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Molecular and Cellular Biology, June 2005, p. 4727-4741, Vol. 25, No. 11
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.11.4727-4741.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

5-Aza-Deoxycytidine Induces Selective Degradation of DNA Methyltransferase 1 by a Proteasomal Pathway That Requires the KEN Box, Bromo-Adjacent Homology Domain, and Nuclear Localization Signal

Kalpana Ghoshal,{dagger}* Jharna Datta,{dagger} Sarmila Majumder, Shoumei Bai, Huban Kutay, Tasneem Motiwala, and Samson T. Jacob*

Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, Ohio

Received 22 December 2004/ Returned for modification 16 January 2005/ Accepted 18 February 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
5-Azacytidine- and 5-aza-deoxycytidine (5-aza-CdR)-mediated reactivation of tumor suppressor genes silenced by promoter methylation has provided an alternate approach in cancer therapy. Despite the importance of epigenetic therapy, the mechanism of action of DNA-hypomethylating agents in vivo has not been completely elucidated. Here we report that among three functional DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), the maintenance methyltransferase, DNMT1, was rapidly degraded by the proteasomal pathway upon treatment of cells with these drugs. The 5-aza-CdR-induced degradation, which occurs in the nucleus, could be blocked by proteasomal inhibitors and required a functional ubiquitin-activating enzyme. The drug-induced degradation occurred even in the absence of DNA replication. Treatment of cells with other nucleoside analogs modified at C-5, 5-fluorodeoxyuridine and 5-fluorocytidine, did not induce the degradation of DNMT1. Mutation of cysteine at the catalytic site of Dnmt1 (involved in the formation of a covalent intermediate with cytidine in DNA) to serine (CS) did not impede 5-aza-CdR-induced degradation. Neither the wild type nor the catalytic site mutant of Dnmt3a or Dnmt3b was sensitive to 5-aza-CdR-mediated degradation. These results indicate that covalent bond formation between the enzyme and 5-aza-CdR-incorporated DNA is not essential for enzyme degradation. Mutation of the conserved KEN box, a targeting signal for proteasomal degradation, to AAA increased the basal level of Dnmt1 and blocked its degradation by 5-aza-CdR. Deletion of the catalytic domain increased the expression of Dnmt1 but did not confer resistance to 5-aza-CdR-induced degradation. Both the nuclear localization signal and the bromo-adjacent homology domain were essential for nuclear localization and for the 5-aza-CdR-mediated degradation of Dnmt1. Polyubiquitination of Dnmt1 in vivo and its stabilization upon treatment of cells with a proteasomal inhibitor indicate that the level of Dnmt1 is controlled by ubiquitin-dependent proteasomal degradation. Overexpression of the substrate recognition component, Cdh1 but not Cdc20, of APC (anaphase-promoting complex)/cyclosome ubiquitin ligase reduced the level of Dnmt1 in both untreated and 5-aza-CdR-treated cells. In contrast, the depletion of Cdh1 with small interfering RNA increased the basal level of DNMT1 that blocked 5-aza-CdR-induced degradation. Dnmt1 interacted with Cdh1 and colocalized in the nucleus at discrete foci. Both Dnmt1 and Cdh1 were phosphorylated in vivo, but only Cdh1 was significantly dephosphorylated upon 5-aza-CdR treatment, suggesting its involvement in initiating the proteasomal degradation of DNMT1. These results demonstrate a unique mechanism for the selective degradation of DNMT1, the maintenance DNA methyltransferase, by well-known DNA-hypomethylating agents.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Methylation of DNA at position 5 of cytosine within a CpG dinucleotide is the predominant covalent modification in the eukaryotic genome (9, 10, 33, 50). This epigenetic modification is essential for mammalian development, genomic imprinting, and silencing of proviral promoters in the genome. Although CpG is usually underrepresented in much of the mammalian genome, short CpG-rich regions (typically 500 to 2,000 bp long), designated CpG islands, are found in the proximal promoter regions of almost 50% of the genes. These regions are usually nonmethylated in normal cells, with the exception of imprinted genes. There have been numerous reports of DNA hypermethylation in disease states, particularly cancer (for reviews, see references 5, 30, and 35). Approximately 1.5% of the CpG islands (an average of 600 CpG islands out of 45,000) in the genome of tumor tissues exhibit aberrant methylation, some showing tumor-specific methylation patterns (5, 6, 18, 45, 46, 52). Many tumor suppressor genes are silenced due to methylation of the CpG islands in their promoter regions. It is therefore conceivable that demethylation and the consequent reactivation of these genes will be a rational approach to the treatment of cancer. In an effort to restore the activity of these genes, 5-azacytidine (5-aza-C) or its congener 5-aza-deoxycytidine or (5-aza-CdR) has been used for cancer therapy (for reviews, see references 4, 16, 17, 29, 32, and 37). Nearly 100 clinical trials with either 5aza-C or 5-aza-CdR alone or in combination with a histone deacetylase inhibitor have been reported in the National Cancer Institute database. While different types of leukemia have been treated with these agents, the most promising results have been achieved in the therapy of myelodysplastic syndrome and leukemia. These drugs have also been used in the treatment of sickle cell anemia and ß-thalassemia (54).

Although considerable efforts have been made in the elucidation of the molecular mechanism(s) by which these potent drugs alter the DNA methylation profile, the exact mechanism of their action remains to be determined. Following its conversion to the nucleoside triphosphate, 5-aza-C is incorporated into RNA and DNA and consequently alters protein synthesis, whereas 5-aza-CdR is incorporated only into DNA. These drugs can also be deaminated into the respective uridines and their triphosphates, which interfere with de novo thymidylate synthesis. A noteworthy mechanism proposed more than two decades ago is based on studies that showed the inability of DNA methyltransferases to methylate DNA following the incorporation of 5-aza-CdR into DNA (19, 36, 53, 56, 59).

Animal cells contain three functional DNA methyltransferases (for reviews, see references 8 and 34). Among these enzymes, Dnmt3a and Dnmt3b exhibit predominant de novo methyltransferase activity, whereas Dnmt1 is exclusively involved in the methylation of hemimethylated DNA. Gene deletion studies with mice have shown that Dnmt1–/– and Dnmt3b–/– are lethal for embryos, whereas Dnmt3a–/– mice die immediately after birth (47). Since DNMT1 requires hemimethylated DNA as the substrate, it is anticipated that the effect of 5-aza-C or 5-aza-CdR will not be fully exerted until the analog-incorporated DNA undergoes replication. Some observations have suggested that the formation of a tight covalent complex between Dnmts and 5-aza-C-substituted DNA alone cannot explain many aspects of these drugs. First, DNA methyltransferase activity decreases much faster than incorporation of 5-aza-C into DNA (19). Second, extensive analysis of the gene expression profile in a colon cancer cell line (HCT116) has shown that the 5-aza-CdR-induced alteration in expression occurs independently of the growth stage and is not due solely to the incorporation of the analog into DNA (25).

While exploring the mechanism of synergistic activation of the methylated metallothionein I promoter by 5-aza-C and trichostatin A (histone deacetylase inhibitor), we made certain observations that led us to believe that 5-aza-CdR and 5-aza-C must have differential effects on mammalian DNA methyltransferases (22). First, Dnmt1 is completely depleted after 5-aza-C exposure, whereas Dnmt3a is significantly less sensitive and Dnmt3b is practically resistant to inhibitor-induced depletion. Second, although all three Dnmts have the conserved PCQ motif in the catalytic domain and can bind to 5-aza-C-incorporated DNA (39, 51), only Dnmt1 is degraded in response to drug treatment. Third, the gene expression profile of 5-aza-CdR-treated cells correlated with that of DNMT1–/– cells and not with that of DNMT3B–/– cells (23). The present study shows that Dnmt1 is rapidly and selectively degraded by the proteasomal pathway in response to these inhibitors and that this process occurs in the nucleus independently of DNA replication and requires the conserved KEN box.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of Dnmt1 mutants. pcDnmt1-KEN/FL-Flag was constructed by cloning mouse Dnmt1 cDNA (–5.2 kb) PCR amplified from pMIG7 [Dnmt1cDNA in pBSK(+)] (7) at the HindIII/XbaI site of p3XFlag-CMV-14 (Sigma). The KEN box of Dnmt1 was mutated to AAA by site-directed mutagenesis of the KEN sequence (43). We performed multiple rounds of PCR by using appropriate primers with altered bases to amplify the 1,580-bp PshAI/NheI fragment of Dnmt1. pDnmt1-KEN/FL-Flag was digested with PshAI/NheI, and the mutated PshAI/NheI fragment was subcloned at the same sites to create pDnmt1-AAA/FL-Flag. The primer pairs used for the KEN-AAA mutation were as follows (underlining indicates the mutated bases): pair 1, KEN-F (5'-ATAAGGAGGACGCGGCAGCTGCCATGAA) and NheI-R (5'-GGGCGTTTCACGGGGCTAGCCACTTTG); and pair 2, KEN-R (5'-TTCATGGCAGCTGCCGCGTCCTCCTTAT) and PshAI-F (5'-GGGTCCTGTCGACACCGGTCTCATTGAGAAG).

