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AtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins

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ABSTRACT

DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome from transposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG binding domain proteins are members of a class of proteins that bind to methylated DNA. The Arabidopsis thaliana genome encodes 13 methyl CpG binding domain (MBD) proteins, but the molecular/biological functions of most of these proteins are still not clear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNA binding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40S ribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physically interacts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component of the RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2 mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci, pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved in RNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 also interacts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.

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References

  • Allain FH, Gilbert DE, Bouvet P and Feigon J 2000 Solution structure of the two N-terminal RNA-binding domains of nucleolin and NMR study of the interaction with its RNA target. J. Mol. Biol. 303 227–241

    Article  CAS  PubMed  Google Scholar 

  • Aufsatz W, Mette MF, van der Winden J, Matzke M and Matzke AJ 2002 HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA. EMBO J 21 6832–6841

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ausin I, Mockler TC, Chory J and Jacobsen SE 2009 IDN1 and IDN2 are required for de novo DNA methylation in Arabidopsis thaliana. Nat. Struct. Mol. Biol. 16 1325–1327

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ausin I, Greenberg MV, Simanshu DK, Hale CJ, Vashisht AA, Simon SA, Lee TF, Feng S, et al. 2012 INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis. Proc. Natl. Acad. Sci. USA 109 8374–8381

  • Barakat A, Szick-Miranda K, Chang IF, Guyot R, Blanc G, Cooke R, Delseny M and Bailey-Serres J 2001 The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome. Plant Physiol. 127 398–415

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bogdanovic O and Veenstra GJ 2009 DNA methylation and methyl-CpG binding proteins: developmental requirements and function. Chromosoma 118 549–565

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Burgers WA, Fuks F and Kouzarides T 2002 DNA methyltransferases get connected to chromatin. Trends Genet. 18 275–277

    Article  CAS  PubMed  Google Scholar 

  • Bycroft M, Grunert S, Murzin AG, Proctor M and St Johnston D 1995 NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5. EMBO J 14 3563–3571

    CAS  PubMed Central  PubMed  Google Scholar 

  • Cao X and Jacobsen SE 2002 Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proc. Natl. Acad. Sci. USA 99 16491–16498

  • Cao X, Aufsatz W, Zilberman D, Mette MF, Huang MS, Matzke M and Jacobsen SE 2003 Role of the DRM and CMT3 methyltransferases in RNA-directed DNA methylation. Curr. Biol. 13 2212–2217

    Article  CAS  PubMed  Google Scholar 

  • Carroll AJ, Heazlewood JL, Ito J and Millar AH 2008 Analysis of the Arabidopsis cytosolic ribosome proteome provides detailed insights into its components and their post-translational modification. Mol. Cell. Proteomics 7 347–369

    Article  CAS  PubMed  Google Scholar 

  • Chakrabarty R, Banerjee R, Chung SM, Farman M, Citovsky V, Hogenhout SA, Tzfira T and Goodin M 2007 PSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions. Mol. Plant Microbe Interact 20 740–750

  • Costa-Nunes P, Pontes O, Preuss SB and Pikaard CS 2010 Extra views on RNA-dependent DNA methylation and MBD6-dependent heterochromatin formation in nucleolar dominance. Nucleus 1 254–259

    Article  PubMed Central  PubMed  Google Scholar 

  • Earley K, Lawrence RJ, Pontes O, Reuther R, Enciso AJ, Silva M, Neves N, Gross M, et al. 2006 Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance. Genes Dev. 20 1283–1293

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Earley KW, Pontvianne F, Wierzbicki AT, Blevins T, Tucker S, Costa-Nunes P, Pontes O and Pikaard CS 2010 Mechanisms of HDA6-mediated rRNA gene silencing: suppression of intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed cytosine methylation. Genes Dev. 24 1119–1132

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Finke A, Kuhlmann M and Mette MF 2012 IDN2 has a role downstream of siRNA formation in RNA-directed DNA methylation. Epigenetics 7 950–960

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gao Z, Liu HL, Daxinger L, Pontes O, He X, Qian W, Lin H, Xie M, et al. 2010 An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation. Nature 465 106–109

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Golemis EA, Adams PD, Bastiaens PIH, Einarson MB, Fisher RJ, Harpur AG, Medaglia MV, Ohh M et al. 2009 Molecular cloning: A laboratory manual. Third edition, (eds) J Sambrook and D W Russell (Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press) 3 18--28

  • He XJ, Hsu YF, Zhu S, Wierzbicki AT, Pontes O, Pikaard CS, Liu HL, Wang CS, et al. 2009 An effector of RNA-directed DNA methylation in Arabidopsis is an ARGONAUTE 4- and RNA-binding protein. Cell 137 498–508

