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
Authors: Shoumei Bai
Tasneem Motiwala
Kalpana Ghoshal
Jharna Datta
Samson T Jacob
Sarmila Majumder
Huban Kutay
Journal: Molecular and Cellular Biology (2005)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: 5-Azacytidine; 5-aza-deoxycytidine; tumor suppressor genes; promoter methylation; epigenetic therapy
Affiliations: Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, OH 43210, USA. , Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, Ohio

Abstract:  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. 

Retracted: No

PubPeer comments:
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 ![file] , 
 2018 correction for figure 2A. No mention of figures 1B, of 3D. http: mcb.asm.org content 38 10 e00539-17.short , 
 Figure 7(A) - lanes 1 and 2. Can we blame compression artefacts? ![file] , 
 #6 Figure 1B. Ku70 panel. How to explain the same  dithering pattern appearing twice? , 
 #6 Also, partial replication of two left-hand lanes and two right-hand lanes in the DNMT3B band. ![file] , 
 #5 Bonus adventures in horizontal flipping: ![file] , 
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 Figure 1A. More similar than you might expect. https: imgur.com A9G6s8m , 
 #5 Embedding my learned colleague s annotation of Fig 1A, for easier access: ![] , 
 Fourth author appears to currently be affiliated with University of Pittsburgh, and has the unfortunate distinction of appearing as coauthor of multiple papers with similar image concerns while at different institutions: https: pubpeer.com search?q=Shoumei+Bai ![file] , 
 12 April 2022 retraction https: journals.asm.org doi 10.1128 mcb.00546-21 The Ohio State University (OSU) informed Molecular and Cellular Biology and ASM that following an investigation of research misconduct, they found u201cfalsified datau201d in Fig. 1A and B, 3D, and 6C-1 and C-2. Based on these findings and an unsatifactory response from the authors, per ASM Publishing Ethics Policies and Procedures, we are abiding by the OSU retraction request. The integrity of the data presented was compromised; therefore, this publication is retracted in its entirety. We apologize to the readers of Molecular and Cellular Biology and regret any inconvenience that this causes. , 
 https: retractionwatch.com 2018 08 17 cancer-researcher-at-osu-up-to-nine-retractions , 
 #14 Seven years from first alert to retraction. ,