For the construction of pcDnmt1-CS-Flag, the PCQ motif of Dnmt1 was mutated to PSQ (CS mutation) by site-directed mutagenesis. By using appropriate primers with altered bases, we performed multiple rounds of PCR to amplify the 1,630-bp XhoI/XbaI fragment. Plasmid Dnmt1-KEN/FL-3XFlag was digested with XhoI/XbaI, and the mutated XhoI/XbaI fragment was subcloned at the same sites to create pcDnmt1-CS-Flag. The primer pairs used for the CS mutation were as follows (underlining indicates the mutated bases): pair 1, PCQ-F (5'-GGTGGGCCACCCAGTCAGGGCTTCAGT) and XbaI-R (5'-AATCTAGAGTCCTTGGTAGCAGCCTCC); and pair 2, PCQ-R (5'-ACTGAAGCCCTGACTGGGTGGCCCACC) and XhoI-F (5'-TACTTCCTCGAGGCCTACAATTCAAAG).

pcDnmt1-{Delta}NLS-Flag was constructed by eliminating the nuclear localization signal (NLS) from Dnmt1 and amplifying the {Delta}NLS-Dnmt1 fragment from Dnmt1-KEN/FL-3XFlag. This fragment was cloned at the HindIII/XbaI site of p3XFlag-CMV-14.

pcDnmt1-KEN (or AAA)/{Delta}CAT-Flag was generated to eliminate the catalytic domain of Dnmt1 from the corresponding full-length plasmids. The catalytic domain was eliminated by amplifying amino acids (aa) 1 to 1112 of the corresponding full-length clones and subcloning at the HindIII/XbaI site of the same vector.

pcDnmt1-{Delta}BAH-Flag was generated by eliminating aa 750 to 1112 from pDnmt1-KEN-FL-Flag by PCR mutagenesis. The PshAI/XhoI fragment (aa 750 to 1112) was amplified from pcDnmt1-KEN-FL-Flag by using the forward primer spanning the PshAI site (PshAI-F, 5'-GGGTCCTGTCGACACCGGTCTCATTGAGAG) and the reverse primer spanning the XhoI site (5'-GGCCTCGAGATTCTCTTCAATCTTCATAGGCTG) and was subcloned at the same site of pcDnmt1-KEN-FL-Flag. All of the clones created were sequenced to confirm their authenticity, and expression was confirmed by Western blot analysis of the expressed protein by using anti-Flag antibody M2 (Sigma).

Cell culture, treatment with various inhibitors, transient transfection assay, and Western blot analysis. HeLa and Cos-7 cells were grown in Dulbecco minimal essential medium plus 10% fetal bovine serum, whereas P1798 cells were grown in RPMI 1640 plus 10% fetal bovine serum. The growing cells were treated with drugs (0.1 to 10 µM) for 2 to 24 h, and whole-cell extracts (WCEs) were prepared by suspending cells in lysis buffer A (50 mM Tris [pH 8.1], 1 mM EDTA, 1% sodium dodecyl sulfate [SDS], and protease inhibitor cocktail) followed by sonication. Western blot analysis was performed with whole-cell extracts (WCEs) and antibodies that specifically recognize human DNMT1 (New England Biolabs) and mouse Dnmt1 (55) and with anti-Dnmt3a and anti-Dnmt3b antibodies that were raised in our laboratory and that detect the respective proteins in mammalian cells (20, 42): ß-tubulin (Santa Cruz), Ku70 (Neomarker), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Chemicon), Cdc20 (Santa Cruz), Cdh1 (Neomarker), and hemagglutinin (HA) (Covance).

For the transfection assay, Cos-7 cells were seeded at a density of 106/plate on the day before transfection and were transfected with Flag-tagged DNMT1 expression vectors by the calcium phosphate precipitation method (23, 44). pTracer-SV40 (Clontech), an expression vector for green fluorescent protein (GFP), was cotransfected to serve as an internal control to determine transfection efficiency. The amounts of the expressed recombinant proteins were determined by Western blot analysis with anti-Flag (Sigma) and anti-GFP (Invitrogen) antibodies. To prepare WCEs, cells were washed with phosphate-buffered saline and solubilized in lysis buffer A (50 mM Tris [pH 8.1], 1 mM EDTA, 1% SDS, and protease inhibitor cocktail). Protein concentrations were determined by using protein assay reagent (Bio-Rad) with bovine serum albumin as a standard.

RNA interference assay. Cdh1 small interfering RNA (siRNA) smart pool and scrambled siRNA were obtained from Dharmacon. HeLa cells were transfected twice at 24-h intervals with 100 nM siRNA along with Lipofectamine 2000 (Invitrogen) as described previously (3). After 24 h, cells were treated with 5-aza-CdR for 6 h, and cell extracts were subjected to Western blot analysis with anti-Cdh1 and anti-Dnmt1 antibodies.

Real-time RT-PCR analysis. Total RNA isolated from HeLa cells by the guanidinium thiocyanate-acid phenol method was treated with Turbo DNase I (Ambion) following the manufacturer's protocol. cDNA was synthesized from total RNA by using random hexamers as primers and murine leukemia virus reverse transcriptase (RT) following the manufacturer's protocol (Applied Biosystem). An aliquot of the cDNA (equivalent to 100 ng of RNA for Dnmt1, 250 ng for each of DNMT3A or DNMT3B, and 10 ng for 18S rRNA) was used for real-time PCR analysis. All real-time PCRs were carried out by using an Mx3000 multiplex quantitative PCR system (Stratagene).

The following PCR primers were used for RT-PCR analysis: Dnmt1, 5'-AGGGAAAAGGGAAGGGCAAG and 5'-AGAAAACACATCCAGGGTCCG; Dnmt3a, 5'-CAGCGTCACACAGAAGCATATCC and 5'-GGTCCTCACTTTGCTGAACTTGG; Dnmt3b, 5'-CCTGCTGAATTACTCACGCCCC and 5'-GTCTGTGTAGTGCACAGGAAAA; and 18S rRNA, 5'-TCAAGAACGAAAGTCGGAGG and 5'-GGACATCTAAGGGCATCACA.

The optimum primer concentration was 150 nM. All PCR amplifications were performed by using brilliant SYBR green QPCR master mix (Stratagene) with ROX as a reference dye in a 20-µl reaction volume. A standard curve for each cDNA was first generated using 10-fold serial dilutions (108 to 102 copies) of the respective cDNAs as templates. To create the standard curve, human 18S rRNA, DNMT1, DNMT3A, and DNMT3B cDNAs were amplified by RT-PCR. The copy number for each cDNA expressed in HeLa cells was calculated from the standard curve and normalized to that for 18S rRNA. PCR cycling conditions were as follows: initial denaturation at 95°C for 10 min; 45 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and dissociation at 95°C for 1 min and 55°C for 30 s (to check for the formation of a primer dimer). The dissociation profile for the amplified products indicated that none of the primer pairs generated a dimer.

RT-PCR analysis. cDNA synthesized from total RNAs of control and 5-az-CdR-treated cells were subjected to RT-PCR analysis with primers specific for mouse MT-I (22) and human MLH1, O6-MGMT, and 18S rRNA. The primers for human MLH1 and O6-MGMT were as follows: human MLH1, 5'-TCACGGTGGAGGACCTTTTTTAC and 5'-ACGGTTGAGGCATTGGGTAGTGTC; and human O6-MGMT, 5'-GCTCTTCACCATCCCGTTTTC and 5'-ATTGCCTCTCATTGCTCCTCCCAC.

