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. 2013 Dec 9;203(5):767–783. doi: 10.1083/jcb.201305027

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© 2013 Bian et al.

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Figure 6. — ChIP-qPCR measurements of context-specific H3K9me3 modifications over PTR1 and HBB BAC transgenes. (a) Map of HBB BAC with location of primers used for ChIP-qPCR; primer spacing was ∼1 kb over PTR1 but ∼5 kb elsewhere. (b–d) ChIP-qPCR measurements of H3K9me3 levels with the percent input values normalized by scaling linearly between 0 (GAPDH promoter) and 1 (IAP transposon; see Results H3K9me3 ChIP over PTR1 and the HBB locus and its correlation with H3K9m3 immunofluorescence and nuclear localization section). (b) Three biological replicates (A, B, and C) show reproducibility of H3K9me3 ChIP over HBB BAC with consistent peak over PTR1. (c) H3K9me3 ChIP values of full-length HBB BAC (green) versus HBBD4 BAC deleted of all PTRs (red) or HBBD4 BAC with PTR1 reinserted at arrow location (blue; inset shows PTR1 values with actual orientation). (d) H3K9me3 ChIP values for HBB BAC by itself or intact HBB BAC flanked by transcriptionally active DHFR BAC transgenes. (e) Mean H3K9me3 ChIP levels are lower for HBBD4 relative to HBB or HBBD4 + PTR1 BACs. (f) Mean H3K9me3 immunofluorescence (IF) levels are similarly reduced for HBBD4 versus HBB transgenes using a normalized, linear scaling of immunofluorescence values between DAPI regions with low immunofluorescence (0) and chromocenter immunofluorescence (1) (see Results section). (g) Normalized mean H3K9me3 ChIP values are decreased over PTR1 in plasmid transgenes relative to HBB, HBBD4 + PTR1, or HBB flanked by DHFR BAC transgenes. (h) Reduced H3K9me3 mean, normalized values for an ∼100-kb HBB region (primer pairs 2–18) without PTRs in HBBD4 BAC lacking PTRs but also in full-length HBB BAC transgenes flanked by DHFR BACs. (c–h) Error bars show SEM. (e–h) Statistical significance: *, P < 0.05; **, P < 0.01.