Microarray Analysis Reveals Potential Biological Functions of Histone H2B Monoubiquitination

Abstract

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.

Fig 1. Overexpression of H2BK120R efficiently reduced H2B monoubiquitination levels.

a. 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. 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.

Fig 2. Microarray analysis of the effects of the loss of H2B monoubiquitination.

a. 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. 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. c. Gene ontology (GO) analysis was performed with the MAS 3.0 online platform launched by the CapitalBio Company. d. Pathway analysis was performed with the MAS 3.0 online platform launched by the CapitalBio Company. e. 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.

Fig 3. Correlation between genes differentially expressed following RNF20 knockdown and overexpression of H2BK120R.

Gene correlation analysis was performed with the online MAS 3.0 software (a molecule annotation system, http://bioinfo.capitalbio.com/mas3/).

Fig 4. Functional analysis of H2B monoubiquitination in embryonic stem cell (ESC) differentiation.

a. 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. 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. c. Microarray analysis of the mouse embryonic stem cells, as treated in b, was performed. d. Gene ontology (GO) and pathway analyses were performed using the MAS 3.0 online service. e. 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.

Fig 5. H2B monoubiquitination is involved in the DNA damage response.

a. 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. 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 []. More than three replicates were used in each analyses. **, P < 0.01 (two-tailed unpaired t test; n > 3). c. 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.

Fig 6. H2B monoubiquitination is essential for chromatin higher-order organization and stability.

a. Gene set enrichment analysis revealed that genes encoding proteins involved in chromatin remodeling and organization were enriched following RNF20 knockdown and H2BK120R overexpression in HEK293T cells. b. Gene set enrichment analysis revealed that genes encoding methyltransferases were enriched following RNF20 knockdown and H2BK120R overexpression in HEK293T cells. The majority of these genes encode histone-related methyltransferases. c. HeLa cells transfected with a control siRNA or an RNF20 siRNA and with an H2BK120R mutant plasmid or a control empty Myc plasmid were harvested and stained with anti-tubulin antibody (green) and DAPI (blue). Cells were analyzed by fluorescence microscopy. 200 anaphase cells were analyzed and quantified as indicated in the bar diagram. d. HeLa cells transfected with a control siRNA or an RNF20 siRNA and with an H2BK120R mutant plasmid or a control empty Myc plasmid were harvested and stained with DAPI (blue). Cells were photographed with fluorescence microscopy. 3000 cell nuclei were analyzed and quantified as indicated in the bar diagram.