BrdUrd incorporation assay. HeLa cells were either left untreated or treated with 20 µg/ml aphidicolin for 24 h followed by treatment with 5-aza-C or 5-aza-CdR for 12 h. The cells were pulse-labeled with bromodeoxyuridine (BrdUrd) (10 µM) (Sigma) for 2 h, fixed with 70% ethanol, denatured with 2 N HCl, and stained with anti-BrdUrd antibody (Sigma) as described previously (2).

Thymidine incorporation assay. Cells were treated with aphidicolin, 5-aza-C, or 5-aza-CdR as described above and incubated with 5 µCi of [3H1]thymidine (MP Biochemicals) for 2 h. Trichloroacetic acid-precipitated DNA was dissolved in buffer containing 3 M NaOH and 0.5% SDS and counted in a scintillation counter.

Pulse-chase experiment. Cos-7 cells transfected with Dnmt1-Flag were labeled with [35S]methionine (1 mCi/ml) (MP Biochemicals) for 2 h in methionine-free medium, washed with phosphate-buffered saline, and then chased in medium containing 2 mM methionine. One group of cells was treated with 5-aza-C (5 µM), and the other group was left untreated. WCEs were made in radioimmunoprecipitation assay buffer from cells harvested at 0, 3, 6, and 9 h and were immunoprecipitated with anti-Flag antibody. The precipitated proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane that was subjected to autoradiography and PhosphorImager analysis. The blot was also subjected to Western blot analysis with anti-Flag antibody.

In vivo ubiquitination assay. Cos-7 cells were transfected with DNMT1-Flag, HA-tagged ubiquitin, and a combination of both by the calcium phosphate precipitation method. At 36 h posttransfection, the cells were treated with lactacystin (20 µM) for 4 h to stabilize the ubiquitinated products. Cells were solubilized in 1% SDS, boiled to inactivate deubiqutinating enzymes, and diluted with buffer to make the final SDS concentration 0.1% (11, 27). Equal amounts of proteins from the extracts were immunoprecipitated with anti-Flag or anti-HA (Covance) antibodies and washed three times with radioimmunoprecipitation assay buffer, and the bound proteins were subjected to Western blot analysis with both antibodies.

Coimmunoprecipitation assay. Cos-7 cells were transfected with Dnmt1-Flag along with plasmids harboring HA-tagged Cdh1, Cdc20, or empty vector (pCMV-HA). After 36 h, cells were harvested, and WCEs in TNN buffer (40 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5% NP-40) were immunoprecipitated with anti-HA or anti-Flag antibodies. Precipitated proteins were subjected to Western blot analysis with anti-Flag and anti-HA antibodies.

Nuclear and cytoplasmic fractions were isolated from HeLa cells as described previously (1). The nuclear pellet was resuspended in lysis buffer A and subjected to sonication to fragment DNA.

DNA methyltransferase activity was measured as described previously (24).

An indirect immunofluorescence assay was performed with HeLa or Cos-7 cells as described previously (23). Anti-DNMT1 antibody used for this purpose was obtained from Imgenex or New England Biolabs.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dnmt1 is selectively degraded in mammalian cells in response to 5-aza-CdR. As a first step to explore the underlying mechanism of the inhibitor-induced degradation of Dnmt1 in mammalian cells we determined the protein levels of Dnmt1, Dnmt3a and Dnmt3b in the extracts of cells treated with 5-aza-CdR for various times. We initially chose lymphosarcoma cells as all three Dnmts are expressed at high levels in these cells (Fig. 1A). A time-dependent decrease in Dnmt1 level following treatment with 5-aza-CdR was observed, as shown by immunoblot analysis of the cell extract. The reduction in the protein level was apparent as early as 2 h of drug treatment, 95% degradation occurring by 12 h (Fig. 1A). Mouse lymphosarcoma cells express one isoform of Dnmt3a and two variants of Dnmt3b that are either unaffected or not significantly altered even after 24 h of 5-aza-CdR exposure (Fig. 1A).



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  		FIG. 1. DNMT1 is selectively degraded by 5-aza-CdR treatment of mammalian cells. (A) Dnmt1, Dnmt3a, and Dnmt3b protein levels in P1798 cell extracts. Western blot analysis of Dnmt1/3a/3b with WCEs from mouse lymphosarcoma cells (100 µg of protein) treated with 5-aza-CdR (2.5 µM) for various times with antibodies specific for these proteins. The blots were reprobed with ß-tubulin to show equal loading of proteins. (B) DNMT1, DNMT3A, and DNMT3B protein levels in extracts from HeLa cells treated with various concentrations of 5-aza-CdR. Western blot analysis with WCEs (250 µg of protein) from HeLa cells. The smaller (70-kDa) subunit of Ku antigen was used as the control to determine equal loading of proteins. (C and D) The 5-aza-CdR concentration required to deplete DNMT1 is proportional to the endogenous DNMT1 level. Immunoblot analysis of DNMT1 with WCEs (200 µg of protein) from wild-type and adenomatous polyposis coli (APC)-mutated colon cancer cells treated with various concentrations of 5-aza-CdR. The blots were reprobed with ß-tubulin to demonstrate comparable levels of proteins in the lanes. For quantitation, the levels in untreated cells were taken as 1. (E) Activation of MT-I, human MLH1, and MGMT genes after 5-aza-CdR treatment. Total RNA was isolated from P1798 cells treated with 5 µM 5-aza-CdR for 24 h. The expression of MT-I, human MLH1, and MGMT mRNAs was analyzed by RT-PCR. 18S RNA was amplified from all of the samples to normalize RNA input. (F) HeLa cells were either left untreated or treated with 10 µM flucytosine (5FC) or 5-fluorodeoxyuridine (5FU) for 12 h followed by treatment with 5-azaC-dR (5 µM) for an additional 12 h. Extracts were subjected to Western blot analysis.

 
We then studied the effect of 5-aza-CdR on DNMT levels in human (HeLa) cells. In these cells DNMT1 level started to decline following treatment with 0.1 µM of the inhibitor and was undetectable after treatment of cells with 5 µM 5-aza-CdR for 12 h (Fig. 1B). DNMT1 could not be recovered from these cells even when DNA was digested by treating the nuclei with DNase 1 before extraction of proteins (Fig. 1B, lane 6). HeLa cells express only one isoform of DNMT3A and two variants of DNMT3B and their levels were not significantly altered by 5-aza-CdR treatment (Fig. 1B).

Next we investigated whether the concentration of 5-aza-CdR needed to induce degradation of DNMT1 is proportional to cellular DNMT1 level. We chose two colon cancer cells DLD1b and RKO that exhibit different constitutive levels of DNMT1 (Fig. 1C). The suppression of DNMT1 promoter by APC/ß-catenin pathway (12) could probably explain relatively high levels of DNMT1 in DLD1b cells where APC is mutated. In APC-positive RKO cells, 5-aza-CdR (1 µM) treatment abolished DNMT1 whereas DNMT1 was detectable in APC-negative DLD1b cells even after treatment with 5 µM 5-aza-CdR for 24 h (Fig. 1C and D). Treatment of mammalian cells of human, mouse and rat origin with 5-aza-CdR at similar concentrations also resulted in selective depletion of Dnmt1 (data not shown). These results demonstrate that selective degradation of DNMT1 by these drugs occurs in mammalian cells and the amount required depends on the level of DNMT1.

To demonstrate that aza-CdR was functional in vivo, we measured mRNA levels of a few genes methylated and silenced in these cells. The RT-PCR data revealed that 5-aza-CdR treatment indeed activated the silenced genes, such as MT-I in P1798 cells, MGMT in HeLa cells, and human MLH1 in RKO cells (Fig. 1E).

To determine whether any modification at C-5 of cytosine can initiate degradation of DNMT1, HeLa cells were treated with flucytosine and 5-fluorodeoxyuridine. These analogs are converted to deoxynucleotide triphosphates and incorporated into DNA by replication machinery. These nucleotides had minimal effect on the endogenous DNMT1 level in HeLa cells and the 5-aza-CdR-induced degradation of DNMT1 continued unabated (Fig. 1F). These results indicate that the aza group at C-5 is essential to induce the degradation of DNMT1.

