A methylation and phosphorylation switch between an adjacent lysine and serine determines human DNMT1 stability

New England Biolabs, Ipswich, Massachusetts, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 01/2011; 18(1):42-8. DOI: 10.1038/nsmb.1939
Source: PubMed


The protein lysine methyltransferase SET7 regulates DNA methyltransferase-1 (DNMT1) activity in mammalian cells by promoting degradation of DNMT1 and thus allows epigenetic changes via DNA demethylation. Here we reveal an interplay between monomethylation of DNMT1 Lys142 by SET7 and phosphorylation of DNMT1 Ser143 by AKT1 kinase. These two modifications are mutually exclusive, and structural analysis suggests that Ser143 phosphorylation interferes with Lys142 monomethylation. AKT1 kinase colocalizes and directly interacts with DNMT1 and phosphorylates Ser143. Phosphorylated DNMT1 peaks during DNA synthesis, before DNMT1 methylation. Depletion of AKT1 or overexpression of dominant-negative AKT1 increases methylated DNMT1, resulting in a decrease in DNMT1 abundance. In mammalian cells, phosphorylated DNMT1 is more stable than methylated DNMT1. These results reveal cross-talk on DNMT1, through modifications mediated by AKT1 and SET7, that affects cellular DNMT1 levels.

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Available from: Pierre-Olivier Estève, Jan 13, 2014
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    • "Uhrf1 knockdown, or mutation of its H3K9me3/me2-binding tandem Tudor domain, also reduced the stability of Dnmt1 during mitosis , suggesting another mechanism whereby DNA maintenance methylation at heterochromatic regions might rely on recognition of histone methylation [83] [105]. It has also been shown that the stability of Dnmt1 is regulated through the cell cycle by a phospho/methyl switch; phosphorylation of Ser143 by AKT1 stabilises Dnmt1 by antagonising methylation of Lys142 by Set7, which targets Dnmt1 for proteasomal degradation [106] [107]. This leads to accumulation of Dnmt1 during Sphase . "
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    ABSTRACT: DNA methylation acts as an epigenetic modification in vertebrate DNA. Recently it has become clear that the DNA and histone lysine methylation systems are highly interrelated and rely mechanistically on each other for normal chromatin function in vivo. Here we examine some of the functional links between these systems, with a particular focus on several recent discoveries suggesting how lysine methylation may help to target DNA methylation during development, and vice versa. In addition, the emerging role of non-methylated DNA found in CpG islands in defining histone lysine methylation profiles at gene regulatory elements will be discussed in the context of gene regulation. This article is part of a Special Issue entitled: Methylation: A Multifaceted Modification looking at transcription and beyond. (C) 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
    Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 12/2014; 1839(12). DOI:10.1016/j.bbagrm.2014.02.007 · 6.33 Impact Factor
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    • "To date, very little is known regarding how PTMs might affect DNMT stability. All we know so far concerns DNMT1 (7), whose methylation at Lys 147 by SET7 and phosphorylation at Ser 143 by Akt kinase have been shown to regulate its stability and degradation, thus affecting DNA methylation (12). "
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    ABSTRACT: DNA methylation is a central epigenetic modification in mammals, with essential roles in development and disease. De novo DNA methyltransferases establish DNA methylation patterns in specific regions within the genome by mechanisms that remain poorly understood. Here we show that protein citrullination by peptidylarginine deiminase 4 (PADI4) affects the function of the DNA methyltransferase DNMT3A. We found that DNMT3A and PADI4 interact, from overexpressed as well as untransfected cells, and associate with each other's enzymatic activity. Both in vitro and in vivo, PADI4 was shown to citrullinate DNMT3A. We identified a sequence upstream of the PWWP domain of DNMT3A as its primary region citrullinated by PADI4. Increasing the PADI4 level caused the DNMT3A protein level to increase as well, provided that the PADI4 was catalytically active, and RNAi targeting PADI4 caused reduced DNMT3A levels. Accordingly, pulse-chase experiments revealed stabilization of the DNMT3A protein by catalytically active PADI4. Citrullination and increased expression of native DNMT3A by PADI4 were confirmed in PADI4-knockout MEFs. Finally, we showed that PADI4 overexpression increases DNA methyltransferase activity in a catalytic-dependent manner and use bisulfite pyrosequencing to demonstrate that PADI4 knockdown causes significant reduction of CpG methylation at the p21 promoter, a known target of DNMT3A and PADI4. Protein citrullination by PADI4 thus emerges as a novel mechanism for controlling a de novo DNA methyltransferase. Our results shed new light on how post-translational modifications might contribute to shaping the genomic CpG methylation landscape.
    Nucleic Acids Research 06/2014; 42(13). DOI:10.1093/nar/gku522 · 9.11 Impact Factor
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    • "Additionally, dysfunctional methylation due to deficiencies in Dnmt3a and Dnmt3b has been reported to be associated with altered expression of Nanog and Oct-4 during differentiation of murine ES cells. In these studies, DNMTs acted as transcriptional repressors of Nanog and Oct-4 genes in ES cells [30], [37], [38]. Accordingly, we herein showed that DNMT3A and DNMT3B also repressed NANOG mRNA levels [30]; however, through a mechanism independent from the miR-720-mediated one (Fig. 7E). "
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    ABSTRACT: Dental pulp cells (DPCs) are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs) have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP) cells from human DPCs and periodontal ligament cells (PDLCs), and performed a locked nucleic acid (LNA)-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP) cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs), which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.
    PLoS ONE 12/2013; 8(12):e83545. DOI:10.1371/journal.pone.0083545 · 3.23 Impact Factor
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