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ito M, Koike A, Koizumi N and Sano H 2003 Methylated DNA-binding proteins from Arabidopsis. Plant Physiol. 133 1747–1754

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Jackson JP, Lindroth AM, Cao X and Jacobsen SE 2002 Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416 556–560

    Article  CAS  PubMed  Google Scholar 

  • Law JA and Jacobsen SE 2010 Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11 204–220

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Law JA, Ausin I, Johnson LM, Vashisht AA, Zhu JK, Wohlschlegel JA and Jacobsen SE 2010 A protein complex required for polymerase V transcripts and RNA- directed DNA methylation in Arabidopsis. Curr. Biol. 20 951–956

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Li CF, Pontes O, El-Shami M, Henderson IR, Bernatavichute YV, Chan SW, Lagrange T, Pikaard CS, et al. 2006 An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126 93–106

    Article  CAS  PubMed  Google Scholar 

  • Lorkovic ZJ, Naumann U, Matzke AJ and Matzke M 2012 Involvement of a GHKL ATPase in RNA-directed DNA methylation in Arabidopsis thaliana. Curr. Biol. 22 933–938

    Article  CAS  PubMed  Google Scholar 

  • Malagnac F, Bartee L and Bender J 2002 An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation. EMBO J. 21 6842–6852

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Maris C, Dominguez C and Allain FH 2005 The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J. 272 2118–2131

    Article  CAS  PubMed  Google Scholar 

  • Matragkou C, Papachristou H, Karetsou Z, Papadopoulos G, Papamarcaki T, Vizirianakis IS, Tsiftsoglou AS and Choli-Papadopoulou T 2009 On the intracellular trafficking of mouse S5 ribosomal protein from cytoplasm to nucleoli. J. Mol. Biol. 392 1192–1204

    Article  CAS  PubMed  Google Scholar 

  • Moore MS and Blobel G 1994 Purification of a Ran-interacting protein that is required for protein import into the nucleus. Proc. Natl. Acad. Sci. USA 91 10212–10216

  • Moy TI and Silver PA 1999 Nuclear export of the small ribosomal subunit requires the ran-GTPase cycle and certain nucleoporins. Genes Dev. 13 2118–2133

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Murashige T and Skoog F 1962 A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant 15 473–497

  • Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN and Bird A 1998 Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393 386–389

    Article  CAS  PubMed  Google Scholar 

  • Pendle AF, Clark GP, Boon R, Lewandowska D, Lam YW, Andersen J, Mann M, Lamond AI, et al. 2005 Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions. Mol. Biol. Cell 16 260–269

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Peters L and Meister G 2007 Argonaute proteins: mediators of RNA silencing. Mol. Cell 26 611–623

    Article  CAS  PubMed  Google Scholar 

  • Pontes O, Li CF, Costa Nunes P, Haag J, Ream T, Vitins A, Jacobsen SE and Pikaard CS 2006 The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126 79–92

    Article  CAS  PubMed  Google Scholar 

  • Pontes O, Costa-Nunes P, Vithayathil P and Pikaard CS 2009 RNA polymerase V functions in Arabidopsis interphase heterochromatin organization independently of the 24-nt siRNA-directed DNA methylation pathway. Mol. Plant 2 700–710

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Preuss SB, Costa-Nunes P, Tucker S, Pontes O, Lawrence RJ, Mosher R, Kasschau KD, Carrington JC, et al. 2008 Multimegabase silencing in nucleolar dominance involves siRNA-directed DNA methylation and specific methylcytosine-binding proteins. Mol. Cell 32 673–684

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Probst AV, Fagard M, Proux F, Mourrain P, Boutet S, Earley K, Lawrence RJ, Pikaard CS, et al. 2004 Arabidopsis histone deacetylase HDA6 is required for maintenance of transcriptional gene silencing and determines nuclear organization of rDNA repeats. Plant Cell 16 1021–1034

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Qi Y, He X, Wang XJ, Kohany O, Jurka J and Hannon GJ 2006 Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443 1008–1012

    Article  PubMed  Google Scholar 

  • Quimby BB, Lamitina T, L'Hernault SW and Corbett AH 2000 The mechanism of ran import into the nucleus by nuclear transport factor 2. J. Biol. Chem. 275 28575–28582

    Article  CAS  PubMed  Google Scholar 

  • Ribbeck K, Lipowsky G, Kent HM, Stewart M and Gorlich D 1998 NTF2 mediates nuclear import of Ran. EMBO J 17 6587–6598