5-aza-CdR-induced degradation of DNMT1 occurs in cells treated with aphidicolin, a potent inhibitor of DNA synthesis. The current notion is that depletion of DNMT1 from the soluble nuclear fraction upon 5-aza-CdR-treatment is due to formation of a covalent complex between the cysteine residue in the PCQ motif of DNMT1 and carbon 6 of 5-aza-CdR incorporated into DNA. If the depletion of DNMT1 from cells treated with inhibitors of DNA methyltransferase occurs solely by this mechanism, one would expect reversal of this process in cells treated with inhibitors of DNA synthesis. To address this issue, HeLa cells were treated with aphidicolin, a potent inhibitor of DNA synthesis, for 24 h followed by treatment with 5-aza-C or 5-aza-CdR for an additional 12 h. To demonstrate that aphidicolin indeed blocked DNA synthesis under this condition we measured BrdUrd incorporation into DNA. In replicating cells the nucleotide analog is incorporated into newly synthesized DNA in place of thymine, which is then detected by anti-BrdUrd antibody. Strong purple staining in the nucleus with pink cell bodies (stained with eosin Y) identified BrdUrd-positive cells (Fig. 2A). The lack of nuclear staining in cells incubated without BrdUrd demonstrated specificity of the assay (Fig. 2A, panel a). A large population of cells was positive for BrdUrd among untreated cells and cells treated with 5-aza-C or 5-aza-CdR (Fig. 2A, panels b to d). In contrast, none of the cells treated with aphidicolin for 24 h were BrdUrd positive, and the nuclei of aphidicolin-treated cells exhibited a staining pattern similar to that of those incubated without BrdUrd (Fig. 2A, panels e to h). Counting of a few hundred cells from multiple plates showed that aphidicolin treatment completely inhibited BrdUrd incorporation in HeLa cells, whereas BrdUrd incorporation was not affected in cells treated with 5-aza-C or 5-aza-CdR alone (data not shown). The DNA replication potential of cells assayed by [3H1]thymidine incorporation showed 95 to 98% inhibition of DNA replication in cells treated with aphidicolin, whereas in 5-aza-C- or 5-aza-CdR-treated cells, it was comparable to that of untreated cells (Fig. 2B).



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  		FIG. 2. 5-Aza-CdR-induced degradation of DNMT1 remains unaffected in HeLa cells when DNA synthesis is blocked with aphidicolin. (A) BrdUrd incorporation is abolished in cells treated with aphidicolin. HeLa cells were treated with aphidicolin (20 µg/ml) for 24 h followed by treatment with 5-aza-C or 5-aza-CdR for an additional 12 h. Cells were incubated with BrdUrd (10 µM) for 2 h, washed, fixed, and stained with anti-BrdUrd antibody and eosin Y (to stain the cell body). (B) Thymidine incorporation is significantly inhibited in cells treated with aphidicolin. Cells were treated as described for panel A and incubated with [3H1]thymidine for 2 h. Tritium incorporation into DNA was measured with a scintillation counter. (C and D) 5-Aza-CdR-induced degradation persists in cells treated with aphidicolin. Western blot analysis of Dnmt1 in extracts from cells treated with drugs as described for panel A. All quantitative results are the mean of three independent experiments.

 
We next measured the level of DNMT1 in cells treated with aphidicolin alone or with aphidicolin followed by 5-aza-CdR treatment by immunoblot analysis. The 5-aza-CdR-induced DNMT1degradation remained unabated in cells pretreated with aphidicolin whereas aphidicolin alone had minimal effect on DNMT1 level (Fig. 2C and D). Similar results were obtained in cells treated with other inhibitors of replication, such as Ara-C and hydroxyurea (data not shown). These results support the notion that DNMT1 can be degraded in response to 5-aza-CdR treatment even when DNA synthesis is blocked.

5-aza-CdR-induced degradation of DNMT1 is a posttranslational event that could be blocked by inhibitors of the proteasomal pathway. To determine whether the decrease in DNMT1 level was due to the down regulation of its expression, we measured mRNA levels of all three DNMTs by real-time PCR (Fig. 3A). As expected, the copy number of DNMT1 was higher than DNMT3A and DNMT3B in untreated HeLa cells (Fig. 3A). A small but significant increase in the mRNA levels of all three DNMTs was observed in cells treated with 5-aza-CdR (2.5 µM) for 24 h (Fig. 3A), as opposed to nearly complete depletion of DNMT1 protein after treatment with the drug (Fig. 1B). At a 5 µM concentration, their levels were comparable to that of the control (Fig. 3A). The expression of DNMT1 did not decrease during 24 to 72 h of exposure to concentrations of 2.5 to 5 µM in a few other cell lines tested (data not shown). These data clearly demonstrated that the decrease in Dnmt1 level upon 5-aza-CdR treatment was not due to decline in the steady-state mRNA level.



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  		FIG. 3. 5-Aza-CdR-induced degradation of DNMT1 occurs at the posttranslational level and can be blocked by proteasomal inhibitors. (A) Expression of DNMT1 mRNA is not reduced in cells upon treatment with 5-aza-CdR. The mRNA levels (copy numbers) of DNMTs normalized to 18S rRNA were analyzed by real time RT-PCR of HeLa cells treated with 5-aza-CdR for 24 h. The bar diagram shows the fold change in the mRNA levels for DNMT1, DNMT3A, and DNMT3B following treatment with various concentrations of 5-aza-CdR. The copy number for each DNMT normalized to 18S rRNA is presented below each bar. The results are the mean ± standard error of three independent experiments performed in triplicate. (B and C) 5-Aza-CdR-induced depletion is not due to inhibition of protein biosynthesis. Western blot analysis of WCEs from cells treated with cycloheximide (20 µg/ml) for 1 h before 5-aza-CdR (5 µM) exposure for 3 and 6 h. (D and E) Decreased half-life of Dnmt1 in cells treated with 5-aza-CdR. Cos-7 cells transfected with pcDnmt1-Flag were labeled with [35S]methionine (for 2 h) at 36 h posttransfection, followed by a chase in complete medium containing an excess of unlabeled methionine for various times. Ectopic Dnmt1 was immunoprecipitated from WCEs (100 µg of protein) with anti-Flag antibody, separated by SDS-polyacrylamide gel electrophoresis, and subjected to autoradiography and phosphorimaging analysis. The level of a nonspecific polypeptide (NS) pulled down by anti-Flag antibody did not significantly change after cycloheximide treatment. (F and G) DNMT1 levels in WCEs from HeLa cells treated with various protease inhibitors 1 h before treatment with 5 µM 5-aza-CdR for 12 h. All quantitative results are the mean of three independent experiments.

 
To investigate whether 5-aza-CdR-induced depletion of DNMT1 is due to inhibition of de novo synthesis of the protein that turns over rapidly or posttranslational degradation of the presynthesized protein we studied the effect of 5-aza-CdR in cells pretreated with cycloheximide. Incorporation of [35S]methionine into proteins was significantly inhibited in cells treated with cycloheximide for 1 h (data not shown). The decrease in the DNMT1 level by 50% in cells treated with cycloheximide alone for 6 h indicates that it has a relatively short half-life (Fig. 3B, lanes 5 and 7, and Fig. 3C). After 5-aza-CdR treatment, the DNMT1 level was significantly reduced with time (Fig. 3B, compare lanes 2 and 6 with lanes 1 and 5, respectively, and Fig. 3C), and pretreatment with cycloheximide had no effect on the degradation process (Fig. 3B, compare lanes 4 and 8 with lanes 3 and 7, respectively, and Fig. 3C). These results demonstrate that 5-aza-CdR-induced degradation of DNMT1 is a posttranslational event.

