Identification of DNMT1 (DNA Methyltransferase 1) Hypomorphs in Somatic Knockouts Suggests an Essential Role for DNMT1 in Cell Survival

Identification of DNMT1 (DNA methyltransferase 1) hypomorphs in somatic knockouts suggests an essential role for DNMT1 in cell survival

Gerda Egger, Shinwu Jeong, Sonia G. Escobar, Connie C. Cortez, Tony W. H. Li, Yoshimasa Saito, Christine B. Yoo, Peter A. Jones*, and Gangning Liang*

Norris Comprehensive Cancer Center, Departments of Urology and Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90089

Edited by Stanley M. Gartler, University of Washington, Seattle, WA, and approved July 28, 2006 (received for review June 5, 2006) Previous studies have shown that DNA methyltransferase (Dnmt) changes in methylation of specific loci, whereas DKO cells lost 1 is required for maintenance of bulk DNA methylation and is Ͼ95% of total DNA methylation. Interestingly, one of the eight essential for mouse development. However, somatic disruption of DKO clones generated (DKO8) retained Ϸ50% of WT meth- DNMT1 in the human cancer cell line HCT116 was not lethal and ylation and exhibited less growth retardation when compared caused only minor decreases in methylation. Here, we report the with the other DKO cell lines (12). The targeting strategy to identification of a truncated DNMT1 protein, which was generated disrupt DNMT1 resulted in deletion of exons 3, 4, and 5 of WT by the disruption of DNMT1 in HCT116 cells. The truncated protein, DNMT1. These deleted exons code for part of the DMAP1 which had parts of the regulatory N-terminal domain deleted but (DNMT1-associated protein) interaction domain, which is a preserved the catalytic C-terminal domain, was present at different transcriptional repressor shown to recruit histone deacetylase levels in all DNMT1 single-knockout and DNMT1͞DNMT3b double- HDAC2 and DNMT1 (13), and the proliferating cell nuclear knockout cell lines tested and retained hemimethylase activity. antigen (PCNA) interaction domain, which is essential in tar- DNMT1 RNAi resulted in decreased cell viability in WT and knock- geting DNMT1 to the replication fork (14). out cells and further loss of DNA methylation in DNMT1 knockout Here, we show that the cell lines generated are not complete cells. Furthermore, we observed a delay in methylation after knockouts for DNMT1 but have retained a hypomorphic allele replication and an increase in hemimethylation of specific CpG sites and express a catalytically active, truncated DNMT1 protein. in cells expressing the truncated protein. Remethylation studies Intriguingly, the DKO8 cell line, which had appeared aberrant in after drug-induced hypomethylation suggest a putative role of the original work in that it maintained a higher level of DNA DNMT1 in the de novo methylation of a subtelomeric repeat, D4Z4, methylation (12), expressed higher levels of this truncated which is lost in cells lacking full-length DNMT1. Our data suggest protein, and our data suggest a function for this variant of that DNMT1 might be essential for maintenance of DNA methyl- DNMT1 for cell viability and maintenance of DNA methylation. ation, proliferation, and survival of cancer cells. Results DNA methylation ͉ epigenetic DNMT1 Transcripts and Protein in HCT116 Knockout Cells. Rhee et al. (11) previously generated a DNMT1 knockout construct in which he biological roles of the major mammalian DNA methyl- exons 3, 4, and 5 of human DNMT1 were replaced with a Ttransferase (Dnmt), DNMT1, have been enigmatic. Al- hygromycin resistance gene. First, we performed RT-PCR anal- though gene-targeting studies in mice have clearly demonstrated yses to identify putative DNMT1 transcripts in HCT116 WT and an essential function of Dnmt1 in embryonic development, cell various knockout cells. We detected PCR products of the survival, and tumorigenesis (1), there have been controversial expected sizes spanning exons 1–6 of DNMT1 in WT and Ϫ/Ϫ reports regarding the function of this enzyme in human cancer DNMT3b cells (Fig. 1 A and B, exons 1–6). Sequencing of cells. In mice, Dnmt1 has been implicated in maintaining the PCR products revealed different shorter transcripts in the majority of bulk DNA methylation, differentiation of ES cells, knockout cell lines, in which the cDNAs had deleted exons 3–5 Ϫ/Ϫ and imprinting (2, 3). Furthermore, deletion of Dnmt1 in mouse in DNMT1 and DKO1 cells, as predicted by the knockout embryonic fibroblasts caused a decrease in genomic methylation, targeting. Interestingly, the transcript detected in the DKO8 cells p53-dependent apoptosis, and deregulation of transcription (4). carried an additional deletion of exon 2, which had most likely Heterozygosity for Dnmt1 in combination with administration of been generated by alternative splicing. This transcript was of the DNMT inhibitor 5-aza-2Јdeoxycytidine (5-aza-CdR) greatly specific interest, because it could potentially give rise to a reduced the number of polyps in a mouse model for intestinal truncated DNMT1 protein in the same reading frame as WT neoplasia (5). DNMT1. To specifically amplify this transcript, we designed a A series of RNAi experiments for DNMT1 have been de- forward RT-PCR primer covering both exon 1 and exon 6 and scribed for various human cancer cell lines, which have shown a reverse primer lying in exon 10 of WT DNMT1. We were able apparently inconsistent results in regard to DNA methylation of tumor suppressor genes (6–10). The differences might be attrib- Author contributions: G.E., P.A.J., and G.L. designed research; G.E., S.J., S.G.E., C.C.C., uted to the techniques used but also to distinct sensitivities of T.W.H.L., Y.S., C.B.Y., and G.L. performed research; G.E., S.J., and G.L. analyzed data; and individual cell lines (10). Furthermore, RNAi may not cause a G.E. and P.A.J. wrote the paper. complete depletion of the protein, so residual protein might still The authors declare no conflict of interest. be available and capable of maintaining DNA methylation. This paper was submitted directly (Track II) to the PNAS office. Rhee et al. (11, 12) generated a widely used series of HCT116 Abbreviations: Dnmt, DNA methyltransferase; DKO, double knockout; 5-aza-CdR, 5-aza- colon cancer cells with homozygous deletions for DNMT1 2Јdeoxycytidine; PCNA, proliferating cell nuclear antigen; DMAP1, DNMT1-associated Ϫ Ϫ Ϫ Ϫ (DNMT1 / ) (11), DNMT3b (DNMT3b / ) (12), or both protein; Ms-SNuPE, methylation-sensitive single-nucleotide primer extension. DNMT1 and DNMT3b (12) [double knockout (DKO)]. Surpris- *To whom correspondence may be addressed. E-mail: jones࿝[email protected] or gliang@ ingly, somatic disruption of DNMT1 resulted in only a 20% usc.edu. decrease in overall genomic methylation with no discernible © 2006 by The National Academy of Sciences of the USA

