RAD6 regulates the expression of cyclin D1 in both the mRNA and protein level.

<p>(<b>A</b>) HL-7702 cells transfected with an empty control or Myc-RAD6 expressing plasmids were lysed and subjected to RT-PCR and western blot assays. (<b>B</b>) HL-7702 cells transfected with control or RAD6 siRNAs were lysed and subjected to RT-PCR and western blot assays. (<b>C</b>) HL-7702 cells transfected with an empty control and Myc-RAD6 plasmids together with or without CCND1 siRNA were harvested and stained with PI, and then cells were subjected to cell cycle assay. The quantification of the cell cycle assay is shown. (<b>D</b>) Soft agar colony formation assays were performed using control, or RAD6 overexpressing, or RAD6 overexpressing together with CCND1 knocking down HL-7702 cells. (<b>E</b>) HL-7702 cells used in the above assays were lysed and subjected to western blot analyses with antibodies against CCND1, Myc-tag and Actin as indicated.</p

RAD6 regulates G1-S transition in human cells.

<p>(<b>A</b>) HL-7702 cells transfected with an empty control or Myc-RAD6 plasmids were stained with propidium iodide (PI) and subjected to cell cycle analysis. The quantification of the cell cycle distribution is shown below. The percentage of each phase cells was employed for the analysis of cell cycle distribution. (<b>B</b>) HL-7702 cells transfected with control or RAD6 siRNAs were stained with PI and subjected to cell cycle analysis. The quantification of the cell cycle distribution is shown on the right. The percentage of each phase cells was employed for the analysis of cell cycle distribution. (<b>C</b>) The expression levels of RAD6 in cells used in the above cell cycle assays (overexpression, upper; knockdown, lower) were analyzed by western blot assay.</p

RAD6 regulates cell proliferation and tumor growth in vitro.

<p>(<b>A</b>) HL-7702 cells transfected with an empty control or Myc-RAD6 plasmids, or a control siRNA or RAD6 specific siRNAs were subjected to MTT assay. OD570 value was examined for the analysis of cell proliferation. Five replicates of each treatment were employed in this assay (n = 5). (<b>B</b>) HL-7702 cells transfected with an empty control or Myc-RAD6 plasmids were subjected to soft agar colony formation assay. The quantification of the colony number is shown below. Three replicates of each treatmen were employed in this assay (n = 3). (<b>C</b>) HL-7702 cells transfected with control or RAD6 shRNAs were subjected to soft agar colony formation assay. The quantification of the colony number is shown on the right. Three replicates of each treatmen were employed in this assay (n = 3).</p

Microarray Analysis Reveals Potential Biological Functions of Histone H2B Monoubiquitination

<div><p>Histone H2B monoubiquitination is a key histone modification that has significant effects on chromatin higher-order structure and gene transcription. Multiple biological processes have been suggested to be tightly related to the dynamics of H2B monoubiquitination. However, a comprehensive understanding of biological roles of H2B monoubiquitination is still poorly understood. In the present study, we developed an efficient tool to disrupt endogenous H2B monoubiquitination levels by using an H2BK120R mutant construct expressed in human cells. Genome-wide microarray analysis of these cells revealed a potential global view of biological functions of H2B monoubiquitination. Bioinformatics analysis of our data demonstrated that while H2B monoubiquitination expectedly affected a number of previously reported biological pathways, we also uncovered the influence of this histone modification on many novel biological processes. Therefore, our work provided valuable information for understanding the role of H2B monoubiquitination and indicated potential directions for its further studies.</p></div

Functional analysis of H2B monoubiquitination in embryonic stem cell (ESC) differentiation.