To prove that 5-aza-CdR-induced depletion of DNMT1 is due to enhanced degradation of the presynthesized protein the half-life of overexpressed Flag-tagged Dnmt1 was measured by pulse-chase study. Cos-7 cells overexpressing Dnmt1-Flag were incubated with [35S]methionine for 2 h to label the newly synthesized proteins followed by chase with excess unlabeled methionine. In untreated cells, the 35S signal in Dnmt1 gradually decreased with time (Fig. 3D, compare lanes 2 to 4 with lane 1, and Fig. 3E). Dnmt1 signal at each time point from the 5-aza-CdR-treated cells was lower than that in control cells (Fig. 3D, compare lanes 5 to 7 with lanes 2 to 4, respectively). Treatment of cells with ZLLL, a proteasomal inhibitor, significantly blocked 5-aza-CdR-mediated decrease in Dnmt1 level (Fig. 3D, compare lanes 8 to 10 with lanes 5 to 7, respectively, and Fig. 3E). 5-Aza-CdR-induced degradation was also blocked by other proteasomal inhibitors such as ALLN, lactacystin but not by inhibitors of proteases involved in the degradation of proteins by lysosomal (chloroquine), cytoplasmic (N{alpha}-p-tosyl-L-lysine chloromethyl ketone, tolylsulfonyl phenylalanyl chloromethyl ketone, and phenylmethylsulfonyl fluoride), and/or apoptotic (ZVAD-FMK) pathways (Fig. 3F and G). It is noteworthy that the half-lives of both endogenous DNMT1 in HeLa cells (Fig. 3B and C) and mouse Dnmt1 ectopically expressed in Cos-7 cells are significantly reduced upon 5-aza-CdR treatment and that pretreatment of cells with proteasomal inhibitors prolonged their half-lives.

5-Aza-CdR-induced degradation of DNMT1 occurs in the nucleus. DNMT1 is a nuclear protein tightly associated with chromatin (8, 40). To investigate whether DNMT1 is degraded within the nucleus in response to 5-aza-CdR treatment or it is translocated to the cytoplasm prior to degradation, nuclear and cytoplasmic fractions from HeLa cells were subjected to immunoblot analysis. The results showed that DNMT1 was predominantly present in the nuclear fraction of untreated cells and disappeared within the first 6 h of 5-aza-CdR treatment and was not detectable after 12 or 24 h (Fig. 4A). Neither full-length DNMT1 nor any low-molecular-weight degradation product was detectable in the cytoplasmic fractions of 5-aza-CdR-treated cells at any time point, indicating that the degradation occurs within the nucleus. Reprobing the blot with antibodies specific for the 70-kDa subunit of Ku antigen (a nuclear marker) and GAPDH (a cytoplasmic protein) showed that Ku70 and GAPDH were exclusively localized in the expected fractions and that their levels were not significantly altered after 5-aza-CdR treatment (Fig. 4A). These results confirmed purity of the fractions and equal loading of the proteins in the lanes.



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  		FIG. 4. 5-Aza-CdR-induced degradation of DNMT1 occurs in the nucleus and can be blocked by a proteasomal inhibitor (lactacystin). (A) Western blot analysis of DNMT1 in nuclear and cytoplasmic fractions. Cells were treated with 20 µM lactacystin for 1 h prior to exposure to 5-aza-CdR (5 µM). Nuclear (100 µg of protein) and cytoplasmic (100 µg of protein) extracts from these cells were subjected to Western blot analysis with anti-DNMT1, anti-Ku70, and anti-GAPDH antibodies. (B) Immunofluorescence staining of DNMT1 and DNMT3A in HeLa cells. Cells grown on coverslips were either left untreated or treated with 5-aza-CdR (5 µM) for various times. The fixed cells were stained with mouse anti-DNMT1 monoclonal antibodies (Imgenex) and rabbit anti-DNMT3A polyclonal antibodies followed by TRITC-conjugated anti-mouse immunoglobulin G (IgG) (for DNMT1) and FITC-conjugated anti-rabbit IgG (for DNMT3A). Nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI) and visualized under a fluorescence microscope (Nikon Eclipse 800). (C) Endogenous nuclear Dnmt1 (Cos-7) but not cytoplasmic ectopic mouse Dnmt1 is sensitive to 5-aza-CdR-induced degradation. Cos-7 cells expressing nuclear localization signal-deleted Dnmt1-Flag ({Delta}NLS) were stained with rabbit anti-DNMT1 antibody (for endogenous enzyme) and mouse anti-Flag monoclonal antibody M2 (for ectopic Dnmt1). FITC-conjugated anti-rabbit IgG and TRITC-conjugated anti-mouse IgG were used to detect the respective proteins.

 
To rule out possible artifacts during fractionation, we monitored the disappearance of DNMT1 from the nuclei of HeLa cells after 5-aza-CdR treatment by an immunofluorescence assay. In untreated cells, DNMT1 localized exclusively in the nuclei (Fig. 4B, panels a and d). The characteristic granular staining indicative of its localization to replication foci, was observed for DNMT1 (Fig. 4B, panel a). Within first 6 h of 5-aza-CdR treatment, the DNMT1 level decreased significantly, and DNMT1 was not detectable after 12 h (Fig. 4B, compare panels f and k with panel a). DNMT3A predominantly localized in the nuclei, but unlike DNMT1, its staining was diffuse (Fig. 4B, panel b). DNMT1 (tetramethyl rhodamine isothiocyanate [TRITC]) and DNMT3A (fluorescein isothiocyanate [FITC]) signal merged in the nucleus, demonstrating their colocalization (Fig. 4B, panel c). In contrast to DNMT1, the reduction in DNMT3A level was negligible even after 12 h of 5-aza-CdR treatment (Fig. 4B, compare panels g and l with panel b).

To visualize potential accumulation of the drug-induced degradation products of Dnmt1 in the cytoplasm we used cells overexpressing a Dnmt1 mutant that is predominantly cytoplasmic due to the lack of an NLS ({Delta}NLS) (Fig. 4C, panels a and f). 5-Aza-CdR treatment of these cells resulted in disappearance of the endogenous wild-type Dnmt1 (FITC) (Fig. 4C, compare panels b and g), whereas the cytoplasmic variant (TRITC) remained unaffected (Fig. 4C, panels a and f). The lack of merge of two signals in the cytoplasm in 5-aza-CdR-treated cells (Fig. 4C, compare panels d and e with panels i and j, respectively) confirms that endogenous DNMT1 in Cos-7 cells is rapidly degraded within the nucleus in response to the drug.

5-Aza-CdR-induced degradation requires a functional ubiquitin-activating enzyme (E1). Proteasomal degradation of intracellular proteins is essential for the clearance of misfolded proteins and proteins that are rapidly turned over. It is a complex process involving multiprotein components and a series of enzymes, E1, E2, and E3 (for reviews, see references 31 and 57). In this ATP-dependent process involving a series of reactions, ubiquitin is transferred to a lysine residue of the target polypeptide to form a polyubiquitinated protein, which is degraded by the 26S proteosome. To confirm that the ubiquitin pathway is essential for the 5-aza-CdR-induced degradation of Dnmt1, we took advantage of a mouse cell line (ts20) harboring a temperature-sensitive mutant of the first enzyme of this pathway, E1 (15). The E1-dependent proteasomal pathway was active in these cells at 34°C (permissive temperature) but was inactivate at 39°C (restrictive temperature). Cells grown at 34°C were shifted to the restrictive temperature (39°C) for 12 h before being treated with 5-aza-CdR (2.5 µM). Cells treated with the analog at 34°C were used as a control. Western blot analysis demonstrated a time-dependent decrease in the Dnmt1 level in cells treated with 5-aza-CdR at 34°C, whereas cells treated at 39°C were unaffected. A significant decrease (~50%) in Dnmt1 occurred within the first 6 h of treatment at 34°C, at which E1 is functional. Under these conditions, no significant decrease occurred at 39°C, at which E1 is inactivated (Fig. 5A and B). The level of endogenous Dnmt1 did not significantly increase at 39°C, a result which was probably due to decreased synthesis of the protein at the higher temperature. The Dnmt3a level under these conditions significantly increased at 39°C, whereas the levels of both isoforms of Dnmt3b were comparable at the two temperatures. The increase in the Dnmt3a level at the higher temperature was probably due to its enhanced synthesis.



