Role of RNA directed DNA methylation in controlling genomic imprinting in arabidopsis thaliana

ROLE OF RNA-DIRECTED DNA METHYLATION IN GENOMIC IMPRINTING IN ARABIDOPSIS THALIANA VU MINH THIET (B. Sc, Vietnam National University, Hanoi) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENT I would like to take this opportunity to thank everyone involved who helped me get to this place in my life and fulfill the long process of persuading the PhD study. I especially would like to express my deepest gratitude to my supervisor Frederic Berger. I would never finished my study without his unending support for the last few years. I believe that I have learnt a tremendous amount from him. I appreciate him for believing in me along the way. I would also like to thank my supervisor Prof Davis Ng for his help that allowed me to stay and work in TLL. I have extremely lucky to work with hearty colleagues Pauline, Pei Qi, Jeanie, Li Jing, Ramesh, Chen Zhong, Sarah, Heike, Nie Xin, Tomo, and also the former lab members Mathieu, Tadashi, Jonathan, Arnold. I would like to thank my colleagues for their sharing, accompanying me all the times during my study. A special thank I give to Pauline for her excellent guidance when I started working in the lab, I have learnt a lot from her discipline in doing experiment and her critical thinking in science. I would like to thank my thesis advisory committee Dr Yu Hao, Dr Toshiro Ito, and Dr Jose Dinneny for their support. I would also like to thank DBS Graduate Office and Ms Reena, Ms Priscilla. They are always there to answer my questions and help me to process my paper works as fast as possible. I also would like to thank Tam, Long, Hang, Hung, Ngoc, Trang for their support and encouragement for last few years in Singapore. i I would like to thank my parents for their support over the years and not complaining me when I could not come home for several TETs. A special thank to my brother for his presence in Singapore and his understanding of my hard time. Thanks for your care of our parents. Last but not least, I acknowledge NUS graduate scholarship and TLL for financial support. Thank you so much everyone. September, 2010 ii TABLE OF CONTENTS SUMMARY . vi LIST OF TABLES x LIST OF ABBREVIATIONS . xi CHAPTER 1: INTRODUCTION Abstract . 1. Introduction of imprinting 1.1.1 First reports of imprinting in plants . 1.1.2 The impact of interploid crosses on imprinting discovery 1.2 Imprinted genes and their function . 1.2.1 Arabidopsis imprinted genes . 1.2.2 Conservation of Polycomb group imprinted genes in cereals . 14 1.3 Conclusion . 15 2. Molecular mechanisms controlling imprinting . 15 2.1 Imprinting by DNA methylation 16 2.1.1 Maintenance of DNA methylation on the silent alleles . 16 2.1.2 Two-step removal of DNA methylation in the central cell 18 2.2 Molecular controls of imprinting by Histone methylation . 20 2.3. Cis-elements controlling imprinting 23 2.3.1 Cis-elements in the promoter . 23 2.3.2. Evidence for imprinting regulation by long distance elements 25 2.4 Genomic imprinting and RNA-directed DNA methylation . 27 2.5 Imprinting, a by-product of the global reprogramming? . 30 iii 3. Biological significance and evolution of imprinting . 31 3.1 Parental conflict . 31 3.2 Maternal control . 33 3.3 Imprinting, a factor of speciation 34 3.4 Transposons as the primer of imprinting evolution in a specific developmental context . 34 Aims of the research 36 CHAPTER 2: GENOMIC IMPRINTING AND RNA-DIRECTED DNA METHYLATION IN ARABIDOPSIS 39 2.1 Introduction . 40 2.2. Results 43 2.2.1 SDC is silenced in somatic tissues and expressed in seeds . 43 2.2.2 SDC is a new imprinted gene in Arabidopsis . 47 2.2.3 Silencing mechanism of SDC paternal allele . 50 2.2.4 Mechanism of activation of SDC maternal allele . 52 2.3 Discussion . 59 2.4 Future work . 65 Material and Methods 66 CHAPTER 3: Accession-dependent Imprinting of HAIKU2 is controlled by the RdDM Pathway . 73 3.1 Introduction . 74 3.2 Results . 78 3.2.1 IKU2 expression in gametes 78 3.2.2 Is IKU2 an imprinted gene? 80 iv 3.2.3 What mechanism controls silencing of the paternal IKU2 allele? 