14080–14085 ͉ PNAS ͉ September 19, 2006 ͉ vol. 103 ͉ no. 38 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604602103 Downloaded by guest on September 27, 2021 A 1 2 5 32 34 A B 30 25 deletion 1KO and DKO1 WT 1KO 3bKO DKO8 deletion DKO8, DKO1 and 1KO 20 hemi meth - + - + - + - + 5-aza-CdR non meth PCNA KEN 15 DNMT1 Replication KG DMAP1 Zn D BAH1 BAH2 Catalytic domain 10 foci targeting HDAC1 NLS 5 0 B D WT 1KO 3bKO DKO8 WT 1KO 3bKO DKO8 O IP Pre 2 WT 1KO 3bKO DKO8 DKO1 RT- H 1.2 ␮ E1-6 Fig. 2. DNMT1 activity. (A) Western blot analysis of cells treated with 0.3 M 1 5-aza-CdR or left untreated for 24 h. The blot was sequentially probed with an 0.8 E1/6-10 antibody against the C terminus of DNMT1 and HDAC1 as a loading control. 0.6 0.4 (B) DNMT1 was immunoprecipitated with the same DNMT1 antibody as in A E32-34 0.2 and incubated with hemimethylated or unmethylated DNA fragments corre-

DNMT1/PCNA 0 sponding to a 428-bp-long p16 intron 1 sequence and 160 ␮M S-adenosyl- WT 1KO 3bKO DKO8 DKO1 Actin methionine for3hat37°C. The percentage of methylation was determined by quantitative Ms-SNuPE (33). IP, immunoprecipitation with DNMT1 antibody; C Pre, control immunoprecipitation with whole rabbit serum. WT 1KO 3bKO DKO8 DKO1 DNMT1 PCNA (1) (Fig. 1A). Thus, the knockout had generated a hypomorphic allele, which yielded a truncated DNMT1 protein in DNMT1Ϫ/Ϫ Fig. 1. DNMT1 expression in different HCT116 cell lines. (A Upper) Genomic cells and two individual DKO cell lines. The availability of the map of WT DNMT1. Black vertical boxes indicate exons; black horizontal bars DKO8 clone, which expressed higher levels of the hypomorphic drawn below indicate deletions determined by direct sequencing of tran- protein than the DKO1 or DNMT1Ϫ/Ϫ cells, allowed us to titrate scripts in different knockout cell lines. (A Lower) DNMT1 protein domain the enzyme levels and facilitated the characterization of the structure. DMAP1 domain, amino acids 1–120; PCNA domain, amino acids potential biological function of the truncated protein. 163–174; NLS, nuclear localization signal domain, amino acids 177–205; DNA DNMT inhibitors such as 5-aza-CdR are incorporated into replication foci-targeting domain, amino acids 331–550; ZN D, zinc ﬁnger region, amino acids 646–692; KEN, KEN box (KENxxxR), amino acids 644–650; DNA and inhibit DNMT activity by forming covalent complexes BAH1 and BAH2, bromo-adjacent homology domains, amino acids 755–880 with the catalytic domains of DNMTs, leading to their depletion and 972–1100, respectively; KG, 6 ϫ 2-aa tandem repeats of K-G, amino acids from protein extracts (15–17). Interestingly, 5-aza-CdR treat- 1109–1120; catalytic domain, amino acids 1139–1616. Note that illustrations ment caused depletion of both the full-length and truncated are not drawn to scale. (B) RT-PCR in different WT and knockout cell lines was DNMT1 proteins, suggesting the presence of a functional cata- performed by using primers located in exons 1 and 6 of WT DNMT1 (E1–6), half lytic domain in the shortened protein (Fig. 2A). Immunopre- exons 1 and 6 and exon 10 of DNMT1 (E1͞6–10), or exons 32 and 34 (E32–34). cipitation experiments and DNMT1 activity assays using hemi- ␤ As a control, primers against -actin were used (Actin). (C) Western blot methylated or unmethylated DNA fragments corresponding to a analysis of various HCT116 cell lines with C-terminal DNMT1 (Upper) or PCNA antibody as a loading control (Lower). (D) Quantitation of the Western blot genomic p16 sequence showed that the truncated enzyme was shown in C for DNMT1 protein levels, normalized to PCNA expression. The WT active (Fig. 2B). Both the WT and truncated proteins showed expression level was arbitrarily set as 1. WT, HCT116 WT; 1KO, DNMT1Ϫ/Ϫ cells; preferences for the naturally occurring hemimethylated DNA 3bKO, DNMT3bϪ/Ϫ cells; DKO8 and DKO1, two independent DNMT1 and target. We could not detect any activity against an unmethylated DNMT3b DKO clones, respectively. sequence in either DNMT1 WT or knockout cell lines. Thus, even though the overall level of Dnmt activity had been highly

