSS of BU-1 as a function of the distance from the TSS to the +3.5 G4. Right panels: specific ChIP signal. Solid black line: wild type; dashed black line: rev1 cells with the Bu-1a +3.5 G4 DNA knocked out on both alleles and then knocked back into its original position on the BU-1A allele; red line: G4 motif knocked back in at +4.5 kb from TSS; blue line: G4 motif knocked back in at +6 kb from TSS. The specific signal was normalised to total H3 and then to the signal at the TSS in wild-type cells, with the exception of H3K9me3 where there was no significant enrichment above IgG control indicated by the grey lines and symbols. H3 density is shown in Supplementary Fig S4. Left panels: ChIP antibody controls. 8.99 and 13.19 are primer pairs located within the constitutive heterochromatin between the ρ-globin and folate receptor genes at the 5′ end of the β-globin locus, and HS4 is an insulator element separating this heterochromatin from the ρ-globin gene and is enriched in ‘active’ histone marks (Sarkies et al, 2010). GAPDH is a fully active locus. For H3K36me3, the controls are two active loci in which enrichment at the promoter is compared with the body of the gene. Results are normalised to the signal from the ρ-globin 8.99 primer pair. Data are presented as an average of three independent immunoprecipitations from separate pools of cells of the indicated genotype with enrichment monitored by qPCR in triplicate for each immunoprecipitation (i.e. each point consists of nine technical replicates). Error bars = SD of the three biological replicates. For the BU-1 ChIP, data points that are significantly different (P < 0.01, Mann–Whitney test) from wild type are indicated with an asterisk. For the controls, significance was tested against the β-globin 8.99 primer pair.