83 3.3 Discussion . 86 3.4 Future work . 90 Material and Methods 91 CHAPTER 4: GENERAL DISCUSSION 94 4.1 Main findings . 95 4.2 Biological significance . 96 4.2.1 Further expansion of the number of imprinted genes . 96 4.2.3 Is imprinting the by-product of the asymmetrical activity of major controls of DNA methylation? 98 4.3. Future perspective . 100 References . 103 Annex: Maternal effect of mutation in RdDM pathway on seed development 116 v SUMMARY In flowering plants and placental mammals, a subset of genes are expressed depending on their parent of origin and defined as imprinted genes. In Arabidopsis, the non-expressed allele of imprinted genes is silenced by either DNA methylation or Histone methylation by Polycomb repressive complex activity. The Arabidopsis imprinted genes are expressed only in endosperm, which nurtures embryo development. Imprinted expression is established in two steps involving the maintenance of DNA methylation. The paternal silenced allele remains marked by DNA methylation during spermatogenesis while gene silencing is released from the maternal allele by a demethylation pathway active during female gametogenesis. After fertilization, the expressed allele remains active and the inactive allele remains silent by maintenance DNA methylation machinery. DNA methylation can be deposited de novo through the RNA-directed DNA methylation pathway (RdDM) or maintained by the methyltransferase MET1. The maintenance methyltransferase MET1 plays a major role and is sufficient to establish monoallelic expression of most imprinted genes identified so far. However, the function of RdDM in genomic imprinting has remained largely unknown. In contrast with MET1 activity, the RdDM pathway results in methylation of cytosine residues in any context and in absence of a hemimethylated template. The RdDM pathway comprises the de novo methyltransferase DRM2 and the RNA polymerases POLIV and POLV. In the course of this thesis, we have identified a role of the RdDM pathway in regulating genomic imprinting in Arabidopsis thaliana. vi First, we identified SDC (suppressor of drm1, drm2, and cmt3) as a maternally imprinted gene. The SDC gene is primarily silenced by the RdDM pathway. We showed that SDC is specifically expressed in endosperm from its maternal allele and silencing of the paternal allele requires the RdDM pathway. The absence of expression of key genes in the RdDM pathway during female gametogenesis while it is maintained during spermatogenesis is sufficient to explain the origin of the imprinted expression of SDC. Second, our results showed that the RdDM pathway is also necessary for silencing the paternal allele of HAIKU2, which is a maternally expressed imprinted gene in endosperm. The imprinted expression of HAIKU2 is observed in a genetic contextdepending manner, relying on the accessions used in the reciprocal crosses. HAIKU2 controls endosperm growth. In conclusion, we described two novel imprinted genes in Arabidopsis. More importantly, we identified RdDM as a new silencing mechanism functioning in imprinting establishment. The loss of RdDM activity during female gametogenesis is predicted to cause a genome-wide demethylation. Our findings suggest that a new class of RdDM-dependent imprinted genes remains to be characterized in plants. vii List of Figures Figure number Figure title Page 1-1 Gametogenesis in flowering plants 1-2 Double fertilization in angiosperms 1-3 Analysis of MEDEA imprinting 1-4 Developmental feature of endosperm 13 1-5 Establishment of DNA methylation-dependent imprinted genes throughout the plant life cycle 17 1-6 DNA methylation dependent mechanisms leading to imprinting of maternally expressed genes in Arabidopsis 19 1-7 Polycomb Repressive Complex (PRC2) dependent mechanisms leading to imprinting of maternally expressed genes in Arabidopsis 21 1-8 Long distance cis-elements of imprinted genes 26 1-9 Regulation of PHERES1 imprinting 26 1-10 RNA-directed DNA methylation pathway 29 2-1 De novo methylation controls SDC expression 45 2-2 Expression of SDC in gametophytes