Ϫ/Ϫ reduced (11, 12), a demonstrable maintenance activity was still to identify such alternative transcripts in both the DNMT1 present in DNMT1Ϫ/Ϫ and DKO cells. and the DKO1 cell lines, although at lower levels than in the ͞ DKO8 cells (Fig. 1B, exons 1 6–10), which were not detected in DNMT1 RNAi. Having demonstrated that the truncated DNMT1 the initial PCR spanning exons 1–6, most likely because there protein possessed in vitro DNMT activity, we asked whether this was a preference for the amplification of the transcript with a protein would have in vivo functions as well. Three independent deletion of exons 3–5. Sequencing of RT-PCR products of exons RNAi knockdown experiments were performed by using three 32–34 showed that mRNAs containing intact catalytic domains rounds of transient transfections with a double-stranded RNA were present in all cell lines (Fig. 1B, exons 32–34, and data not oligonucleotide whose sequence had been previously known to shown). Therefore, all knockout cell lines expressed truncated specifically knock down DNMT1 (7). We obtained substantial DNMT1 transcripts, which could give rise to a shortened reductions of DNMT1 protein levels in both DNMT1 WT and DNMT1 protein. Indeed, we were able to detect a faster knockout cell lines (Fig. 3A), which were accompanied by a migrating protein as compared with full-length DNMT1 on reduction in cell number, with Ͻ50% viable cells in WT, Western blots of nuclear extracts of the different knockout cell DNMT1Ϫ/Ϫ, and DNMT3bϪ/Ϫ cells (Fig. 3B). The DKO8 cells lines probed with an antibody directed against the C terminus of were less affected by the additional reduction of DNMT1 GENETICS DNMT1 (Fig. 1C Upper). We quantified the levels of the protein, probably because of their slow proliferation rates or Ϫ Ϫ truncated protein to Ϸ20% of WT DNMT1 in the DNMT1 / because they had already been selected to survive with low levels and DKO1 cell lines and to Ϸ40% in the DKO8 cells (Fig. 1D), of Dnmts. Furthermore, after the reduction of DNMT1 protein which was in line with the amount of transcript detected in the levels, only DNMT1Ϫ/Ϫ cells sustained a significant loss in DNA different cell lines (Fig. 1B, exons 1͞6–10). The deletion of Ϸ17 methylation at specific loci, whereas a mild effect was observed kDa encoded part of the DMAP1 interaction domain and the in DNMT1 WT and DKO8 cells (Fig. 3C). These results indicate PCNA interaction domain. However, the truncated protein that even trace amounts of DNMT1 may therefore be sufficient retained the nuclear localization signal and other regulatory to maintain some methylation at specific loci. The observed N-terminal domains, as well as the catalytic C-terminal domain decrease in DNA methylation in the DNMT1Ϫ/Ϫ cells after RNAi

Egger et al. PNAS ͉ September 19, 2006 ͉ vol. 103 ͉ no. 38 ͉ 14081 Downloaded by guest on September 27, 2021 70 A B 60 100 WT 50 1KO1 80 40 3bKO WT 1KO 3bKO DKO8 30 DKO8 60 20 DKO1 (h/(h+f)) 0 C RNAi 0 C RNAi 0 C RNAi 0 C RNAi 10 DNMT1 40 0 20 % hemimethylation PCNA % viable cells H1 H2 H3 0 WT 1KO 3bKO DKO8 Fig. 4. Hemimethylation assay. Hemimethylation was assessed in the different HCT116 cell lines at three different sites within the p16 gene (H1, H2, and D4Z4 NS RUNX1 NS C100 NS NS 100 NS H3). H1 is located in the promoter, H2 is in a CpG-poor region within intron 1, P=0.001 P=0.011 80 80 and H3 is in a CpG-rich region of intron 1. Percentage of hemimethylation was 60 deﬁned as h͞(h ϩ f), where h represents hemimethylated sites (only one 60 * * 40 40 strand methylated) and f represents fully methylated sites (both strands 20 20 methylated).

NS % methylation % methylation 0 0 C RNA 0 C RNAi 0 C RNAi 0 C RNAi 0 0 C RNAi 0 C RNA 0 C R 0 C RNA NAi i i i WT 1KO 3bKO DKO8 WT 1KO 3bKO DKO8 site within the enzyme substantially increased the percentage of MAGE A1 TIMP3 sites that were hemimethylated within the hypomorphic cells. 100 100 NS NS 80 NS NS P=0.67 80 P=0.024 Sequence Specificity of Residual Methylation. 60 T 60 We next measured the 40 40 * residual methylation within CpG-poor regions and CpG islands NS 20 20 of the genome and separated out the analysis of imprinted loci

