A role for the Werner syndrome protein in epigenetic inactivation of the pluripotency factor Oct4

Abstract

Werner syndrome (WS) is an autosomal recessive disorder, the hallmarks of which are premature aging and early onset of neoplastic diseases (Orren, 2006; Bohr, 2008). The gene, whose mutation underlies the WS phenotype, is called WRN. The protein encoded by the WRN gene, WRNp, has DNA helicase activity (Gray et al., 1997; Orren, 2006; Bohr, 2008; Opresko, 2008). Extensive evidence suggests that WRNp plays a role in DNA replication and DNA repair (Chen et al., 2003; Hickson, 2003; Orren, 2006; Turaga et al., 2007; Bohr, 2008). However, WRNp function is not yet fully understood. In this study, we show that WRNp is involved in de novo DNA methylation of the promoter of the Oct4 gene, which encodes a crucial stem cell transcription factor. We demonstrate that WRNp localizes to the Oct4 promoter during retinoic acid-induced differentiation of human pluripotent cells and associates with the de novo methyltransferase Dnmt3b in the chromatin of differentiating pluripotent cells. Depletion of WRNp does not affect demethylation of lysine 4 of the histone H3 at the Oct4 promoter, nor methylation of lysine 9 of H3, but it blocks the recruitment of Dnmt3b to the promoter and results in the reduced methylation of CpG sites within the Oct4 promoter. The lack of DNA methylation was associated with continued, albeit greatly reduced, Oct4 expression in WRN-deficient, retinoic acid-treated cells, which resulted in attenuated differentiation. The presented results reveal a novel function of WRNp and demonstrate that WRNp controls a key step in pluripotent stem cell differentiation.

Fig. 1. Enrichment of WRNp at the Oct4 promoter

(A) Q-ChIP analysis of WRNp levels at the Oct4 promoter in undifferentiated pluripotent cells and cells stimulated to differentiate with either 1 or 10 µM RA for three days. WRNp was immunoprecipitated from NCCIT lysates and the amount of associated Oct4 promoter DNA was measured by real-time PCR using the Oct4 B probe and primers (See Supplementary Table 2). Left – raw data, Oct4 promoter amplicons detected in samples with 0, 1, or 10 µM RA, immunoprecipitated with a WRN antibody (WRNp), or without antibody to determine extent of background signal (Background). Error bars indicate standard deviation. Right – ratio of Oct4 promoter amplicons detected in lysates immunoprecipitated with the antibody over the signal detected in no antibody control samples. (B) WRNp enrichment at the Nanog, CD4 and β-globin promoters in the presence and absence of RA (fold enrichment over background). Cells were treated as in A.

Fig. 2. shRNA-mediated knockdown of WRNp in NCCIT cells

NCCIT cells were transfected with plasmids encoding one of two types of shRNA against WRN, or shRNA against G9a, or one of two control plasmids (C_V – empty vector, C_S – vector control expressing a scrambled, non-targeting sequence, G(−) – G9a-deficient clone, 31–34, 61, and 62 – WRNp deficient clones, see Supplementary Table 3). Transfected cells were selected in puromycin and individual clones were isolated and subjected to western blotting analysis with a WRNp antibody or a γ-tubulin control.

Fig. 3. WRNp knockdown does not affect changes in histone methylation during pluripotent cell differentiation

Q-ChIP analysis of Oct4 promoter DNA associated with H3K4me2 (dimethylated lysine 4 of H3) and H3K9me3 (trimethylated lysine 9 of H3) in undifferentiated and RA treated cells. Results are presented as a ratio of amplicons (Oct4 B primer/probe set, see Supplementary Table 2) detected in differentiated over non-differentiated samples. Cells were treated with 10 µM RA for 72 hrs, when histone methylation changes were shown to peak (Feldman et al. 2006).

Fig. 4. Deficient DNA methylation of the Oct4 promoter in WRNp-deficient clones

During the differentiation of NCCIT cells, Oct4 promoter DNA is progressively methylated and thus becomes resistant to the methylation-sensitive enzymes HpaII and HhaI. Cells were stimulated with 10 µM RA for 7 days then harvested, DNA extracted and digested with the methylation-sensitive enzymes HpaII or HhaI. The digested DNA was then subjected to real-time PCR with primers targeting segments of the Oct4 promoter that flank the restriction sites. Error bars indicate standard deviation. (A) Map of the Oct4 promoter with the location of the HpaII and HhaI restriction sites and DNA methylation sites (GeneBank No. AJ297527) and amplified Oct4 segments (B, D, E, and F). (B) CpG methylation status in the Oct4 B region of undifferentiated and RA treated WRNp-deficient, G9a-deficient and control cells (see Fig. 2 for terminology). The results are shown as the number of amplicons obtained with DNA from undifferentiated cells (−) as well as cells that were stimulated to differentiate with RA (+) (left panels). Right – a ratio of the number of amplicons detected in stimulated vs. non-stimulated samples (after adjustment to DNA input according to qPCR signal of undigested samples). (C) CpG methylation status in the Oct4 D region. (D) CpG methylation status in the Oct4 E region. (E) CpG methylation status in the Oct4 F region. (F) CpG methylation status in the Oct4 B region of undifferentiated and RA treated WRNp-deficient clones 61 and 62 and control cells. Cells were treated as in Fig. 4A-E. (G) CpG methylation status in the Oct4 D region of 61 and 62 clones. (H) CpG methylation status in the Oct4 E region. (I) CpG methylation status in the Oct4 F region of 61 and 62 clones. See Supplementary Figs. 2 and 3 for additional information.

