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.
Highlights
DNA methylation is a key mechanism of epigenetic regulation in eukaryotes
To learn more about how post-translational modifications (PTMs) affect DNA methyltransferases (DNMTs) function, we focused on the de novo DNA methyltransferase DNMT3A, about which very little is known
293T cells have often been employed for peptidylarginine deiminase 4 (PADI4) overexpression studies, while U2OS cells have been used for PADI4 overexpression and in endogenous/RNAi experiments.) As shown in Figure 1B, when 293T cells were transfected with GAL4-tagged DNMT3A and HA-tagged PADI4, we found DNMT3A to interact with PADI4 (Figure 1B, lane 3)
Summary
DNA methylation is a key mechanism of epigenetic regulation in eukaryotes. DNA methyltransferases (DNMTs) establish and maintain DNA, methylation of the fifth carbon of cytosine residues within CG dinucleotides [1]. DNMT1 is primarily a maintenance methyltransferase ensuring the inheritance of proper DNA methylation patterns in differentiated somatic cells [2]. De novo DNA methylation is mediated essentially by DNMT3A and DNMT3B, which knockout studies in mice have shown to be essential to embryonic development [3]. DNA methylation plays a crucial role in maintaining cell pluripotency, X-chromosome inactivation and genomic imprinting [4], while aberrant DNA methylation is the best-characterized epigenetic hallmark of several pathologies, including cancers [5]. Dysregulated expression of DNMTs has been reported in various human cancers [6]
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