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A tumor suppressor function of Smurf2 associated with controlling chromatin landscape and genome stability through RNF20

In addition to allelic mutations, cancers are known to harbor alterations in their chromatin landscape. Here, we show that genomic ablation of Smurf2, a HECT-domain E3 ubiquitin ligase, results in dysregulation of DNA damage response and genomic stability, culminating to increased susceptibility to various types of cancers in aged mice. We demonstrate that Smurf2 regulates histone H2B monoubiquitination as well as histone H3 tri-methylation at K4 and K79 by targeting RNF20 to proteasomal degradation in both mouse and human cells. We further show that Smurf2 and RNF20 are co-localized at the γ-H2AX foci of double-stranded DNA breaks in the nucleus. Thus, Smurf2 has a tumor suppression function that normally maintains genomic stability by controlling the epigenetic landscape of histone modifications through RNF20.

. Chromatin compaction and acetylation pattern of histones in Smurf2-deficient cells (a) Assessment of global chromatin compaction in primary mouse dermal fibroblasts (MDFs) derived from WT and Smurf2 -/-(KO) animals. Whole cell nuclei were treated with MNase for indicated period of time. DNA was then extracted and visualized in agarose gelelectrophoresis. (b) Quantification of (a). N1-N7 indicates the number of nucleosomes in each oligonucleosome. (c) Analysis of acetylation pattern of histones in B6-and BL-derived WT and KO MEFs. Nucleosomal histones were extracted from cells using acid-based extraction protocol 1 hr following cell treatment with HDAC inhibitor Trichostatin A (500 ng/ml). Acetylation pattern of core histones was then analyzed in Western blot using indicated antibodies. (b) Validation of specificity of the anti-RNF20 antibody in immunofluorescence. MEFs, grown on coverslips, were transfected with either non-silencing (NS) siRNA or siRNA against murine RNF20. Fourty-eight hours later cells were immunostained with anti-RNF20 antibody, counterstained with DAPI and analyzed under a confocal microscope. All images were obtained at equal microscope and camera settings. Bars = 20 m. (c) RT-PCR analysis of RNF20 mRNA expression in immortalized WT and Smurf2KO MEFs. (d) Western blot analyses of RNF20 turnover rate in MEFs after cycloheximide (CHX) treatment (50 g/ml). The quantification of RNF20 level relative to that of actin is shown in the bottom panel.
(e) Immunohistochemical staining of RNF20 in tumor and adjacent normal tissues derived from Smurf2 -/mice. Figure S7. Smurf2 interacts with RNF20 in cells and leads to RNF20 degradation (a) Reciprocal co-immunoprecipitation experiments showing interaction between MYC-Smurf2 and FLAG-RNF20 expressed in HEK293 cells. WCL, whole cell lysate. (b) Co-localization of Smurf2 with RNF20 in cell nuclei. GFP-tagged Smurf2 was expressed in U2OS cells by transient transfection. Endogenous RNF20 was detected with anti-RNF20 antibody, and the images were taken using a confocal microscope. Bars = 10 m. (c) Overexpression of Smurf2 decreases intracellular levels of endogenous RNF20 (arrow). Bars = 20 m. Figure S8. Inactivation of RNF20 increases chromatin compaction and reduced the levels of ubH2B in different human cancer cells (a) MNase digestion of whole nuclei isolated from U2OS cells following RNF20 knockdown (72 hrs). Quantification of N1-N3 nucleosome is shown in the right. (b) MNase digestion of whole nuclei isolated from HCT116 cells following RNF20 knockdown (72 hrs). Quantification of N1-N3 nucleosome is shown in the right. (c) Depletion of RNF20 significantly reduces the levels of ubH2B in the panel of different human tumor cell lines. The expression of RNF20 and the levels of ubH2B were monitored 72 hrs later on the whole cell lysates and chromatin fractions prepared from these cells that transfected with indicated siRNA.

Figure S9. Proposed mechanism of Smurf2 action as a regulator of chromatin compaction and genomic integrity
Blank M et al., Table S1 Supplemental

Cell growth and transformation assay
To assess cell growth, cells were counted and plated into 60 mm plates at equal density and allowed to grow for the indicated period of time. Then the cells were trypsinized, combined with their medium supernatants, centrifuged, and stained with 0.2% Trypan blue.
Cell number and viability were analyzed in a Cellometer TM Auto T4 Cell Counter (Nexcelom Bioscience).
The ability of cells to undergo transformation was evaluated in a foci formation assay 23 . Immortalized MEFs were seeded at equal density of 100, 200 or 500 cells into 60 mm plates. After 11 days, colonies were visualized by crystal violet staining. Only colonies consisting of at least 50 cells were considered for the scoring. The transformed nature of the colonies was also verified and photographed under a light microscope.

Cell cycle and cell death analysis
Cell cycle progression was analyzed in two-dimensional flow cytometry. The analysis of cells in active M-phase with anti-phospho-Serine10 Histone H3 antibody (Upstate) was performed as described 40 . For assessment of cell cycle progression through the S-phase, cells were pulsed with BrdU (10 µM; 45 min), fixed, and treated with 2M HCl and 0.1 M sodium borate (pH 8.5). The cells were then immunostained with mouse anti-BrdU antibody (Chemicon), followed by labeling with FITC-conjugated anti-mouse IgG secondary antibody. Following treatment with DNase-free RNase (5 µg/ml; 37 o C for 30 min) and staining with propidium iodide (PI), the samples were analyzed in biparametric FACS. All FACS data were collected using a FACSCalibur equipped with BD Cell Quest TM Pro software (BD Biosciences). Cell cycle gating and analysis were carried out using WinMDI 2.8 software.