Fig. 4. Deficient DNA methylation of the Oct4 promoter in WRNp-deficient clones

During the differentiation of NCCIT cells, Oct4 promoter DNA is progressively methylated and thus becomes resistant to the methylation-sensitive enzymes HpaII and HhaI. Cells were stimulated with 10 µM RA for 7 days then harvested, DNA extracted and digested with the methylation-sensitive enzymes HpaII or HhaI. The digested DNA was then subjected to real-time PCR with primers targeting segments of the Oct4 promoter that flank the restriction sites. Error bars indicate standard deviation. (A) Map of the Oct4 promoter with the location of the HpaII and HhaI restriction sites and DNA methylation sites (GeneBank No. AJ297527) and amplified Oct4 segments (B, D, E, and F). (B) CpG methylation status in the Oct4 B region of undifferentiated and RA treated WRNp-deficient, G9a-deficient and control cells (see Fig. 2 for terminology). The results are shown as the number of amplicons obtained with DNA from undifferentiated cells (−) as well as cells that were stimulated to differentiate with RA (+) (left panels). Right – a ratio of the number of amplicons detected in stimulated vs. non-stimulated samples (after adjustment to DNA input according to qPCR signal of undigested samples). (C) CpG methylation status in the Oct4 D region. (D) CpG methylation status in the Oct4 E region. (E) CpG methylation status in the Oct4 F region. (F) CpG methylation status in the Oct4 B region of undifferentiated and RA treated WRNp-deficient clones 61 and 62 and control cells. Cells were treated as in Fig. 4A-E. (G) CpG methylation status in the Oct4 D region of 61 and 62 clones. (H) CpG methylation status in the Oct4 E region. (I) CpG methylation status in the Oct4 F region of 61 and 62 clones. See Supplementary Figs. 2 and 3 for additional information.

Fig. 5. Methylation on Nanog, CD4 and β-globin promoters

Cells were treated with RA as described in Fig. 4. DNA was extracted and digested with HpaII. The digested DNA was then subjected to real-time PCR with primers targeting segments of the Nanog and CD4 promoter that flank the HpaII restriction sites in these promoters. No such restriction site was found in the β-globin promoter. Control cells – C_V (see Fig. 4), WRNp-deficient cells – clone 61 (see Fig. 4).

Fig. 6. WRNp is in a complex with and is required for Dnmt3b recruitment to the Oct4 promoter

(A) Dnmt3b is localized in the chromatin fraction of control and WRNp-deficient cells. C_V and WRNp-deficient cells (clone 61) were treated with 10 µM RA for 3 days. Chromatin fraction was then separated and both chromatin-lacking lysates (L) and the chromatin fraction (Ch) were analyzed by western blotting. H3 – histone H3, loading control for the chromatin fraction, Trim5α – cytoplasmic fraction loading control. (B) Dnmt3b is present in WRNp immunoprecipitates. Top: WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp then was immunoprecipitated from the chromatin-lacking lysates and from the chromatin fraction of undifferentiated and RA treated cells. WRNp immunoprecipitates were resolved on SDS-PAGE and Dnmt3b was detected by western blotting analysis. ChI – whole chromatin input lysate of RA-treated cells (1/10 of the immunoprecipitation input), D3 – chromatin input lysate immunoprecipitated with the Dntm3b antibody, W – lysates immunoprecipitated with a WRNp antibody, I – lysates immunoprecipitated with normal rabbit IgG. Bottom: Control cells and WRNp-deficient cells (clone 61) were stimulated with 10 µM RA for 3 days. WRNp then was immunoprecipitated from the chromatin fraction of undifferentiated and RA treated cells. (C) WRNp is present in Dnmt3b immunoprecipitates. WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp and Dnmt3b then were immunoprecipitated from the chromatin fraction. Immunoprecipitates were resolved on SDS-PAGE and WRNp was detected by western blotting analysis. ChI – whole chromatin input lysate of RA-treated cells (1/10 of the immunoprecipitation input), W – lysates immunoprecipitated with a WRNp antibody, D3 - lysates immunoprecipitated with a Dnmt3b antibody, I – lysates immunoprecipitated with normal rabbit IgG. (D) Effect of EtBr and DNAse on Dnmt3b and WRNp co-immunoprecipitation. WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp was immunoprecipitated from the chromatin fraction as in Fig. 6B and C, except cell lysates were treated with EtBr (100 µg/ml) or DNAse prior to immunoprecipitation (see Methods). Immunoprecipitates were resolved on SDS-PAGE and Dnmt3b was detected by western blotting analysis. Et – EtBr-treated lysates, Ds – DNAse-treated lysates, M – mock-treated lysates, W – lysates immunoprecipitated with a WRNp antibody, I – lysates immunoprecipitated with normal rabbit IgG. (E) Q-ChIP analysis of Dnmt3b levels at the Oct4 promoter regions of control and WRNp-deficient cells. Cells were treated with RA for three days as described above and then subjected to Q-ChIP analysis using a Dnmt3b antibody.