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  		FIG. 5. 5-Aza-CdR-induced degradation of Dnmt1 occurs in ts20 cells only when E1 is active at a permissive temperature (34°C). (A) 5-Aza-CdR-induced degradation is blocked in E1 mutant cells at a nonpermissive temperature. ts20 cells grown at 34°C were incubated for 24 h at 39°C or 34°C followed by treatment with 5 µM 5-aza-CdR for various times. WCEs (100 µg of protein) were subjected to Western blot analysis with anti-Dnmt1/3a/3b and anti-GAPDH antibodies. (B) Quantitative analysis of the data in panel A. Error bars indicate standard errors. (C) DNA methyltransferase activity is sensitive to 5-aza-CdR treatment only in cells with active E1. DNA methyltransferase activity was measured in nuclear extracts from ts20 cells by using poly(dI-dC) as a substrate. Cells were treated with ZLLL (25 µM) 1 h before the drug treatment. The numbers in the lower panel represent 3H1 incorporation into each extract (100 µg of protein). The results are the mean ± standard error of three independent assays. (D) 5-Aza-CdR sensitivity of Dnmt1 is restored at a nonpermissive temperature in ts20 cells expressing wild-type Dnmt1. Western blot analysis of Dnmt1 in WCEs (100 µg of protein) from ts20 and H38-5 cells (ts20 cells transfected with wild-type E1) exposed to 34°C or 39°C before drug treatment. (E) Quantitative analysis of the data in panel D. All quantitative results are the mean ± standard error of three independent experiments. For quantitation, the levels of Dnmt1 in untreated cells were assigned a value of 1.

 
We also measured DNA methyltransferase activity in the nuclear extracts prepared from these cells. The results showed that the activity at permissive temperature (34°C) decreased after treatment with 5-aza-CdR and the activity recovered by pretreatment of cells with the proteasomal inhibitor ZLLL (Fig. 5C). The enzyme activity increased nearly three folds when the cells were shifted to 39°C, which is probably due to increased stability/activity at higher temperature. The resistance of the enzyme to 5-aza-CdR-induced degradation correlated with the stabilization of Dnmt1 at this temperature (Fig. 5A and B). The increase in DNA methyltransferase activity by ZLLL even in control cells implicates the involvement of proteasomal pathway in the degradation of Dnmt1 under normal physiological conditions.

To confirm that the resistance of ts20 cells to 5-aza-CdR-induced degradation at 39°C is indeed due to inactivation of E1, we treated H38-5 cells (ts20 cells that stably express wild-type E1) with the inhibitor (15). In H38-5 cells the degradation of Dnmt1 occurred at both temperatures (Fig. 5D and E), which indicated requirement of the functional E1 for protein degradation. Inhibition of the degradation in H38-5 cells by pretreatment with ZLLL at both temperatures further confirms the role of proteasomal pathway in the degradation process (data not shown). A more pronounced degradation at 39°C is probably due to higher activity of E1 at this temperature. Based on the results from these series of experiments we can conclude that only Dnmt1 is sensitive to 5-aza-CdR-induced degradation and this process requires a functional ubiquitin-activating enzyme.

A conserved N-terminal KEN box, a BAH domain, and an NLS, but not a C-terminal catalytic domain, are essential for 5-aza-CdR-induced nuclear degradation of Dnmt1. Next we investigated the nature of the degradation signal unique to Dnmt1 that makes it susceptible to proteasomal degradation upon 5-aza-CdR treatment. Dnmt1 is a very large protein that harbors the catalytic domain in its C terminus and various regulatory domains in its N terminus (Fig. 6A). Analysis of mammalian Dnmt1 identified a KEN box located near the N terminus of the protein (Fig. 6B). The KEN box is a signature sequence (KENxxxN) in which the last amino acid is not absolutely conserved for the proteasomal degradation of many cell cycle regulatory proteins (3, 28, 48, 58). The conserved KENxxxR box in the mammalian Dnmt1 but not in Dnmt3a and Dnmt3b provided us the impetus to explore its potential role in the proteasomal degradation of this protein. To address whether The KEN box is required for proteasomal degradation of Dnmt1, KEN was mutated to AAA (Fig. 6A). To delineate the role of other domains of Dnmt1, if any, on the degradation process we generated additional mutants. The cysteine in the catalytic site PCQ motif that is involved in covalent bond formation with C-6 of 5-aza-CdR incorporated DNA (53) was mutated to serine. Dnmt1 mutants with deletions in the NLS ({Delta}NLS), the bromo-adjacent homology (BAH) domain ({Delta}BAH), and the catalytic domain ({Delta}CAT) were also generated to elucidate their role in 5-aza-CdR-induced degradation (Fig. 6A).




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  		FIG. 6. Mutation in the KEN box and deletions in the BAH domain or the NLS stabilize Dnmt1 against 5-aza-CdR-induced degradation, whereas mutation of cysteine in the PCQ motif cannot protect it from degradation. (A) Schematic representation of various mutants of mouse DNMT1 used in the present study. (B) Conserved KEN box in mammalian DNMT1. (C and D) The wild type and CS and catalytic domain deletion mutants are sensitive to 5-aza-CdR-induced degradation. Cells were cotransfected with 0.5 nmol of each of the Dnmt1 expression vectors along with GFP. Cells were distributed equally into three plates at 24 h posttransfection and treated with 5-aza-CdR (5 µM) for 6 and 12 h. WCEs (100 µg of protein) were subjected to Western blot analysis with anti-Flag (for ectopic), anti-Dnmt1 (for endogenous), and anti-GFP antibodies. All quantitative results are the mean ± standard error of three independent experiments. (E) Dnmt3a and Dnmt3b (wild type and CS mutant) are resistant to degradation by 5-aza-CdR. Cos-7 cells were transfected with the expression vectors or the pcDNA3.1 vector alone followed by treatment with the inhibitor as described for panel B. WCEs (25 µg of protein) were subjected to Western blot analysis with antibodies against Dnmt3a and Dnmt3b. These proteins were not detected in extracts of Cos-7 cells transfected with the vector alone (data not shown). WCEs (100 µg of protein) were subjected to Western blot analysis with anti-Dnmt1 antibody. (F) The wild type and CS and AAA mutants of Dnmt1 localize predominantly in the nucleus, whereas {Delta}NLS-Dnmt1 and {Delta}BAH-Dnmt1 localize exclusively in the cytoplasm. Cells transfected with the expression vectors were stained with anti-Flag antibody (to detect Dnmt1) and 4',6'-diamidino-2-phenylindole (DAPI).

 
Dnmt1 expression vectors along with GFP were transfected into Cos-7 cells. Cells were split into three plates after 24 h and treated with 5-aza-CdR for various times. The results showed that the expression of both the wild type and the CS mutant of Dnmt1 were comparable and both were markedly degraded with time (Fig. 6C-1, lanes 1 to 6, and Fig. 6D). In contrast, mutation of the KEN box to AAA significantly increased basal level of Dnmt1 and imparted resistance to 5-aza-CdR-induced degradation (Fig. 6C-1, lanes 7 to 9, and Fig. 6D). Similarly, deletion of the catalytic domain increased expression of Dnmt1 but did not abolish 5-aza-CdR-induced degradation, as evident by the susceptibility of KEN/{Delta}CAT, but not AAA/{Delta}CAT, to degradation (Fig. 6C-2, lanes 1 to 6, and Fig. 6D). These experiments were repeated three times and reproducible results were obtained. Unlike endogenous Dnmt1 (Fig. 6C1-3, lower panel), ectopic Dnmt1 (Fig. 6C1-3, upper panel) was not completely degraded by 5-aza-CdR treatment for 12 h probably because of overexpression. We were compelled to study the effect of the inhibitor on transiently expressed Dnmt1 as a result of unsuccessful attempts to generate stable cell lines overexpressing the wild type and mutant full-length Dnmt1. The results clearly demonstrate that the KEN box but not the catalytic domain plays a critical role in stabilizing the basal Dnmt1 level by preventing its degradation in response to 5-aza-CdR treatment. Further, Dnmt1 with {Delta}NLS and {Delta}BAH located downstream of the KEN box rendered the protein completely resistant to 5-aza-CdR-induced degradation (Fig. 6C-3, lanes 1 to 6, and Fig. 6D). Neither the wild type nor CS mutants of Dnmt3a and Dnmt3b were susceptible to 5-aza-CdR-induced degradation following overexpression in Cos-7 cells (Fig. 6E). In contrast, endogenous DNMT1 decreased with time after 5-azaC-dR treatment of these cells (Fig. 6E). These results demonstrate that among three functional Dnmts only Dnmt1 is degraded in response to the drug.