% methylation NS % methylation 0 0 C RNAi 0 C RNAi 0 C RNAi 0 C RNAi 0 0 C RNAi 0 C RNA 0 C RNAi 0 C RNAi and repeat sequences in the different HCT116 cell lines to study

i whether WT or truncated DNMT1 was required for the main- WT 1KO 3bKO DKO8 WT 1KO 3bKO DKO8 tenance of methylation at specific loci (Fig. 5). Quantitative Fig. 3. DNMT1 siRNA. (A) Western blot of HCT116 WT and knockout cells Ms-SNuPE analyses of specific CpG-poor regions in the untransfected (0) or transiently transfected with negative control siRNA (C) or DNMT1Ϫ/Ϫ cells showed that these sequences lost Ϸ30% of their DNMT1 siRNA (RNAi) and probed with a DNMT1 or PCNA antibody. (B) methylation in the hypomorphic cells independently of whether Percentage of viability was calculated as the ratio of the number of DNMT1 they were located within the promoters or transcribed regions of RNAi-transfected cells to control-transfected cells. (C) Ms-SNuPE assays of A untransfected, control, and DNMT1 RNAi cells for different loci. D4Z4, subte- genes (Fig. 5 , nos. 1–8). On the other hand, CpG islands were lomeric repeat, CpG island; RUNX1, CpG-poor region in intron 1; MAGE A1, less sensitive to DNMT1 depletion, and most of these islands CpG-poor promoter region; TIMP3, CpG island promoter region. Data shown were maintained at Ϸ90% of the level found in the WT cells (Fig. are representatives of at least three independently repeated experiments. NS, 5A, nos. 9–20). Once again, there were no clear differences in the P value not significant; *, P value significant; T, P value shows a tendency decreased methylation levels within promoters relative to non- toward significance. promoter regions. The imprinted loci examined were hardly affected by depletion (Fig. 5A, nos. 21 and 22), and the methylation of repeats, including long interspersed nuclear elements was accompanied by a weak induction of genes normally silenced and Alus in general and specific Alu sequences located within by hypermethylation, such as the tumor suppressor TIMP3 or the genes and the subtelomeric region D4Z4, showed an Ϸ10% testis-specific antigen MAGE A1 (data not shown).

Hemimethylation in HCT116 Cells. Many experiments have demon- A 1KO B 3bKO CpG poor CpG island imprinted repeat CpG poor CpG island imprinted repeat strated that DNMT1 preferentially methylates hemimethylated 100 100 DNA and functions at the replication fork to copy methylation 80 80 patterns while anchored to PCNA (1, 14, 18, 19). Because the 60 60 DNMT1 hypomorphic cell line had presumably lost the ability to 40 40 20 20 interact with PCNA and therefore might not be able to fully % methylation of WT 0 % methylation of WT 0 methylate its target sequences during S phase, it might be 1 5 10 15 20 25 1 5 10 15 20 25 anticipated that the cells would show an increased level of C DKO8 D DKO1 hemimethylation after depletion of WT DNMT1. We therefore CpG poor CpG island imprinted repeat CpG poor CpG island imprinted repeat measured the level of hemimethylation within three regions of 100 100 the p16 locus by using an assay we have previously described that 80 80 60 60 couples methylation-sensitive restriction enzyme digestion with 40 40 bisulfite treatment and methylation-sensitive single-nucleotide 20 20 % methylation of WT % methylation of WT 0 0 primer extension (Ms-SNuPE) analysis (20, 21). The ability of 1 5 10 15 20 25 1 5 10 15 20 25 the assay to detect hemimethylation at three sites [within the 5Ј region of the p16 gene (H1), at a CpG-poor site (H2), and at a Fig. 5. Methylation of various regions in HCT116 WT and knockout cells. CpG-rich site in intron 1 (H3)] was first validated by using T24 Methylation levels (average of two to three CpG sites at each locus) were determined by Ms-SNuPE analyses and graphed as a percentage of WT meth- cells after 24-h treatment with 5-aza-CdR (20). Results shown in ylation levels. Open bars represent promoter regions, and ﬁlled bars indicate Fig. 4 clearly demonstrate an increase in the proportion of nonpromoter regions. The numbering of regions is as follows. CpG-poor hemimethylated sites in cells that were hypomorphic for regions: 1, RUNX3 promoter; 2, MAGE A1 promoter; 3, NANOG promoter; 4, DNMT1. The increase in hemimethylation was especially prom- p16 intron 1; 5, PAX3 intron; 6, RUNX1 intron; 7, BDNF intron; 8, PCNA intron. inent at the CpG-poor site tested and seemed to correlate with CpG islands: 9, p16 promoter; 10, ENDRB promoter; 11, ATBF1 promoter; 12, the levels of the truncated DNMT1 protein, demonstrating the XIST promoter; 13, TIMP3 promoter; 14, TPEF promoter; 15, DNMT3a2 promoter; 16, microRNA-127; 17, p16 exon 1; 18, p16 intron 4; 19, M4-4 single- importance of DNMT1 for the maintenance of methylation of copy sequence, chromosome 16q22; 20, p16 exon 2. Imprinted regions: 21, CpG-poor regions (compare also Fig. 5). Thus, failure to localize H19; 22, SNRPN. Repeats: 23, D4Z4; 24, p53-Alu; 25, p16-Alu; 26, LINE global; DNMT1 to the replication fork by deletion of the PCNA-binding 27, Alu global.

