An Unmethylated 3′ Promoter-Proximal Region Is Required for Efficient Transcription Initiation (original) (raw)
Figure 5
Promoter-Proximal DNA Methylation Leads to Hypoacetylation of H3K9/14 across the Transgene and Local H3K9 Trimethylation
Chromatin was generated from clones 6− and 9P, and ChIP was conducted using antibodies specific for H3 acetylated on K9/K14, H3 trimethylated on K9 or control rabbit IgG. Quantitative real-time PCR was carried out on the immunoprecipitated DNA using primers specific for the endogenous Gnas and/or β-maj genes as internal controls and the TATA box or GFP regions of the transgene (shown in Figure 4A). For each antibody, values shown represent the mean percent (+/− standard deviation) of bound material/total input chromatin for three independent experiments.
(A) Analysis of the TATA box and GFP regions of the transgene reveals enrichment of H3K9/14Ac (H3Ac) exclusively in the highly transcribed unmethylated cassette. The level of enrichment of H3K9/14Ac in the β-maj gene promoter region was similar between the two clones, indicating that the immunoprecipitations worked with similar efficiencies.
(B) Analysis of the TATA box and GFP regions of the transgene reveals a significantly higher level of H3K9me3 (H3K9m) enrichment exclusively in the GFP region of clone 9P. As this region shows a high density of DNA methylation, these results reveal that DNA methylation is sufficient to promote H3K9 trimethylation in this euchromatic region. The levels of enrichment of H3K9me3 at Gnas and β-maj was similar between the two clones, indicating that the immunoprecipitations worked with similar efficiencies.