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sasaki T, Lorković ZJ, Liang S-C, Matzke AJM and Matzke M 2014 The Ability to Form Homodimers Is Essential for RDM1 to Function in RNA-Directed DNAMethylation. PLoS ONE. 9, e88190. doi:10.1371/journal.pone.0088190

    Article  PubMed Central  PubMed  Google Scholar 

  • To TK, Kim JM, Matsui A, Kurihara Y, Morosawa T, Ishida J, Tanaka M, Endo T, et al. 2011 Arabidopsis HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1. PLoS Genet. 7, e1002055

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wassenegger M, Heimes S, Riedel L and Sanger HL 1994 RNA-directed de novo methylation of genomic sequences in plants. Cell 76 567–576

    Article  CAS  PubMed  Google Scholar 

  • Wierzbicki AT, Ream TS, Haag JR and Pikaard CS 2009 RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nat. Genet. 41 630–634

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Xie M, Ren G, Costa-Nunes P, Pontes O and Yu B 2012 A subgroup of SGS3-like proteins act redundantly in RNA-directed DNA methylation. Nucleic Acids Res. 2012 1–10

    Google Scholar 

  • Yano A, Kodama Y, Koike A, Shinya T, Kim HJ, Matsumoto M, Ogita S, Wada Y, et al. 2006 Interaction between methyl CpG-binding protein and ran GTPase during cell division in tobacco cultured cells. Ann. Bot. 98 1179–1187

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ye R, Wang W, Iki T, Liu C, Wu Y, Ishikawa M, Zhou X and Qi Y 2012 Cytoplasmic Assembly and Selective Nuclear Import of Arabidopsis ARGONAUTE4/siRNA Complexes. Mol. Cell 46 859–870

    Article  CAS  PubMed  Google Scholar 

  • Zemach A and Grafi G 2003 Characterization of Arabidopsis thaliana methyl-CpG-binding domain (MBD) proteins. Plant J 34 565–572

    Article  CAS  PubMed  Google Scholar 

  • Zemach A, Li Y, Wayburn B, Ben-Meir H, Kiss V, Avivi Y, Kalchenko V, Jacobsen SE, et al. 2005 DDM1 binds Arabidopsis methyl-CpG binding domain proteins and affects their subnuclear localization. Plant Cell 17 1549–1558

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zhao Q, Leung S, Corbett AH and Meier I 2006 Identification and characterization of the Arabidopsis orthologs of nuclear transport factor 2, the nuclear import factor of ran. Plant Physiol. 140 869–878

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zheng X, Zhu J, Kapoor A and Zhu JK 2007 Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing. EMBO J 26 1691–1701

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zheng Z, Xing Y, He XJ, Li W, Hu Y, Yadav SK, Oh J and Zhu JK 2010 An SGS3-like protein functions in RNA-directed DNA methylation and transcriptional gene silencing in Arabidopsis. Plant J 62 92–99

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zheng Q, Rowley MJ, Bohmdorfer G, Sandhu D, Gregory BD and Wierzbicki AT 2013 RNA polymerase V targets transcriptional silencing components to promoters of protein-coding genes. Plant J 73, 179--189

  • Zubko E, Gentry M, Kunova A, Meyer P 2012 De novo DNA methylation activity of methyltransferase 1 (MET1) partially restores body methylation in Arabidopsis thaliana. Plant J 71 1029--1037

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Acknowledgements

We acknowledge Dr Jefferson, CAMBIA, Australia, for providing the pCAMBIA1301 vector. APP acknowledges the Council of Scientific and Industrial Research, India, for providing the research fellowship. AKS acknowledges the Department of Science and Technology, India, for the Purse Grant. The SAP grant of University Grant Commission to the Department of Plant Molecular Biology for support of infra-structure is also acknowledged. This work was supported by the Purse Grant of the Department of Science and Technology, India. The work was also supported by infrastructure support provided by University Grants Commission (UGC) in the form of Special Assistance Program to Department of Plant Molecular Biology.

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  1. Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi, 110021, India

    Adwaita Prasad Parida, Amrapali Sharma & Arun Kumar Sharma

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  1. Adwaita Prasad Parida
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Corresponding editor: Man Mohan Johri

[Parida AP, Sharma A and Sharma AK 2017 AtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins. J. Biosci.]

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Parida, A.P., Sharma, A. & Sharma, A.K. AtMBD6, a methyl CpG binding domain protein, maintains gene silencing in Arabidopsis by interacting with RNA binding proteins. J Biosci 42, 57–68 (2017). https://doi.org/10.1007/s12038-016-9658-1

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