14082 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604602103 Egger et al. Downloaded by guest on September 27, 2021 decrease in DNA methylation (Fig. 5A, nos. 23–27). These data A B were quite different from data obtained from studies in mice (2, 100 100 3), indicating a potential role of the truncated protein in 80 80 maintaining the methylation of certain regions. Overall, DNMT1 60 60 appeared to play a more prominent role in maintaining the 40 40 20 20 % methylation

methylation of CpG-poor regions rather than CpG island re- % methylation gions, because the decrease in methylation was significant in 0 0 0' 0' 0' 0' 0' 0' 0' 0' 8h 8h 8h 8h 8h 8h 8h 8h 15' 30' 15' 30' 15' 30' 15' 30' CpG-poor regions as compared with CpG islands (P ϭ 0.003). 15' 30' 15' 30' 15' 30' 15' 30' WT 1KO 3bKO DKO8 WT 1KO 3bKO DKO8 Interestingly, there was little effect of knocking out DNMT3b with respect to the level of residual methylation in any of the Fig. 6. Methylation kinetics of newly synthesized DNA. The different cell sequences analyzed (Fig. 5B). Of particular interest was the fact lines were pulsed for 1 h with BrdU and subsequently chased with thymidine that D4Z4, a subtelomeric repeat, was hardly affected by dis- for 0, 15, or 30 min or 8 h. DNA that had incorporated BrdU was isolated by ruption of this enzyme even though it was shown to be demeth- anti-BrdU immunoprecipitation. Methylation levels were determined by Ms- ylated in patients with ICF syndrome (immunodeficiency, cen- SNuPE analyses at the indicated time points. (A) Methylation kinetics of a tromeric instability, and facial anomalies syndrome), which is CpG-poor region within the RUNX1 intron. (B) Methylation levels of the CpG-rich subtelomeric repeat D4Z4 at different time points of the pulse– characterized by mutations in DNMT3b (22). chase. By far the most dramatic effects on residual DNA methylation were seen in the two DKO strains (Fig. 5 C and D). Interestingly, the amount of residual truncated DNMT1 protein seemed to in a CpG island, showed substantial delays in the DNMT1Ϫ/Ϫ correlate with the level of residual DNA methylation, in such a cells, whereas the sequence was almost completely methylated way that the DKO8 cells, which expressed Ϸ2-fold the amount immediately after replication in the WT and DNMT3bϪ/Ϫ cells of the truncated DNMT1 protein as the DKO1 cells, also (Fig. 6B). Because this region was not methylated in the DKO8 retained an overall higher level of DNA methylation at most loci cells, no change in methylation was seen after DNA synthesis. examined, which has been observed previously for the total Therefore, in cells lacking a fully functional DNMT1, neither the methylation content of these cells (12) (compare Fig. 5 C and D). RUNX1 nor the D4Z4 region acquired its full level of methylation Ϸ It has been reported previously that the DKO1 cells lose 95% until a considerable time after the DNA had left the replication of their 5-methylcytosine content (12), and a methylation screen fork. We interpret this result to suggest that, in the absence of revealed a massive loss in hypermethylation of CpG islands in the an adequate level of DNMT1 at the replication fork, because of same cells (23). Although we observed a loss in methylation both the lack of the PCNA interaction domain, the truncated enzyme in CpG-poor regions and CpG islands, we also detected several completes the methylation process on DNA, which is already regions that had still maintained up to 50% of WT methylation assembled into chromatin. levels in DKO1 cells and up to 90% in the higher DNMT1- expressing DKO8 cells (Fig. 5 C and D). Of particular interest Remethylation of Target Regions After 5-aza-CdR Treatment. We next was the complete demethylation of the CpG island promoter of treated the different cell lines for a 24-h period with the DNMT3a2 (Fig. 5, no. 15), a DNMT3a variant, which is ex- demethylating agent 5-aza-CdR to depress the methylation level pressed predominantly during embryogenesis (24) and which and then grew the cells in the absence of further drug treatment might be expected to be up-regulated in DKO cells. As had been and measured the kinetics of remethylation. We examined the reported earlier (12, 25), we found that imprinted regions and D4Z4 and p16 exon 2 regions before and after drug treatment by repetitive elements such as D4Z4 were strongly demethylated in genomic bisulfite sequencing to assess in a precise manner how DKO cells, although specific Alu sequences located in the p53 the methylation patterns were reestablished (Fig. 7). Both WT and p16 genes retained substantial residual methylation. Overall, Ϫ/Ϫ the methylation pattern in these cells, which was maintained and DNMT1 cells showed almost complete methylation of the D4Z4 sequence before treatment, with very substantial demeth- predominantly by DNMT3a together with the truncated ␮ DNMT1, was determined by factors other than the sequence ylation being induced 4 days after exposure to 0.3 M 5-aza-CdR itself. The correlation between the amount of residual DNMT1 (Fig. 7A). The methylation increased 32 days after treatment in protein and the level of methylation retained in the DKO cells the WT cells but was far more sporadic than that seen in the suggests a strong impact of the truncated protein on mainte- untreated controls, showing that it was difficult for this region to nance of methylation at most loci tested. The methylation of regain methylation once it had been removed. In the DNMT1 regions that lost all of their methylation in DKO cells might have knockout cells, however, many copies of the D4Z4 sequence been maintained by the cooperative action of both WT DNMT1 examined remained completely unmethylated even 32 days after and DNMT3b, as suggested before (12). treatment. The fact that some strands were fully methylated 32 days after treatment in the DNMT1Ϫ/Ϫ cells suggests that the Kinetics of Methylation of Newly Synthesized DNA. Methylation of methylation in this particular sequences was inherited and newly synthesized DNA has to be synchronized with the process indeed copied by some process. The slow remethylation in DNMT1 WT cells as compared with the almost complete lack of of DNA replication and chromatin assembly and is an inherently Ϫ Ϫ complex process. We used BrdU pulse–chase experiments to remethylation in the DNMT1 / cells suggests that full-length follow the kinetics of methylation of newly synthesized DNA as DNMT1 might be able to bring about some sporadic de novo described in ref. 20. methylation in DNMT1 WT cells, whereas the truncated protein GENETICS The methylation of a CpG-poor region (RUNX1) was almost has lost this ability. Similarly, almost complete methylation was complete after a 30-min pulse with BrdU in both WT and seen in untreated WT or DNMT1 knockout cells at the p16 exon DNMT3bϪ/Ϫ cells (Fig. 6A). In contrast, in cells lacking a fully 2 region (Fig. 7B). Substantial demethylation was apparent functional DNMT1 protein, the methylation of this region was shortly after drug treatment, and almost complete remethylation substantially delayed with respect to DNA synthesis in both was present 32 days after treatment in the WT cells. Cells lacking DNMT1Ϫ/Ϫ and DKO8 cells. Interestingly, the delay was greater a full-length DNMT1 protein also showed substantial remethy- in the DNMT1Ϫ/Ϫ cells compared with the DKO8 cells, which lation, suggesting that this region was maintained by the trun- might have been due to the different levels of the truncated cated DNMT1 protein, perhaps in addition to another protein protein present in the two cell lines. Similarly, the methylation such as DNMT3a. It also appeared that the single-knockout cells of the subtelomeric D4Z4 repeat sequence, which is embedded were demethylated to a higher degree after 5-aza-CdR treatment