To address further the mechanism for differential sensitivity of various mutants of Dnmt1 to 5-azaC-dR in vivo, we determined their subcellular localization. Immunostaining of the overexpressed Dnmt1 variants with anti-Flag antibody showed that the wild type and the CS mutant localized predominantly in discrete foci characteristic of replication origins (7) whereas AAA mutant predominantly localized in the nucleoplasm (Fig. 6F, panels a, d, and g). This differential subnuclear localization of AAA mutant may explain its enhanced stability and resistance to 5-aza-CdR-induced degradation. Surprisingly, {Delta}BAH also failed to localize in the nucleus, as observed for {Delta}NLS (Fig. 6F, panels j and m). These results revealed that both {Delta}NLS- and {Delta}BAH-Dnmt1 are refractory to 5-aza-CdR-induced degradation due to their inability to localize in the nucleus and that the KEN box is essential for the nuclear degradation of Dnmt1.

APC/CCdh1 is the ubiquitin ligase involved in both physiological turnover and 5-aza-CdR-induced degradation of DNMT1. Most of the proteins destined for degradation by proteasomal pathway are generally polyubiquitinated although there are a few exceptions (for reviews, see references 31, 53, and 57). Therefore, it was logical to investigate whether Dnmt1 is indeed ubiquitinated in vivo. For this purpose, Cos-7 cells were transiently transfected with HA-ubiquitin, Dnmt1-Flag or both. Cell extracts prepared in buffer containing 1% SDS, were diluted to 0.1% SDS and subjected to immunoprecipitation with anti-Flag (for Dnmt1) or anti-HA (for ubiquitin) antibodies. Detection of Dnmt1 precipitated by anti-Flag antibody when coexpressed with HA-ubiquitin in immunoblot analysis using both antibodies (Fig. 7A, lanes 5, 6, 9, and 10) demonstrates that Dnmt1 is indeed ubiquitinated in vivo. The ubiquitinated ladder of Dnmt1 was pronounced in cells incubated with lactacystin (Fig. 7A, compare lanes 6 and 10 with lanes 5 and 9, respectively). Inability of anti-HA antibody to detect Dnmt1-Flag when expressed alone (Fig. 7A, lanes 7 and 8) confirmed specificity of the antibody. Similarly, ubiquitinated Dnmt1 was pulled by anti-HA antibody only in cells expressing both Dnmt1 and ubiquitin (Fig. 7A, lanes 15, 16, 19, and 20). Significant increase in the level of polyubiquitinated Dnmt1 in cells treated with lactacystin (Fig. 7A, compare lanes 18 and 20 with lanes 17 and 19, respectively) further demonstrated the involvement of proteosome in the turn over of the protein under normal physiological conditions.



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  		FIG. 7. Dnmt1 is ubiquitinated in vivo and interacts with Cdh1, the substrate recognition subunit of APC/CCdh1 (E3 ligase) which is involved in its degradation and which is dephosphorylated upon 5-aza-CdR treatment. (A) Dnmt1 is ubiquitinated in vivo. Cos-7 cells were transfected with wild-type pcDnmt1-Flag, HA-ubiquitin, or both. At 36 h posttransfection, cells were treated with lactacystin (20 µM) for 4 h. WCEs (250 µg of protein) from these cells were immunoprecipitated with anti-Flag or anti-HA antibodies and subjected to immunoblot analysis with both antibodies. (B and C) Overexpression of Cdh1 decreases basal Dnmt1 levels and enhances 5-aza-CdR-induced degradation. Cos-7 cells were transfected with wild-type pcDnmt1-Flag and increasing amounts of pcCdh1, pcCdc20, or pCMV-HA (empty vector). After 24 h, cells were split into two; 12 h later, cells were either left untreated or treated with 5-aza-CdR for an additional 6 h. WCEs (100 µg of protein) were subjected to Western blot analysis with anti-Flag, anti-HA, or anti-Ku70 antibodies. (D and E) DNMT1 levels are elevated in cells (untreated or 5-aza-CdR treated) depleted of Cdh1 by RNA interference. HeLa cells were transfected with Cdh1 siRNA or nonspecific siRNA (NS) followed by 5-aza-CdR exposure for 6 h (see Materials and Methods for details). Western blot analysis (100 µg of protein) was performed with anti-DNMT1, anti-Cdh1, or anti-Cdc20 antibodies. (F) Dnmt1 associates with Cdh1. Cells were cotransfected with Dnmt1-Flag, pcCdh1-HA, or pcCdc20-HA. After 32 h, cells were treated with lactacystin (20 µM) for 4 h. Cell extracts prepared in TNN buffer were subjected to immunoprecipitation with anti-Flag or anti-HA antibodies followed by Western blot analysis with both antibodies. (G and H) Phosphorylation of Cdh1 decreases whereas that of Dnmt1 is not significantly altered in cell treated with 5-aza-CdR. Cos-7 cells were transfected with pcCdc20, pcCdh1, or pcDnmt1-Flag. After 36 h, cells were either left untreated or treated with 5-aza-CdR (5 µM) followed by labeling with 32P-labeled orthophosphate (1 mCi/ml) for 2 h. WCEs (250 µg of protein) were immunoprecipitated with either anti-HA (for Cdc20 and Cdh1) or anti-Flag (for Dnmt1) antibodies. Precipitated proteins were transferred to a nitrocellulose membrane, which was subjected to phosphorimaging analysis followed by Western blot analysis to measure specific protein levels. The 32P signal in each band was quantified by using ImageQuant software (Molecular Dynamics). The quantitative results are the mean of three independent experiments.

 
We then explored the identity of the E3 ligase involved in proteasomal degradation of Dnmt1. KEN box proteins are usually recognized by anaphase promoting complex (APC/CCdh1) ligase that is involved in late mitotic or early G1 degradation of cell cycle regulatory proteins (for reviews, see references 3, 28, and 49). In contrast, APC/CCdc20, in general, recognizes proteins with "destruction box" that are degraded in early mitosis. To identify the ligase, we overexpressed Cdh1 or Cdc20 in Cos-7 cells and measured the level of cotransfected Dnmt1 with or without 5-aza-CdR treatment. The results showed that overexpression of Cdh1 but not Cdc20 resulted in dose-dependent decrease in basal Dnmt1 level, which decreased further upon drug treatment compared to those transfected with Cdc20 or vector alone (Fig. 7B, compare lanes 9 to 12 with lanes 1 to 8, and Fig. 7C). Western blot analysis with anti-HA antibody showed that both Cdc20 and Cdh1 were expressed at a comparable level (Fig. 7B). These results suggest that APC/CCdh1 is the ligase involved in regulating the physiological turn over of Dnmt1 and its 5-aza-CdR-induced degradation. To demonstrate further the involvement of APC/CCdh1 in regulating the DNMT1 level Cdh1 was depleted in HeLa cells using siRNA (Fig. 7D, compare lanes 3 and 4 with lanes 1 and 2, respectively). A significant increase in DNMT1 levels in both untreated and 5-aza-CdR-treated cells transfected with Cdh1 siRNA (compared to those transfected with nonspecific siRNA) confirmed the role of APC/CCdh1 in DNMT1 degradation (Fig. 7D, compare lanes 3 and 4 with lanes 1 and 2, respectively, and Fig. 7E).

Next we investigated whether Cdh1 can interact with Dnmt1. To address this issue we expressed Dnmt1-Flag and HA-Cdh1 or Cdc20 and pulled down the proteins with respective antibodies. Significantly reduced Dnmt1 level in cells overexpressing Cdh1 compared to cells overexpressing Cdc20 (Fig. 7F, lanes1 and 2) further reinforces the role of Cdh1 in the regulation of Dnmt1. Anti-Flag antibody pulled down significantly less Dnmt1 from Cdh1-overexpressing cells than from Cdc20-transfected cells, a result which correlated with the levels of ectopic Dnmt1 in these cells (Fig. 7F, lanes 1 to 4). Specific pull down of Dnmt1 with anti-HA antibody only from cells overexpressing Cdh1 (Fig. 7F, lanes 5 and 6) clearly demonstrated an interaction between these two proteins. Similarly, anti-Flag antibody pulled down not only Dnmt1 but also Cdh1, suggesting an interaction between the two proteins (Fig. 7F, lane 4).