Fig. 6. WRNp is in a complex with and is required for Dnmt3b recruitment to the Oct4 promoter

(A) Dnmt3b is localized in the chromatin fraction of control and WRNp-deficient cells. C_V and WRNp-deficient cells (clone 61) were treated with 10 µM RA for 3 days. Chromatin fraction was then separated and both chromatin-lacking lysates (L) and the chromatin fraction (Ch) were analyzed by western blotting. H3 – histone H3, loading control for the chromatin fraction, Trim5α – cytoplasmic fraction loading control. (B) Dnmt3b is present in WRNp immunoprecipitates. Top: WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp then was immunoprecipitated from the chromatin-lacking lysates and from the chromatin fraction of undifferentiated and RA treated cells. WRNp immunoprecipitates were resolved on SDS-PAGE and Dnmt3b was detected by western blotting analysis. ChI – whole chromatin input lysate of RA-treated cells (1/10 of the immunoprecipitation input), D3 – chromatin input lysate immunoprecipitated with the Dntm3b antibody, W – lysates immunoprecipitated with a WRNp antibody, I – lysates immunoprecipitated with normal rabbit IgG. Bottom: Control cells and WRNp-deficient cells (clone 61) were stimulated with 10 µM RA for 3 days. WRNp then was immunoprecipitated from the chromatin fraction of undifferentiated and RA treated cells. (C) WRNp is present in Dnmt3b immunoprecipitates. WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp and Dnmt3b then were immunoprecipitated from the chromatin fraction. Immunoprecipitates were resolved on SDS-PAGE and WRNp was detected by western blotting analysis. ChI – whole chromatin input lysate of RA-treated cells (1/10 of the immunoprecipitation input), W – lysates immunoprecipitated with a WRNp antibody, D3 - lysates immunoprecipitated with a Dnmt3b antibody, I – lysates immunoprecipitated with normal rabbit IgG. (D) Effect of EtBr and DNAse on Dnmt3b and WRNp co-immunoprecipitation. WRNp-proficient control cells (C_V, see Fig. 2) were stimulated with 10 µM RA for 3 days. WRNp was immunoprecipitated from the chromatin fraction as in Fig. 6B and C, except cell lysates were treated with EtBr (100 µg/ml) or DNAse prior to immunoprecipitation (see Methods). Immunoprecipitates were resolved on SDS-PAGE and Dnmt3b was detected by western blotting analysis. Et – EtBr-treated lysates, Ds – DNAse-treated lysates, M – mock-treated lysates, W – lysates immunoprecipitated with a WRNp antibody, I – lysates immunoprecipitated with normal rabbit IgG. (E) Q-ChIP analysis of Dnmt3b levels at the Oct4 promoter regions of control and WRNp-deficient cells. Cells were treated with RA for three days as described above and then subjected to Q-ChIP analysis using a Dnmt3b antibody.

Fig. 7. Oct4 expression and Oct4 promoter methylation status in RA-treated WRNp-deficient cells

(A) RA-treated WRNp-deficient cells continue to express Oct4. WRNp-deficient (31–62), G9a-deficient [G(−)] and control (C_V) cells were treated with 10 µM RA for 7 days, after which cells were harvested and Oct4 expression analyzed by western blotting (+, cells treated with RA; −, untreated, control samples). (B) Rbl2 knockdown stimulates Dnmt3b expression and reverses the Oct4 promoter methylation defect of WRNp-deficient cells. The day following RA addition, cells were treated with siRNA targeting Rbl2. Left – Dnmt3b expression in WRNp-deficient cells (61) treated with control (Ci) or Rbl2 siRNA (Ri) at 5 days after RA addition. Right – DNA methylation in the Oct4 promoter B region. (C) Oct4 expression in differentiated (+RA) and undifferentiated (−RA) control and WRNp-deficient cells treated with siRNA. Cells were treated as in B. Oct4 expression was analyzed by western blotting 7 days after RA addition.

Fig. 8. Expression of stem cell markers in WRNp-deficient cells

(A) Control (C_V, C_S) and WRNp-deficient cells (31) were treated with RA for 7 days. Cells were then harvested and the presence of stem cell markers was analyzed by western blotting (+, cells treated with RA; −, untreated control samples). TRA-1-60, TRA-1-81 and EpCAM bands are indicated. γ-tubulin served as a loading control. (B) WRNp-deficient cells (31) were treated with RA for 7 days. Cells were then transfected with Oct4 siRNA (Oi) or control siRNA (Ci). Three days after transfection, cells were harvested and the expression of stem cell markers analyzed as described above (−RA – untreated cells, +RA − RA treated cells).

Fig. 9. Model of the WRNp role in the regulation of Oct4 inactivation in differentiating pluripotent cells

For details see text.