Egger et al. PNAS ͉ September 19, 2006 ͉ vol. 103 ͉ no. 38 ͉ 14083 Downloaded by guest on September 27, 2021 Ϫ Ϫ A B The slightly smaller protein present in DNMT1 / and DKO WT WT cells corresponded to the size predicted by the mRNA. The untreated untreated deletion covered most of the DMAP1 interaction domain, a day 4 after region that is also deleted in the murine oocyte-specific isoform treatment day 4 after Dnmt1o. Deletion of this region produced no abnormal pheno- treatment type in mice, which expressed the shortened protein in all day 32 after treatment somatic tissues instead of full-length Dnmt1 (27). In fact, this day 32 after treatment protein could maintain normal levels of DNA methylation and 1KO 1KO appeared to be more stable than the full-length form of Dnmt1 untreated untreated containing the DMAP1 interaction domain (27). The truncated protein showed preferential DNMT activity in day 4 after day 4 after vitro against a hemimethylated target, suggesting that it might treatment treatment also be active in vivo. However, the fact that a substantial day 32 after increase in the percentage of hemimethylation was seen in the treatment day 32 after knockout cells suggests that there was not sufficient DNMT1 treatment protein to complete maintenance methylation. The deletion of DKO8 the PCNA interaction domain in the hypomorphic protein could untreated account for a failure of tethering the truncated protein to the replication machinery and for a loss of immediate methylation of day 4 after hemimethylated DNA (14, 18, 20). Indeed, a considerable delay treatment in methylation was seen, suggesting that the reduced amount of non-PCNA-targeted enzyme acted over a longer period to day 32 after achieve methylation. Interestingly, Ϸ50% of the newly synthe- treatment sized DNA was methylated immediately after replication (time 0), which might be due to the fact that the truncated protein still Fig. 7. Remethylation after 5-aza-CdR treatment. HCT116 WT and knockout possesses a replication foci-targeting domain (amino acids 331– cells were treated with 0.3 ␮M 5-aza-CdR for 24 h and released from the drug thereafter. Methylation levels were determined by bisulfite sequencing in 550 of WT DNMT1) and therefore might be targeted to regions untreated cells and on days 4 and 32 after treatment. Each line with circles of active replication. The observed delay could therefore simply indicates an individual DNA molecule. Open circles represent unmethylated be a consequence of limited amounts of DNMT1 protein, which cytosines, and filled circles represent methylated cytosines within a CpG is also strengthened by the fact that the DKO8 cells, with double Ϫ Ϫ dinucleotide context. (A) Bisulfite sequencing of D4Z4.(B) Sequencing results the amount of truncated DNMT1 compared with the DNMT1 / for a p16 exon 2 region. cells, show higher levels of methylation immediately after replication. In their original work, Rhee et al. (12) described eight on day 4 at both loci tested, which might account to some extent independent DKO clones, seven of which grew very slowly and for the lower levels of methylation in the single-knockout cells had lost Ϸ95% of their 5-methylcytosine content. However, one at day 32. clone, DKO8, retained higher methylation levels and prolifer- We also analyzed the kinetics of remethylation of the D4Z4 ated more rapidly. Our analyses revealed that this clone ex- and p16 exon 2 region for 50 days after 5-aza-CdR treatment by presses higher levels of the truncated DNMT1 protein, both at quantitative Ms-SNuPE (Fig. 8, which is published as supporting the RNA and protein levels, and retains considerably higher information on the PNAS web site). As seen before with bisulfite levels of DNA methylation at various loci tested. Likewise, sequencing, the remethylation of D4Z4 occurred very slowly Ϫ Ϫ further depletion of the truncated protein by RNAi caused a after treatment in HCT116 WT cells. DNMT3b / cells showed measurable decrease in methylation of various loci in the remethylation kinetics similar to WT cells, whereas remethyla- Ϫ/Ϫ Ϫ Ϫ DNMT1 cells and a decrease in cell viability in both WT and tion was completely inhibited in DNMT1 / cells, indicating a knockout cell lines. This finding suggests that a certain threshold strong dependency of this region on WT DNMT1. On the other of DNMT1 protein is necessary to confer survival of the cells. hand, p16 exon 2 was remethylated rapidly and with similar The DKO cells have been selected for survival with low levels of kinetics both in DNMT1 WT and knockout cell lines, most likely DNMT1 and might also compensate the loss with expression of because of a strong influence of DNMT3a on this region. a different methyltransferase. A candidate would be the embryonic isoform DNMT3a2, which is expressed from a CpG island Discussion that was methylated in all cell lines except the DKOs. Taken The generation of HCT116 colon cancer cells with disruption of together, the data suggest to us that the truncated protein is DNMT1, DNMT3b, or both has been an important step in indeed functional in vivo and might help to maintain DNA elucidating the role of DNMTs in cancer cells. The finding that methylation and also play a role in cell proliferation or even unique truncated DNMT1 isoforms, which were generated by survival. the knockout strategy (11, 12), are expressed in all independent Although DNMT1 is viewed as a maintenance enzyme, it has DNMT1 and DKO cell lines suggests that DNMT1 expression been speculated that it might confer some de novo methyltrans- may in fact be essential for cell survival. We detected transcripts ferase activity (1, 28). In vitro, DNMT1 shows a clear preference with deletions of exons 2–5 in all DNMT1 knockout cell lines. for hemimethylated DNA; nevertheless, it can methylate com- Interestingly, these transcripts are very similar to a naturally pletely unmethylated CpG pairs with a higher affinity than the occurring, alternative DNMT1 mRNA (GenBank accession no. classic de novo enzymes DNMT3a and DNMT3b (29, 30). Yet AF180682), which splices from exon 1 to exon 5 of WT DNMT1, this finding is still questionable in vivo. Our data suggest that although no corresponding protein has been described as yet. DNMT1 can confer some de novo activity against a demethylated Previous studies have demonstrated that a deletion of up to 580 subtelomeric repeat, D4Z4. After treatment with 5-aza-CdR, aa from the N terminus of human DNMT1 did not inhibit its WT and DNMT3bϪ/Ϫ cells slowly regain their methylation, methyltransferase activity in vitro (26). It will be interesting to whereas DNMT1Ϫ/Ϫ cells are impaired in their remethylation. test in the future whether these transcripts are present and This failure could be caused by loss of cooperativity with translated into a protein in a natural setting in vivo. DNMT3b or by direct loss of de novo activity. Because DNMT3b

