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. 2015 Oct;22(10):1630-40.
doi: 10.1038/cdd.2015.6. Epub 2015 Feb 20.

JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein

L Zhao  1 , Y Zhang  2 , Y Gao  1 , P Geng  2 , Y Lu  2 , X Liu  1 , R Yao  1 , P Hou  2 , D Liu  3 , J Lu  2 , B Huang  1
Affiliations

JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein

L Zhao et al. Cell Death Differ. 2015 Oct.

Abstract

Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which can stabilize the state of senescence. The retinoblastoma (RB) protein has an important role in SAHF; cells that lack active RB pathway fail to form SAHF. It has been known that the posttranslational modifications of RB, for example, phosphorylation, regulate its function. To date, whether methylation of RB impacts on the SAHF formation is unknown. Here we report that JMJD3, a histone demethylase catalyzing the tri-methylation of H3K27 (H3K27me3), can demethylate the non-histone protein RB at the lysine810 residue (K810), which is a target of the methyltransferase Set7/9. We detected a significant upregulation of JMJD3 during cellular senescence and SAHF formation in WI38 cells induced by H-RasV(12), and we found that ectopic expression of JMJD3 promoted cellular senescence and SAHF formation in WI38 cells. Furthermore, during the process of SAHF assembly, JMJD3 was transported to the cytoplasm and interacted with RB through its demethylase domain JmjC. Significantly, our data demonstrated that the JMJD3-mediated demethylation of RB at K810 impeded the interaction of RB with the protein kinase CDK4 and resulted in reduced level of phosphorylation of RB at Serine807/811 (S807/811), implicating an important role of the interplay between the demethylation and phosphorylation of RB in SAHF assembly. This study highlights the role of JMJD3 as a novel inducer of SAHF formation through demethylating RB and provides new insights into the mechanisms of cellular senescence and SAHF assembly.

