The Ubiquitin-like with PHD and Ring Finger Domains 1 (UHRF1)/DNA Methyltransferase 1 (DNMT1) Axis Is a Primary Regulator of Cell Senescence

Abstract

As senescence develops, cells sequentially acquire diverse senescent phenotypes along with simultaneous multistage gene reprogramming. It remains unclear what acts as the key regulator of the collective changes in gene expression at initiation of senescent reprogramming. Here we analyzed time series gene expression profiles obtained in two different senescence models in human diploid fibroblasts: replicative senescence and H₂O₂-induced senescence. Our results demonstrate that suppression of DNA methyltransferase 1 (DNMT1)-mediated DNA methylation activity was an initial event prior to the display of senescent phenotypes. We identified seven DNMT1-interacting proteins, ubiquitin-like with PHD and ring finger domains 1 (UHRF1), EZH2, CHEK1, SUV39H1, CBX5, PARP1, and HELLS (also known as LSH (lymphoid-specific helicase) 1), as being commonly down-regulated at the same time point as DNMT1 in both senescence models. Knockdown experiments revealed that, among the DNMT1-interacting proteins, only UHRF1 knockdown suppressed DNMT1 transcription. However, UHRF1 overexpression alone did not induce DNMT1 expression, indicating that UHRF1 was essential but not sufficient for DNMT1 transcription. Although UHRF1 knockdown effectively induced senescence, this was significantly attenuated by DNMT1 overexpression, clearly implicating the UHRF1/DNMT1 axis in senescence. Bioinformatics analysis further identified WNT5A as a downstream effector of UHRF1/DNMT1-mediated senescence. Senescence-associated hypomethylation was found at base pairs -1569 to -1363 from the transcription start site of the WNT5A gene in senescent human diploid fibroblasts. As expected, WNT5A overexpression induced senescent phenotypes. Overall, our results indicate that decreased UHRF1 expression is a key initial event in the suppression of DNMT1-mediated DNA methylation and in the consequent induction of senescence via increasing WNT5A expression.

Keywords: DNA methylation; cellular senescence; gene expression; gene regulation; microarray.

FIGURE 1.

Decreased DNMT1-mediated DNA methylation activity is involved in senescence of HDF. A–C, HS of primary HDFs was developed as described under “Experimental Procedures.” A, representative images of the SA-β-gal assay. C, control; d, days. B, Western blotting analyses for DNMT1 and DNMT3A (left) and their quantifications (right). MW, molecular weight. C, messenger RNA levels by qRT-PCR. **, p < 0.01 versus control primary HDFs by Student's t test. D–F, RS of primary HDFs was generated as described under “Experimental Procedures,” and HDFs of DT2 (black columns) and DT14 (white columns) were used. D, quantifications (left) and representative images (right) of the SA-β-gal assay. E, Western blotting analysis (left) and their quantifications (right). F, messenger RNA levels by qRT-PCR. **, p < 0.01 versus DT2 by Student's t test. G, HDFs (DT2) were exposed to the indicated concentrations of 5-AzC (Sigma) for 5 days. Representative images (top panel) and quantifications (bottom left panel) of the SA-β-gal assay are shown. Also shown is a Western blotting analysis (bottom right panel). Con, control. **, p < 0.01 versus Con by Student's t test. H, HDFs (DT2) were transfected with siRNA for DNMT1 for 5 days. Representative images (top panel) and quantification data (bottom left panel) of the SA-β-gal assay are shown. Also shown is a Western blotting analysis (right bottom panel). **, p < 0.01 versus siRNA for negative control (siNC) or control by Student's t test.

FIGURE 2.

Expression of DNMT1-interacting proteins commonly regulated in both RS and HS of HDFs. A, time series HDFs obtained from the HS model were subjected cDNA microarray. A heat map of the time series gene expression profile is shown. C, control; d, days. B, progressively up-regulated (HS_UP, 310 genes) and down-regulated genes (HS_DOWN, 404 genes) were matched with four different modular genes (G1∼G4) identified from the time series RS model in our previous report (5). C, enrichment score indicating the −log10-transformed p values calculated from the gene set enrichment analysis. D, Venn diagram showing the number of the overlapping genes among the gene signatures for DIPs and RS and HS models. Seven genes were identified to be commonly regulated in the progress of the two HDF senescence models. Also shown are heat maps of time series gene expression profiles of the seven DIPs (top panels) and SA-β-gal assay (bottom panels) in the two HDF senescence models. **, p < 0.01 versus DT2 (left graph) or C (control, right graph) by Student's t test. E, the protein expression levels of the seven DIPs were validated by Western blotting analysis. MW, molecular weight.

FIGURE 3.