14084 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604602103 Egger et al. Downloaded by guest on September 27, 2021 cells show the same remethylation kinetics as WT cells, we 3 h at 37°C. DNA methylation was determined after bisulfite speculate that the latter might be the case. conversion and PCR amplification by Ms-SNuPE analysis. A In summary, the knockout cells generated by Rhee et al. (11, detailed protocol for the assay can be found in Supporting 12) are useful tools for the study of DNMT function in cancer Materials and Methods. cells. However, the fact that they are hypomorphs for DNMT1 masks the real phenotype that DNMT1 disruption might have, Quantitation of DNA Methylation Levels. Genomic DNA was bisul- and it might be anticipated that a complete null will not produce fite-converted by using high-speed conversion as described in ref. viable cells, as seen before in mouse studies. 32. Methylation levels were measured by Ms-SNuPE (33) or bisulfite sequencing (see details in Supporting Materials and Materials and Methods Methods). Tissue Culture and Drug Treatment. HCT116 cells were kept in McCoy’s 5A modified medium supplemented with 10% FCS. DNMT1 RNAi. RNAi experiments were performed by transiently ␮ For 5-aza-CdR treatment, cells were incubated with 0.3 M transfecting 100 nM, 21-nt-long double-stranded RNA oligonu- 5-aza-CdR for 24 h; then, the medium was changed, and cells cleotides (Silencer Custom siRNA; Ambion, Austin, TX) into were harvested at the indicated time points. the different HCT116 cell lines by using Oligofectamine (In- vitrogen). (For details, see Supporting Materials and Methods). RNA Isolation and RT-PCR. Total RNA was isolated by using TRIzol ␮ reagent (Invitrogen, Carlsbad, CA). Total RNA (2–5 g) was Hemimethylation Assay. Hemimethylation was measured as de- transcribed with SuperScript III (Invitrogen). Primer sequences scribed in ref. 20. can be found in Table 1, which is published as supporting information on the PNAS web site. BrdU Pulse–Chase. BrdU pulse–chase experiments were performed as described in ref. 20. For details, also see Supporting Protein Extraction and Western Blotting. Nuclear extracts were Materials and Methods. prepared as described in ref. 31; see also Supporting Materials and Methods, which is published as supporting information on the Statistics. For details about statistical calculations, see Supporting PNAS web site. Antibodies used were DNMT1 (Santa Cruz Materials and Methods. Biotechnology, Santa Cruz, CA), histone deacetylase HDAC1 (Upstate Biotechnology, Lake Placid, NY), and PCNA (Santa We thank Drs. Bert Vogelstein (Howard Hughes Medical Institute, The Cruz Biotechnology). For quantitation of Western blots, we used Johns Hopkins University, Baltimore, MD) and Stephen Baylin (The Johns a Fluor-S MultiImager (Bio-Rad, Hercules, CA). Hopkins University) for kindly providing the HCT116 cell lines and constructive discussions and Dong Yun Yang (Norris Comprehensive In Vitro DNMT1 Assay. Immunoprecipitated DNMT1 was incu- Cancer Center Statistics Core Facility) for performing statistical analyses. bated with 1 ␮l (30 ng) of hemimethylated or unmethylated This work was supported by National Institutes of Health Grants R01 CA oligonucleotide (corresponding to an endogenous p16 se- 82422 and R01 CA 83867, National Institutes of Health Training Grant quence), 0.5 ␮l of BSA, and 0.25 ␮lofS-adenosylmethionine for 5251143701 (to T.W.H.L.), and the Max Kade Foundation (G.E.).

