H2B is monoubiquitylated cotranscriptionally. (A) HeLa cells were treated with 100 μM DRB for the indicated times, and cell extracts were subjected to Western blot analysis with the indicated antibodies. (B) HeLa cells were either not treated (no DRB) or treated with 100 μM DRB for 3 h, and harvested at the indicated time points after DRB removal. Cell extracts were subjected to Western blot analysis with the indicated antibodies. (C) qRT-PCR analysis of TTC17 pre-mRNA in HeLa cells in proximal (1 kb downstream from the TSS) and distal (92 kb downstream from the TSS) regions of the gene, following 3 h of 100 μM DRB treatment (DRB) and at the indicated time points after DRB removal. All values were normalized to 16S rRNA in the same sample. (D–F) Treatment as in C, but cells were subjected to ChIP analysis with antibodies against H2Bub1 (D), H3K36me3 (E), and H3K79me2 (F). Bars indicate ChIP readings normalized to input in the same treatment and represent averages from duplicate qPCR reactions; error bars, SD. Similar results were obtained in four independent experiments.
