Re-introduction of Lsh into Lsh−/− cells restores Pol II stalling and requires the presence of Dnmt3b.
(A) Western analysis using anti-Lsh antibodies or anti-Flag antibodies to detect Lsh protein expression in Lsh−/− MEFs stably transfected with the tet-off repressor Lsh vector before and after 24 hrs of tetracycline treatment. Note, that traces of Lsh protein were visible after longer exposure of the blot indicating a slight leakiness of the repressor system. (B) RT-PCR analysis for detection of HoxC6 and HoxC8 and Lsh mRNA comparing extracts derived from Lsh−/−MEFs before and after 24 hours of tetracycline treatment. (C) Genomic DNA derived from Lsh−/− MEFs (before and after 24 hrs of tetracycline treatment) was examined by bisulphite sequencing at regions downstream of TSS at the HoxC6 (Left) and the HoxC8 (Right) genes. (D) Ratio of Ser 5 Pol II ChIP signals for the TSS region to the downstream region and the ratio of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1 comparing chromatin derived from Lsh−/− MEFs before and after 24 hours of tetracycline treatment to induce Lsh. Error bars represented the standard errors of the mean of three independent experiments. Western Blot (E) and Real-time RT-PCR analysis (F) for detection of Dnmt3b mRNA from Lsh inducible cells treated with control siRNA (siControl) or siRNA targeted against Dnmt3b (siDnmt3b) for 48 hrs. (G) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh. (H) Summary of bisulphite sequencing results derived from ten clones for each sample (Figure S10) to illustrate the percentage of CpG methylation loss at the first 10 (HoxC6, left) or 10 (HoxC8, right) CpG sites downstream of TSS in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh. (I) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 genes in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh.
