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P68/Ddx5 RNA Helicase Interacts and Co-Localizes in Vivo with the De Novo DNA Methyltransferases Dnmt3a1 and Dnmt3a2 Anastasia Mpakali1,2#, Andriana G

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P68/Ddx5 RNA Helicase Interacts and Co-Localizes in Vivo with the De Novo DNA Methyltransferases Dnmt3a1 and Dnmt3a2 Anastasia Mpakali1,2#, Andriana G ics om & B te i ro o P in f f o o r l m a Journal of a n t r i Mpakali et al., J Proteomics Bioinform 2012, 5:1 c u s o J DOI: 10.4172/jpb.1000207 ISSN: 0974-276X Proteomics & Bioinformatics Research Article Article OpenOpen Access Access P68/Ddx5 RNA Helicase Interacts and Co-Localizes In vivo with the De Novo DNA Methyltransferases Dnmt3a1 and Dnmt3a2 Anastasia Mpakali1,2#, Andriana G. Kotini1,2#, Magda Spella3, Marina Kouyialis2, Angelliki Tserga2, Leonidas Fragkos-Livanios4, Martina Samiotaki4 and Theodora Agalioti2,3* 1National and Kapodistrian University of Athens-Medical School 2BSRC-Alexander Fleming-Institute of Molecular Biology and Genetics 3Laboratory for Molecular Respiratory Carcinogenesis- Department of Physiology, Faculty of Medicine, University of Patras 4BSRC-Alexander Fleming-Institute of Molecular Oncology #These authors contributed equally Abstract The 5-methyl cytosine (5meC) genomic methylation patterns play crucial roles in mammalian development and are altered in cancer. The enzymes that create, maintain and modify the DNA methylation patterns are the DNA methyltransferases (Dnmts) which are all encoded by essential genes. The de novo Dnmts -Dnmt3a and Dnmt3b- establish the DNA methylation patterns early in mammalian development by introducing DNA methylation marks where no previous methylation exists. These enzymes do not exhibit affinity for specific DNA sequences, thus their recruitment to specific DNA loci and their activities must be tightly regulated. In particular, Dnmt3a2 –one of the two protein isoforms produced by the Dnmt3a locus- is the most abundant DNA methyltransferase in mouse Embryonic Stem Cells. To identify Dnmt3a (and DNA methylation) regulators we have searched for Dnmt3a2 interacting proteins in mESCs by pull down and Mass Spectrometry. The DEAD box p68/Ddx5 RNA helicase was identified to directly interact with Dnmt3a1 and Dnmt3a2 in vitro and in vivo. We have created a mutant Ddx5 (Ddx5MUT) protein exhibiting an altered nuclear localization pattern compared to the wt protein. Both wt and mutant Ddx5 interact directly in vitro and co localize in vivo with Dnmt3a proteins. Our data suggest that the Dnmt3a/Ddx5 interaction might be significant for modulating the DNA methylation/demethylation dynamics in vivo. Introduction P68/Ddx5 is a prototypical member of the DEAD box family of RNA helicases exhibiting ATPase and RNA unwinding activities. DNA methylation in mammals is associated with important The DEAD box family is named after the amino acid sequence of its developmental phenomena such as genomic Imprinting and conserved Motif II (also known as the Walker B motif) containing the X-inactivation and is inversely correlated with transcription initiation [1]. The DNA methylation patterns, thought to characterize each cell amino acids asp-glu-ala-asp (D-E-A-D). Ddx5 is a multifunctional type as a molecular fingerprint, are established early in mammalian protein involved in transcription regulation, mRNA splicing, miRNA development and are altered in cancer [2-5]. The mouse DNA biogenesis and in DNA damage repair pathways [13]. In particular, methyltransferases (Dnmts), the enzymes that create, maintain and in DNA repair, Ddx5 is associated with Thymidine DNA glycosylase modify the DNA methylation patterns are all encoded by essential (TDG), a protein which is essential for active DNA demethylation [14]. genes. These enzymes are categorized according to their ability to However, the Ddx5 roles in this context are not well understood. introduce new DNA methylation marks into genomic regions where New, exciting findings point towards the notion that the DNA no previous DNA methylation exists (de novo Dnmts) or to copy methylation patterns, thought to characterize each cell type as a pre-existing methylation into newly synthesized DNA strands in the context of the genomic CpG nucleotides (maintenance enzyme, molecular fingerprint are dynamically built up and erased during Dnmt1) [6-9]. The de novo Dnmts belong to the Dnmt3 family which development and in adult tissues [15-17]. Currently, our understanding contains three members, namely Dnmt3a, Dnmt3b and Dnmt3L. of the DNA methylation/demethylation dynamics involves the actions Dnmt3a and Dnmt3b are active enzymes while Dnmt3L is an auxiliary of several groups of enzymes such as a) the DNA methyltransferases molecule enhancing the enzymatic activity of Dnmt3a/3b proteins (Dnmts) which introduce and maintain the 5-methylcytosine (5meC), [10]. The Dnmt3a locus (together with Dnmt3L) is responsible for b) the ten-eleven translocation (TET) family of enzymes that modifies establishing the differential DNA methylation marks associated with genomic Imprinting, which results into the “parent of origin”- specific monoallelic expression of ~100 autosomal genes [11]. Additionally, *Corresponding author: Theodora Agalioti, Laboratory for Molecular Respiratory DNMT3A is associated with cancer pathogenesis because it is found Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Basic Biomedical Sciences Research Building, 2nd Fl, University Campus, mutated in 20% of acute myeloid leukemia patients [12]. 