Figure 4. Promoter-Proximal Methylation Affects RNAPII Recruitment and TBP Binding.

Chromatin isolated from unmethylated and patch-methylated clones 6− and 9P, respectively, was immunoprecipitated using antibodies (Abs) specific for the N-terminal domain of RNAPII (sc-899), the unphosphorylated CTD of RNAPII (8WG16), TBP, or control rabbit IgG. Quantitative real-time PCR was conducted using the transgene specific primers shown (A), or a control primer pair specific for the endogenous β-maj gene. The mean percent (+/− standard deviation) of bound material/total input chromatin for two to three independent chromatin preparations is shown for each ChIP experiment. No enrichment of RNAPII was detected in the silent β-maj gene in either clone with sc-899 (B) or 8WG16 (C), demonstrating the low level of background detected under the conditions used. Analysis of the TATA box and GFP regions of the transgene reveals significant enrichment of RNAPII exclusively in the unmethylated, highly transcribed clone, particularly at the 5′ end of the transgene. Similarly, TBP binding is detected exclusively in the promoter region of the unmethylated clone (D), suggesting that inhibition of transcription of the patch-methylated cassette occurs as the level of transcription initiation.