1. Goll MG, Bestor TH (2005) Annu Rev Biochem 74:481–514. 17. Santi DV, Norment A, Garrett CE (1984) Proc Natl Acad Sci USA 81:6993– 2. Li E, Bestor TH, Jaenisch R (1992) Cell 69:915–926. 6997. 3. Li E, Beard C, Forster AC, Bestor TH, Jaenisch R (1993) Cold Spring Harbor 18. Iida T, Suetake I, Tajima S, Morioka H, Ohta S, Obuse C, Tsurimoto T (2002) Symp Quant Biol 58:297–305. Genes Cells 7:997–1007. 4. Jackson-Grusby L, Beard C, Possemato R, Tudor M, Fambrough D, Csank- 19. Jeltsch A (2006) Curr Top Microbiol Immunol 301:203–225. ovszki G, Dausman J, Lee P, Wilson C, Lander E, Jaenisch R (2001) Nat Genet 20. Liang G, Chan MF, Tomigahara Y, Tsai YC, Gonzales FA, Li E, Laird PW, 27:31–39. Jones PA (2002) Mol Cell Biol 22:480–491. 5. Laird PW, Jackson-Grusby L, Fazeli A, Dickinson SL, Jung WE, Li E, 21. Cheng JC, Weisenberger DJ, Gonzales FA, Liang G, Xu GL, Hu YG, Marquez Weinberg RA, Jaenisch R (1995) Cell 81:197–205. VE, Jones PA (2004) Mol Cell Biol 24:1270–1278. 6. Robert MF, Morin S, Beaulieu N, Gauthier F, Chute IC, Barsalou A, MacLeod 22. Kondo T, Bobek MP, Kuick R, Lamb B, Zhu X, Narayan A, Bourc’his D, AR (2003) Nat Genet 33:61–65. Viegas-Pequignot E, Ehrlich M, Hanash SM (2000) Hum Mol Genet 9:597–604. 7. Leu YW, Rahmatpanah F, Shi H, Wei SH, Liu JC, Yan PS, Huang TH (2003) 23. Paz MF, Wei S, Cigudosa JC, Rodriguez-Perales S, Peinado MA, Huang TH, Cancer Res 63:6110–6115. Esteller M (2003) Hum Mol Genet 12:2209–2219. 8. Suzuki M, Sunaga N, Shames DS, Toyooka S, Gazdar AF, Minna JD (2004) 24. Chen T, Ueda Y, Xie S, Li E (2002) J Biol Chem 277:38746–38754. Cancer Res 64:3137–3143. 25. Cui H, Onyango P, Brandenburg S, Wu Y, Hsieh CL, Feinberg AP (2002) 9. Ting AH, Jair KW, Suzuki H, Yen RW, Baylin SB, Schuebel KE (2004) Nat Cancer Res 62:6442–6446. Genet 36:582–584. 10. Ting AH, Jair KW, Schuebel KE, Baylin SB (2006) Cancer Res 66:729–735. 26. Pradhan S, Esteve PO (2003) Biochemistry 42:5321–5332. 11. Rhee I, Jair KW, Yen RW, Lengauer C, Herman JG, Kinzler KW, Vogelstein 27. Ding F, Chaillet JR (2002) Proc Natl Acad Sci USA 99:14861–14866. B, Baylin SB, Schuebel KE (2000) Nature 404:1003–1007. 28. Hermann A, Gowher H, Jeltsch A (2004) Cell Mol Life Sci 61:2571–2587. 12. Rhee I, Bachman KE, Park BH, Jair KW, Yen RW, Schuebel KE, Cui H, 29. Fatemi M, Hermann A, Pradhan S, Jeltsch A (2001) J Mol Biol 309: Feinberg AP, Lengauer C, Kinzler KW, et al. (2002) Nature 416:552–556. 1189–1199. 13. Rountree MR, Bachman KE, Baylin SB (2000) Nat Genet 25:269–277. 30. Okano M, Xie S, Li E (1998) Nat Genet 19:219–220. 14. Chuang LS, Ian HI, Koh TW, Ng HH, Xu G, Li BF (1997) Science 277:1996–2000. 31. Fatemi M, Pao MM, Jeong S, Gal-Yam EN, Egger G, Weisenberger DJ, Jones 15. Bouchard J, Momparler RL (1983) Mol Pharmacol 24:109–114. PA (2005) Nucleic Acids Res 33:e176. 16. Taylor SM, Constantinides PA, Jones PA (1984) Curr Top Microbiol Immunol 32. Shiraishi M, Hayatsu H (2004) DNA Res 11:409–415. GENETICS 108:115–127. 33. Gonzalgo ML, Jones PA (1997) Nucleic Acids Res 25:2529–2531.

Egger et al. PNAS ͉ September 19, 2006 ͉ vol. 103 ͉ no. 38 ͉ 14085 Downloaded by guest on September 27, 2021