UHRF1 is an upstream regulator of DNMT1 expression. A–C, HDFs (DT2) were transfected with siRNAs for the indicated targets for 3 days. A, Western blotting analyses. The bands of knockdown (KD) targets were obtained at the same position as shown in Fig. 2E. NC, negative control; MW, molecular weight. B, Western blotting analyses (top panel) and their quantification (bottom panel). ex, exposure. **, p < 0.01 versus siNC. C, messenger RNA levels by qRT-PCR. **, p < 0.01 versus siNC. D, an HDF (DT7) was infected with a recombinant retrovirus (rRV) harboring the indicated target cDNA for 3 days. Shown are messenger RNA levels by qRT-PCR (left panel) and Western blotting analyses (right panel). **, p < 0.01 versus RFP by Student's t test. AU, arbitrary unit. **, p < 0.01 versus siNC. E, HDFs (DT2) were transfected with siUHRF1 for 5 days. Intracellular ROS levels were monitored by flow cytometric analysis after staining cells with DCF-DA fluorescence dye (DCF fl). *, p < 0.05 versus siNC; **, p < 0.01 versus siNC. F, after an HDFs (DT2) was transfected with siRNA for UHRF1 (siUHRF1) for 24 h, the cells were transfected again with the pGL3-DNMT1-pro plasmid for 2 days and then subjected to a promoter assay. G, HDFs (DT2) were exposed to the indicated dose of H₂O₂ for 2 days, and intracellular ROS levels were monitored. **, p < 0.01 versus no H₂O₂ treatment. H, after an HDF (DT2) was transfected with the pGL3-DNMT1-pro or pGL3-basic plasmid (pGL3) for 24 h, the cell was exposed to the indicated dose of H₂O₂ for 2 days and then subjected to intracellular ROS level analysis using DCF-DA fluorescence dye. *, p < 0.05 versus siNC or no H₂O₂ treatment by Student's t test.

FIGURE 4.

Knockdown of UHRF1 effectively induces senescence via DNMT1 suppression. A–C, an HDF (DT2) was transfected with siRNAs for the indicated targets for 5 days. A, quantification (top panel) and representative images (bottom panel) of the SA-β-gal assay are shown. B, knockdown levels of the targets were confirmed by Western blotting analysis. **, p < 0.01 versus siNC by Student's t test. MW, molecular weight. C, Western blotting analysis. NC, negative control. D–F, an HDF (DT2) was transfected with siRNAs for the indicated targets and on the next day infected with an rRV harboring the indicated target cDNA for 4 days. DCF-fl, DCF-DA fluorescence dye. D, intracellular ROS levels using DCF-DA fluorescence dye (DCF fl). AU, arbitrary unit. **, p < 0.01 versus siNC/GFP; ##, p < 0.01 versus siUHRF1/GFP. E, cell growth rate by counting cell numbers. **, p < 0.01 versus siNC/GFP; ##, p < 0.01 versus siUHRF1/GFP. F, Western blotting analysis.

FIGURE 5.

WNT5A is a downstream effector of the UHRF1/DNMT1 axis. A, an HDF (DT2) was transfected with siRNAs for DNMT1 or UHRF1 for 3 days, and total cellular RNAs were subjected to a cDNA microarray and bioinformatics analysis. Up-regulated genes by DNMT1 knockdown and UHRF1 knockdown were matched with the up-regulated genes in the two HDF senescence models (RS and HS). The Venn diagram shows the number of the overlapping genes among DNMT1_UP, UHRF1_UP, RS_UP, and HS_UP. Six commonly up-regulated genes were identified. B and C, an HDF (DT2) was exposed to the indicated concentration of 5-AcZ for 5 days. B, messenger RNA levels were monitored by qRT-PCR. Con, control. *, p < 0.05 versus Con; **, p < 0.01 versus Con. C, Western blotting analysis for WNT5A protein expression. MW, molecular weight. D, Western blotting analysis (top panel) and qRT-PCR (bottom panel) for WNT5A in the progress of RS. **, p < 0.01 versus PD24 by Student's t test. E, Western blotting analysis (top panel) and qRT-PCR (bottom panel) for WNT5A in the progress of HS. **, p < 0.01 versus control (C). d, days. F, an HDF (DT2) was transfected with siRNAs for the indicated targets for 4 days and subjected to Western blotting analysis. The bands of knockdown (KD) targets were obtained at the same position as shown in Fig. 2E.

FIGURE 6.

Hypomethylation in a specific region (from −1569 to −1363) of the WNT5A promoter in the process of senescence. A, schematic for potential CpG islands of WNT5A promoter regions and the selected regions used for the MSS and MSP. B, MSS of region A was performed with HDFs (DT2 and DT14) as described under “Experimental Procedures.” Fourteen CpG dinucleotides exist within the region, and methylated (Met) CpG dinucleotides are shown as black and non-methylated (Unmet) ones as white. C, MSP was performed using the primer set for the confirmed CpG methylation hot spots (−1490, −1483, and −1476 bp from the WNT5A transcription start) within the WNT5A promoter in the progress of replicative HDF senescence. M, DNA size marker. D and E, an HDF (DT2) was infected with the rRV encoding WNT5A cDNA for 6 days. D, cell growth rate by cell number counting. **, p < 0.01 versus RFP by Student's t test. E, Western blotting analysis.