Figure 6.

EZH2 inhibition alters germline transcript expression due to a decrease in H3K27me3 on the Vβ segment promoter and recombination signal sequence regions. The SCIET27 cell line was treated overnight with the specific inhibitor GSK126 at a concentration of 2 µM. Both the mRNA and histones were extracted, and ChIP was performed. (A) To evaluate the efficiency of GSK126 inhibition, the H3K27me3 level was quantified by Western blotting. The right panel shows one representative Western blot out of three independent experiments. The left panel presents the relative expression b of H3K27me3 normalized to histone H3 used as a loading control. (B) The presence of trimethylated H3K27 on the promoter and the RSS regions of four Vβ segments was analyzed by ChIP. Graphics present the enrichment of H3K27me3 on the eight different chromatin regions. Symbols correspond to three independent experiments. Median is indicated as a black line. (C) Expression of four Vβ germline transcripts evaluated by semiquantitative PCR. Left panel, representative experiment out of three independent experiments. Right panel, relative intensity of the Vβ germline transcripts’ amplification normalized to PPIA intensity. Data are mean ± SD of three independent experiments. Mann–Whitney tests (A,C) or t-tests (B) were used to reveal statistically significant differences. * p < 0.05, ** p < 0.01, *** p < 0.001, t indicates a tendency.