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Figures

Figure 1
Figure 1
JMJD3 was required for Ras-induced SAHF assembly. (a and b) Reverse transcriptase-PCR (a) and western blotting (b) analysis of the expression levels of JMJD3 in Ras-induced SAHF. (c) Western blots of JMJD3 protein expression in WI38 cells infected with JMJD3 shRNA (shJMJD3#1/#2/#3) or the control vector shRNA (shCtrl). (d) SA-β-gal staining of WI38 cells. Cells were infected with shJMJD3#2/#3; after 2 days cells were infected with H-RasV12. The percentage of SA-β-gal positive cells was calculated (Left). Scale bars: 200 μm. Western blotting (Right) was performed to confirm the expression of JMJD3 and H-RasV12 after retroviral infection. (e) Cells from panel (d) were stained with 4,6-diamidino-2-phenylindole (DAPI) to calculate the percentage of cells with DAPI foci. (f) Cells from panel (d) were pulse-labeled with 5′-BrdU (bromodeoxyuridine) for 24 h and then immunofluorescence stained to detect the 5′-BrdU. Error bar represent mean±S.D.
Figure 2
Figure 2
Ectopic JMJD3 promoted SAHF formation in WI38 cells. (a) Western blotting confirmation of the ectopic expression of JMJD3 and JMJD3H1390A after retroviral infection in WI38 cells. (b) WI38 cells were infected with JMJD3 and JMJD3H1390A for 6 days and then stained with SA-β-gal. Cells positive of SA-β-gal activity were calculated (Right). (c) WI38 cells infected with either JMJD3 or JMJD3H1390A were stained with 4,6-diamidino-2-phenylindole (DAPI), and the percentage of cells with DAPI foci was calculated. (d) WI38 cells from panel (c) were pulse-labeled with 5′-BrdU (bromodeoxyuridine) for 24 h and then immunofluorescence stained to detect the 5′-BrdU. A total of 100 cells were scored for 5′-BrdU incorporation. (e) Confocal fluorescence microscopy of SAHF markers (H3K9me3 and HMGA1) in WI38 cells and WI38 cells infected JMJD3 or JMJD3H1390A. Scale bars: 5 μm. Error bars represent the mean S.D. of triplicate experiments
Figure 3
Figure 3
JMJD3 was located in cytoplasm in the process of SAHF assembly. (a) Western blots of the methylation levels of H3K27me3/me2/me1 and H3K9me3 in the process of Ras-induced SAHF formation. (b) Confocal fluorescence microscopy of JMJD3 in SAHF formation. Scale bars: 5 μm. (c) The presence of JMJD3 protein in cytoplasmic and nuclear extracts
Figure 4
Figure 4
JMJD3 interacted with RB by its Jmjc domain. (a and b) Co-IP assays showing the relationship between JMJD3 and RB. 293T cells were cotransfected with a His-JMJD3 expression vector, a Flag-RB expression vector or a mock control vector. The interaction of His-JMJD3 and Flag-RB was examined by immunoprecipitation using anti-FLAG and anti-His antibodies (a). Endogenous JMJD3 and RB were coimmunoprecipitated from WI38 cells infected with H-RasV12 (b); the histogram at the right shows the data of densitometric analysis. (c) Purified GST-RB was pulled down with cell lysate of HEK-293T expressing JMJD3. (d) Schematic representation of the JmjC domain. (e) Purified GST-JmjC was pulled down with cell lysate of HEK-293 overexpressing RB. (f) Model of truncated fragments of RB. (g) Purified GST-tagged mutant RB was pulled down with JMJD3. (h) JmjC domain of JMJD3 directly interacted with RB(659-840) in vitro. GST pull-down assays was performed with the indicated GST-fused JmjC domain and purified Flag-RB(659-840) in HEK-293 cells
Figure 5
Figure 5
JMJD3 demethylated RB at K810 residue. (a) WI38 cells were infected with H-RasV12 for the days indicated and coimmunoprecipitated with anti-RB, and then the levels of Lys-methylation and phosphorylation of RB (Ser807/811) were determined. (b) In vitro demethylation by JmjC and JmjC mut was performed using purified Flag-RB(659–840) recombinant protein from HEK-293 cells and immunoblotted with anti-Lys-me antibody. (c) In vitro methylation–demethylation of GST-fused RB(659–840). The protein was methylated by methyltransferase Set7/9 and then demethylation by JmjC or JmjC mut. The top blot shows the validation of the anti-Lys-me antibody. (d) In vitro methylation–demethylation assay. The methylated proteins were visualized with fluorography. Densitometric analysis is shown on the right. (e) In vitro demethylation assay of RB peptide containing methylated K810 was performed using JmjC or JmjC mut. Anti-Lys-me antibody was used for dot blotting. (f) WI38 cells were infected with Flag-RB/Flag-RB(K810R), then infected with or without H-RasV12, coimmunoprecipitated with Flag and the level of methylated Lysine was detected. (g) WI38 cells were infected with a truncated mutation of Flag-RB (659-840K810R), then infected with or without H-RasV12, immunoprecipitated with Flag and the level of methylated Lysine was tested
Figure 6
Figure 6
JMJD3-mediated RB demethylation reduced the level of RB phosphorylation. (a) Determination of phosphorylation of RB (Ser807/811) in WI38 cells ectopically expressing JMJD3 or JMJD3H1390A. (b) WI38 cells were infected with shJMJD3 to knock down endogenous JMJD3 and then infected with H-RasV12. Cell lysis was immunoblotted with anti-phospho-RB(Ser807/811) antibody. (c) WI38 cells were infected with Flag-RB, Flag-RBK810R, co-infected with Flag-RB and JMJD3 or with Flag-RB and H-RasV12, coimmunoprecipitated with anti-Flag antibody and immunoblotted with anti-phospho-RB(S807/811) antibody. (d) Cell extracts from WI38 cells ectopically expressing JMJD3 were coimmunoprecipitated with anti-RB antibody and immunoblotted with anti-CDK4 antibody. (e) WI38 cells were infected with shJMJD3, and the binding of CDK4 to RB was examined using co-IP. (f) mRNA expression levels of E2F targets were assessed by real-time quantitative PCR in WI38 cells ectopically expressing RB or RB(K810R). The experiments were repeated at least three times. Error bars represent mean±S.D. *P<0.05. (g) Cells infected with Flag-RB or Flag-RB(K810R) were stained with 4,6-diamidino-2-phenylindole (DAPI) to calculate the percentage of cells with DAPI foci. (h) Immunofluorescence of Flag and H3K9me3/HMGA1 of WI38 cells infected with Falg-RB or Flag-RBK810R. Scale bars: 5 μm
Figure 7
Figure 7
A proposed working model for JMJD3 function in regulation of cellular senescence. In normal growing cells, RB is phosphorylated by CDK4, releases E2F, promotes the transcription of its target genes and results in cell cycle progression. When cells are exposed to the senescence signals, such as oxidative stress, DNA damage or hyperoncogenic signaling pressure, JMJD3 demethylates RB at K810, represses CDK4 binding to RB, reduces the level of phosphorylation of RB at S807/811 and induces senescence and SAHF formation
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