It is likely that 5-aza-C or 5-aza-CdR activates the proteasomal degradation of Dnmt1 by activating posttranslational modification of either Dnmt1 or Cdh1. Because phosphorylation/dephosphorylation is the most common modification of proteins we studied the phosphorylation status of these two proteins in absence and presence of 5-aza-CdR. Cdh1 was heavily phosphorylated in vivo and its phosphorylation level was reduced significantly upon the drug treatment. In contrast, Cdc20 was poorly phosphorylated and was not significantly affected by 5-aza-CdR treatment (Fig. 7G). Comparable levels of Cdh1and Cdc20 were pulled down by HA antibody (Fig. 7G). Dnmt1 was also heavily phosphorylated in vivo and its reduced phosphorylation correlated with its decreased level upon 5-aza-CdR treatment (Fig. 7H), suggesting that 5-aza-CdR treatment does not dramatically alter the level of phosphorylation of Dnmt1. These results also suggest that dephosphorylated Cdh1 probably interacts with Dnmt1 and 5-aza-CdR treatment facilitates their interaction by dephosphorylating Cdh1, which is then degraded by the proteasomal machinery.


   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The commonly accepted mechanism of action of 5-aza-CdR or 5-aza-C is based on the initial report (53) that incorporation of these cytidine analogs into DNA inhibits the capacity of DNMTs to methylate DNA. This has been attributed to replacement of C-5 of cytosine with N-5 and covalent complex formation between DNMT and 5-aza-CdR-incorporated DNA that results in depletion of functional enzyme. The unavailability of functional DNMTs prevented methylation of the newly replicated strand leading to DNA demethylation and subsequent gene activation. Several observations led us to conclude that this may not be the only mechanism of DNA demethylation by these anticancer drugs. First, DNMT activity decreases much faster than its incorporation into DNA (19). Second, among the three DNMTs, only DNMT1 is rapidly and selectively degraded in mammalian cells in response to DNA methyltransferase inhibitors, such as 5-aza-C (22) or zebularine (14). Third, unlike bacterial methyltransferase, mutation of cysteine at the catalytic site, which participates in covalent bond formation, exerts only a minimal effect on mammalian DNA methyltransferase activity (51). Fourth, other cytosine analogs modified at C-5 cannot activate the degradation of Dnmt1; these data indicate a specific requirement for the 5-aza group in cytosine or cytidine to achieve enzyme degradation. Fifth, the gene expression profile of aza-CdR-treated cells is very similar to that of DNMT1–/– cells (23). The depletion of DNMT1 even in the presence of a potent inhibitor of DNA synthesis indicates that 5-aza-CdR-induced degradation occurs independently of DNA replication. It appears that the proteasomal degradation of DNMT1 starts earlier than the incorporation of 5-aza-CdR into DNA and the subsequent covalent complex formation with Dnmt1. These data are substantiated by the observation that neither the wild type nor CS mutants of Dnmt3a and Dnmt3b are susceptible to inhibitor-induced degradation (Fig. 6E). However, the association of Dnmt1 with DNA may be necessary for degradation, as the KEN box mutant that localizes predominantly in the nucleoplasm is resistant (Fig. 6F). Recently, it was shown that Dnmt1 was loaded onto chromatin not only during S phase but also throughout G2 and M phases (21); these data further support the replication-independent effect of 5-aza-CdR (25).

It may be argued that DNMT1 could be degraded due to activation of apoptotic pathway induced by 5-aza-CdR. The morphology of HeLa cells treated with the drug for as long as 24 h did not show signs of apoptosis (unpublished data) and 5-aza-CdR-induced depletion of DNMT1 could not be prevented by the caspase inhibitors. Further, thymidine and BrdUrd-incorporation assays showed that DNA replication was not blocked in 5-aza-C or 5-aza-CdR-treated cells. Fluorescence-activated cell sorting analysis showed similar cell cycle profiles for both control and inhibitor-treated HeLa cells (data not shown). These data support the conclusion that the degradation of DNMT1 is not due to the induction of apoptosis by the drug.

A recent study showed that the treatment of cells with 5-aza-CdR results in DNA damage, which activates p53, resulting in p21/WAF1 induction and cell cycle arrest (61). DNA damage appears to be a late event, as it could be detected only after 72 h, whereas the degradation of DNMT1 occurs as early as 6 h in cells independent of their p53 status (unpublished data). The persistent degradation even in the absence of DNA synthesis and the lack of degradation by other pyrimidine analogs, such as Ara-C, flucytosine, or 5-fluorodeoxyuridine, further reinforce the notion that the degradation process is not secondary to DNA damage.

Finally, the requirement of the recognition signals within the primary structure of DNMT1 for its degradation merits discussion. In cell lines expressing all three DNMTs we observed much faster and pronounced degradation of DNMT1 compared to DNMT3A and 3B in response to 5-aza-CdR. Enhanced degradation of cellular proteins also occur in response to toxic agents. Recently, it was shown that the arsenite-induced degradation of Cdc25c is also mediated through a KEN box (13). It is evident from our study that the KEN box is essential for the degradation of DNMT1, whereas the catalytic domain is not essential for its degradation. Cell cycle-dependent expression of Dnmt1 occurs both at transcriptional and posttranslational level (41). It is likely that KEN box-dependent proteasomal degradation plays an important role in the cell cycle-dependent regulation of Dnmt1. Clearly, the lack of the KEN box in Dnmt3a and Dnmt3b prevented their degradation after exposure to 5-aza-CdR. Surprisingly, both the BAH domain and the NLS are required for the nuclear localization of Dnmt1, which explains the requirement of both of these domains for 5-aza-CdR-induced degradation. Deletion of either of these two results in cytoplasmic localization of the protein. It was shown earlier that BAH domain-deleted Dnmt1 fused to EGFP cannot bind to DNA when allowed to localize in the nucleus with the simian virus 40 NLS (40). The role of the BAH domain in the nuclear transport of Dnmt1 could not be identified because the endogenous NLS of Dnmt1 was replaced by the very strong NLS of simian virus 40. It would be of interest to determine whether BAH domains of other proteins, such as Rsc1 and Orc1p, play similar roles (26, 38, 60).

An interesting observation is the polyubiquitination of Dnmt1 in vivo and its activation by 5-aza-CdR. Further, this study has shown involvement of the ubiquitin ligase, APC/CCdh1 in the degradation process that is facilitated by the significant dephosphorylation of Cdh1 in response to 5-aza-CdR. It will be of interest to identify other proteins that are degraded by 5-aza-C treatment. Studies with yeasts have shown that Cdh1 is inactivated by cell cycle-dependent kinases, such as Cdc28, and activated by dephosphorylation with Cdc14, a dual-specificity phosphatase (49). It would be of interest to investigate whether 5-aza-CdR treatment inactivates a specific kinase or activates a phosphatase in mammalian cells. It is possible that Cdh1 can interact with Dnmt1 upon dephosphorylation by a specific phosphatase.

In conclusion, this study offers a rational explanation for the rapid demethylation and reactivation of silenced genes, such as tumor suppressor genes, by 5-aza-C or 5-aza-CdR. It also provides an impetus to explore other cytidine analogs that are capable of inducing proteasomal degradation of Dnmt1 at a much lower concentration and that therefore may exhibit significantly greater efficacy in the epigenetic therapy of cancer.


   ACKNOWLEDGMENTS
 
We thank Harvey Ozer for the ts20 and H38-5 cell lines, Kristian Helin for the Cdh1 and Cdc20 expression vectors, Timothy Bestor for Dnmt1 cDNA, Chin-Lin Heish for the Dnmt3a and Dnmt3b expression vectors, Shoji Tajima for anti-Dnmt1 antibody, Michelle Pagano and Zeev Ronai for the ubiquitin cDNAs.

This study was supported in part by grants ES 10874, CA 81024, and CA 86978 from the National Institutes of Health.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, OH 43210. Phone: (614) 688-5494. Fax: (614) 688-5600. E-mail for Samson T. Jacob: jacob.42{at}osu.edu. E-mail for Kal-pana Ghoshal: ghoshal.1{at}osu.edu. Back

{dagger} K.G. and J.D. contributed equally to the experimental procedures. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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