Inhibition of transcription does not affect the total amount of ubiquitinated histone 2A in chromatin.

Using a polyclonal anti-ubiquitin antibody in Western blotting experiments, we detected three antibody-binding components in a HeLa cell extract: ubiquitin, a ubiquitin-histone 2A conjugate (uH2A) and a 17 kD protein, probably corresponding to an additional ubiquitin conjugate. Since ubiquitination of histone 2A (H2A) has been invoked in the transcription process, the amount of uH2A was studied after inhibition of ribosomal RNA (rRNA) synthesis with actinomycin D and of heterogeneous nuclear RNA (hnRNA) synthesis with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). The amount of uH2A did not change, suggesting that the overall level of ubiquitination of histone 2A is not directly coupled to on-going transcription of either rRNA or hnRNA. Since the uH2A content of protein coding genes constitutes a considerable portion of total chromatin uH2A, it seems also likely that there is no major change in the degree of ubiquitination on the templates of the protein-coding genes themselves upon cessation of transcription. It is proposed that the pattern of ubiquitination of histone 2A is established on a long-term basis and that it is related to the overall organization and distribution of the chromatin material in the interphase nucleus.