<p><b>a.</b> Gene set enrichment analysis revealed that genes encoding proteins involved in cell differentiation were enriched following RNF20 knockdown and H2BK120R overexpression in HEK293T cells. <b>b.</b> Mouse embryonic stem cells (mESC) transfected with H2BWT or H2BK120R mutant plasmids were treated with or without LIF and RA, as indicated, to maintain ES status (LIF+) or to induce differentiation (RA+). Cells were lysed and analyzed by western blot with the specified antibodies. <b>c.</b> Microarray analysis of the mouse embryonic stem cells, as treated in b, was performed. <b>d.</b> Gene ontology (GO) and pathway analyses were performed using the MAS 3.0 online service. <b>e.</b> H2BK120R mutant ES cells showed impaired differentiation potential compared with the H2BWT ES cells. H2BWT or H2BK120R ES cells were treated with or without LIF and RA, as indicated, for 5 days. Cells were then subjected to alkaline phosphatase (AP) staining assay (upper panel). RNF20 knockdown inhibits ES cell differentiation. Control or RNF20-specific shRNA transfected ES cells were treated with or without LIF and RA, as indicated, for 5 days. The morphology of ES colonies was then analyzed by microscopy.</p

Overexpression of H2BK120R efficiently reduced H2B monoubiquitination levels.

<p><b>a.</b> HEK293T cells transfected with an RNF20-specific siRNA or a control siRNA and with a GFP-tagged H2BK120R mutant plasmid or an empty GFP plasmid were harvested after 48 h of transfection. Cell extracts were prepared and analyzed by western blot with the specified antibodies. <b>b.</b> HeLa cells were transfected with an RNF20-specific siRNA or a control siRNA and with a GFP-tagged H2BK120R mutant plasmid or an empty GFP plasmid. Cells were harvested after 48 h of transfection, and cell extracts were prepared. Western blot analysis was performed with the specified antibodies.</p

Correlation between genes differentially expressed following RNF20 knockdown and overexpression of H2BK120R.

<p>Gene correlation analysis was performed with the online MAS 3.0 software (a molecule annotation system, <a href="http://bioinfo.capitalbio.com/mas3/" target="_blank">http://bioinfo.capitalbio.com/mas3/</a>).</p

Microarray analysis of the effects of the loss of H2B monoubiquitination.

<p><b>a.</b> HEK293T cells transfected with an RNF20-specific siRNA and with a Myc-tagged H2BK120R plasmid or an empty Myc plasmid were harvested after 48 h of transfection and lysed for western blot analysis with the specified antibodies. <b>b.</b> HEK293T cells transfected with an RNF20-specific siRNA and with a Myc-tagged H2BK120R plasmid or an empty Myc plasmid were prepared for microarray analysis. Heatmap of the differentially expressed genes following RNF20 knockdown and H2BK120R overexpression is shown in the upper left panel. The numbers of differentially expressed genes are shown in the bar and Venn diagrams. <b>c.</b> Gene ontology (GO) analysis was performed with the MAS 3.0 online platform launched by the CapitalBio Company. <b>d.</b> Pathway analysis was performed with the MAS 3.0 online platform launched by the CapitalBio Company. <b>e.</b> A series of differentially expressed genes following RNF20 knockdown and H2BK120R overexpression were randomly selected and analyzed by real-time PCR using gene-specific primers. The RT-PCR assays were repeated independently for three times, and five replicates were used in each time. Error bars indicate SD, n = 5.</p

H2B monoubiquitination is involved in the DNA damage response.

<p><b>a.</b> Gene set enrichment analysis revealed that genes coding for components of the DNA damage repair were enriched following RNF20 knockdown and H2BK120R overexpression in HEK293T cells. <b>b.</b> Effects of RNF20 knockdown and H2BK120R overexpression on HR and NHEJ mediated DNA DSB repair. The DNA DSB repair efficiency was calculated as we previously described [<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133444#pone.0133444.ref027" target="_blank">27</a></b>]. More than three replicates were used in each analyses. **, <i>P <</i> 0.01 (two-tailed unpaired <i>t</i> test; <i>n</i> > 3). <b>c.</b> H2BWT and H2BK120R mutant plasmids transfected HEK293T cells were subjected to X-Ray irradiation (80 Kv for 5 min), and then cells were cultured for the indicated times. Cells were harvested and analyzed by western blot with the specified antibodies.</p