1 Asklepiou Str, GR-26504 Patras-Greece, Tel: +30 2610 969116; Fax: +30 2610 997215; E-mail: [email protected], [email protected] The Dnmt3a genomic locus produces two proteins differing in that Received December 04, 2011; Accepted January 10, 2012; Published January a 219aa unique N-terminal “tail” lacking from the Dnmt3a2 is present 16, 2012 in the longer isoform Dnmt3a1. Dnmt3a1 and Dnmt3a2 proteins are Citation: Mpakali A, Kotini AG, Spella M, Kouyialis M, Tserga A, et al. (2012) P68/ expressed in mouse Embryonic Stem Cells (mESCs) and in early stages Ddx5 RNA Helicase Interacts and Co-Localizes In vivo with the DNA of mouse development which are thought to be enriched in Stem and Methyltransferases Dnmt3a1 and Dnmt3a2. J Proteomics Bioinform 5: 007-014. precursor cells. Dnmt3a1 is expressed in low levels in adult somatic doi:10.4172/jpb.1000207 cells. In contrast, the Dnmt3a2 mRNA and protein expression ceases to Copyright: © 2012 Mpakali A, et al. This is an open-access article distributed under undetectable levels in later stages of development, in adult somatic cells the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and and upon mESCs differentiation [6]. source are credited. J Proteomics Bioinform ISSN:0974-276X JPB, an open access journal Volume 5(1) : 007-014 (2012) - 007 Citation: Mpakali A, Kotini AG, Spella M, Kouyialis M, Tserga A, et al. (2012) P68/Ddx5 RNA Helicase Interacts and Co-Localizes In vivo with the DNA Methyltransferases Dnmt3a1 and Dnmt3a2. J Proteomics Bioinform 5: 007-014. doi:10.4172/jpb.1000207 5meC by successive hydroxylation and oxidation towards 5-hydroxy- His-Ddx5 or with His-Ddx5MUT proteins. The beads were washed twice methyl cytosine (5hmC), c) the activation induced cytidine deaminases with PBS+0.01% Tween and five times with PBS+0.01% Tween plus 0.3 (AID and Apobec 1-3) which are thought to deaminate (5meC or M NaCl. The beads were subsequently boiled in SDS PAGE buffer and 5hmC) and d) the DNA glycosylases of the Base Excision Repair (BER) analyzed in Western blots. pathway initiating the replacement of modified cytosines (5meC or Fluorescence and confocal microscopy 5hmC) with unmodified ones thus, effecting DNA demethylation. After 24h the cover slips bearing the transfected cells were washed To get insights on how the DNA methylation/demethylation twice with PBS and the cells were fixed in 4% PFA for 10 min at RT. The dynamics are regulated we sought to identify Dnmt3a2 interacting cells were washed twice for 5min with PBS. Excess fluid was removed proteins in Mouse Embryonic Stem Cells (mESCs). We have identified and the cover slips were placed “face down” on a drop of ProLong Gold that the p68/Ddx5 DEAD box RNA helicase is specifically pulled down anti-fade reagent with DAPI (Invitrogen) on objective slides, so as the from mESCs nuclear extract preparations by a GST-tagged Dnmt3a2 fixed cells to be immersed into the DAPI containing solution. Excess bait protein. We show that the interaction between the two proteins fluid was removed and the preparations were left to dry for 30 min is direct. To explore the functional significance of this interaction in at RT in the dark. Direct fluorescence (figure 3 of this manuscript) vivo, we have created a Ddx5 mutation that is expected to be an active was monitored on the Observer D1 fluorescence microscope (Zeiss). ATPase with a compromised Helicase activity. We have expressed Areas of interest were scanned with x63 objective lens and images fluorescently tagged (GFP and RFP) versions of Dnmt3a1/3a2 and wt were captured by the AxioVision Rel. 4.8.2 program (Zeiss). The co- and mutant Ddx5 and examined their sub-cellular localization. We localization studies of the co-transfected protein constructs were find that unlike wt Ddx5, the mutant protein does not form nuclear conducted with the use of a Leica TCS SP5 with the dual (Tandem) speckles. Both wt and mutant Ddx5 proteins co-localize better with Scanner (Leica). Green and Red direct fluorescence were excited by Dnmt3a2 and to a lesser extent with Dnmt3a1 in the cell nucleus. laser beams at 488nm and 555nm respectively. Emissions were then sequentially acquired. Areas of interest were scanned with a x40 oil Materials and Methods immersion objective lens. Cell lines, plasmids and transfections Silver staining HEK 293T were maintained in ES medium [DMEM supplemented The following modified silver staining protocol which is compatible with 10% Fetal Bovine Serum (PAN Biotech), 50U/ml Penicillin/ with the LC-MS/MS analysis, was used for the SDS PAGE gel treatment. Streptomycin, 2mM L-Glutamine and 0.1 mM β-Merkaptoethanol. Briefly, the gels were immersed in a solution of 50% Methanol, 5% The cells were grown on round cover slips placed in tissue culture Acetic Acid for 20 min, followed by a 10 min wash in 50% methanol dishes and coated with 0.1% gelatin.
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