The role of H2B monoubiquitination in cellular differentiation

Histones, subjected to post-translational modifications, are important regulators of the cellular processes. One of these modifications is monoubiquitination of histone H2B (H2Bub1). H2Bub1 is associated with the actively transcribed genes. Moreover, H2Bub1 is required for the proper DNA repair and was recently reported to be lost during tumor progression. The levels of H2Bub1 in the cell are tightly regulated. In mammals, ubiquitination is mediated by the E3 ubiquitin ligase RNF20/40. Another important upstream regulator of H2Bub1 is the CDK9 enzyme that promotes transcriptional elongation. It, together with an adaptor protein WAC, facilitates the RNF20/40 recruitment to the chromatin. Differentiation of the cell is a process that results in cellular specialization and acquiring a physiological function. It is accompanied by the significant changes in gene expression and in histone modification patterns. This project aimed to understand the role of H2Bub1 in cellular differentiation. Investigating human mesenchymal stem cells (hMSCs) it was observed that the H2Bub1 levels increase during differentiation into osteoblasts and adipocytes. Depletion of the H2Bub1 regulators RNF40, WAC and CDK9 resulted in inhibition of the hMSC differentiation suggesting that H2Bub1 is required for the correct progression of this process. Mechanistically, H2Bub1 was shown to participate in the activation of the “bivalent” genes that carry activatory and inhibitory histone marks. H2Bub1 deposition is required for removal of the repressive H3K27me3 from the differentiation-dependent genes. Taken together, these observations for the first time demonstrate the involvement of H2Bub1 in cellular differentiation. The proposed model suggests that H2Bub1 executes its function via promoting the resolution of bivalency on the differentiation-specific genes. These results give additional insights into H2Bub1 function during transcription of the certain subsets of genes. The obtained knowledge increases our understanding of the transcriptional regulation, carcinogenesis and stem cell biology

MDM2 Associates with Polycomb Repressor Complex 2 and Enhances Stemness-Promoting Chromatin Modifications Independent of p53

SummaryThe MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of lineage-specific genes, independent of p53

Polyadenylation of histone H2B genes assigned using polyadenylation and alternative polyadenylation (APA) map.

<p>Number of reads at polyA sites on different replication- dependent histone H2B genes which are mapped using xPAD server (<a href="http://johnlab.org/xpad/" target="_blank">http://johnlab.org/xpad/</a>) in (A) MCF7 (human breast cancer cell line), (B) MCF10A (immortalized human mammary epithelial cell line) cells and (C) normal and breast tumor tissues.</p

Comparison of stem-loop sequences in H2B genes.

<p>Alignment of the stem-loop sequences of H2B genes performed with ClustalW2 multiple sequence alignments tools (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). Highly expressed histones are marked in grey. Stem loop sequence is shown in orange, bases that are different from canonical are underlined.</p

Expression of the histone H2B gene complement in different cell lines.

<p>Expression of different H2B genes in the indicated cell lines was analyzed by qRT-PCR. Relative expression values between the individual genes were normalized using diploid genomic DNA (see materials and methods) and indicated as “Rel. gDNA units”. Mean±SD, n = 3.</p

Expression of spliced histone transcripts are increased during committed osteoblast differentiation.

<p>(A) hFOB 1.19 cells were differentiated into osteoblasts for 7 days. Alkaline phosphatase (<i>ALPL</i>) expression in undifferentiated (undiff.) and differentiated (OB) cells was analyzed by qRT-PCR. Values were normalized to <i>HNRNPK</i> expression. Mean±SD, n = 3. (B, C) Samples shown in (A) were examined for (B) spliced <i>HIST1H2BD</i> and (C) <i>HIST1H2AC</i> expression. Values were normalized to total <i>HISTH2BD</i> and <i>HISTH2AC</i> respectively. Mean±SD, n = 3. P-values were calculated and statistical significance is represented as follows (*P≤0.05; **P≤0.01).</p