and seeds 46 2-3 Allele specific RT-PCR analysis of maternal SDC expression 49 2-4 SDC imprint is controlled by RdDM pathway 51 2-5 Maternally expressed SDC is not controlled by DEMETER 54 2-6 Expression of NRPD2 gametophytes and developing seed 55 2-7 NRPD2a-RFP construct recues the loss of endogenous nrpd2a function 56 2-8 Expression of NRPD1b-RFP (PolV) in gametophytes and developing seed 57 2-9 A proposed model for mechanism activating SDC 58 viii expression in central cell 2-10 A proposed model for RdDM function in controlling SDC imprinting 63 3-1 Loss of IKU2 causes small seed size phenotype 77 3-2 HAIKU genetic pathway controls seed size 77 3-3 RT-PCR analysis of IKU2 expression 79 3-4 Allele-specific RT-PCR results show maternal expression of IKU2 81 3-5 Origin parental expression of IKU2 in combination of different Arabidopsis accessions 82 3-6 Expression of IKU2 in mutation of DNA methylation and Polycomb group 85 3-7 Silencing of paternal IKU2 allele is controlled by a DNAdependent RNA polymerase IV 85 ix Haun W.J., Laoueille-Duprat S., O'Connell M J., Spillane C., Grossniklaus U., Phillips A.R., Kaeppler S.M., Springer N.M. (2007) Genomic imprinting, methylation and molecular evolution of maize Enhancer of zeste (Mez) homologs. Plant J 49:325-37. Henderson I.R., Jacobsen S.E. (2007) Epigenetic inheritance in plants. Nature 447:418-24. Henderson I.R., Jacobsen S.E. (2008) Tandem repeats upstream of the Arabidopsis endogene SDC recruit non-CG DNA methylation and initiate siRNA spreading. Genes Dev 22:1597-606. Hennig L., Derkacheva M. (2009) Diversity of Polycomb group complexes in plants: same rules, different players? Trends Genet 25:414-23. Hermon P., Srilunchang K.O., Zou J., Dresselhaus T., Danilevskaya O.N. (2007) Activation of the imprinted Polycomb Group Fie1 gene in maize endosperm requires demethylation of the maternal allele. Plant Mol Biol 64:387-95. Herr A.J., Jensen M.B., Dalmay T., Baulcombe D.C. (2005) RNA polymerase IV directs silencing of endogenous DNA. Science 308:118-20. Honys D., Twell D. (2004) Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol 5:R85. Hsieh T.F., Ibarra C.A., Silva P., Zemach A., Eshed-Williams L., Fischer R.L., Zilberman D. (2009) Genome-wide demethylation of Arabidopsis endosperm. Science 324:1451-4. Ikeda Y., Kobayashi Y., Yamaguchi A., Abe M., Araki T. (2007) Molecular basis of late-flowering phenotype caused by dominant epi-alleles of the FWA locus in Arabidopsis. Plant Cell Physiol 48:205-20. Ingouff M., Haseloff J., Berger F. (2005a) Polycomb group genes control developmental timing of endosperm. Plant J 42:663-74. Ingouff M., Fitz Gerald J.N., Guerin C., Robert H., Sorensen M.B., Van Damme D., Geelen D., Blanchoin L., Berger F. (2005b) Plant formin AtFH5 is an evolutionarily conserved actin nucleator involved in cytokinesis. Nat Cell Biol 7:374-80. Jahnke S., Scholten S. (2009) Epigenetic resetting of a gene imprinted in plant embryos. Curr Biol 19:1677-81. 107 Jia Y., Lisch D.R., Ohtsu K., Scanlon M.J., Nettleton D., Schnable P.S. (2009) Loss of RNA–Dependent RNA Polymerase (RDR2) Function Causes Widespread and Unexpected Changes in the Expression of Transposons, Genes, and 24-nt Small RNAs. PLoS Genet 5:e1000737. Johnson L.M., Law J.A., Khattar A., Henderson I.R., Jacobsen S.E. (2008) SRAdomain proteins required for DRM2-mediated de novo DNA methylation. PLoS Genet 4:e1000280. Johnston A.J., Matveeva E., Kirioukhova O., Grossniklaus U., Gruissem W. (2008) A dynamic reciprocal RBR-PRC2 regulatory circuit controls Arabidopsis gametophyte development. Curr Biol 18:1680-6. Josefsson C., Dilkes B., Comai L. (2006) Parent-dependent loss of gene silencing during interspecies hybridization. Curr Biol 16:1322-8. Jullien P.E., Berger F. (2009) Gamete-specific epigenetic mechanisms shape genomic imprinting. Curr Opin Plant Biol 12:637-42. Jullien P.E., Berger F. (2010) Parental Genome Dosage Imbalance Deregulates Imprinting in Arabidopsis. PLoS Genet 6:e1000885. Jullien P.E., Kinoshita T., Ohad N., Berger F. (2006a) Maintenance of DNA Methylation during the Arabidopsis Life Cycle Is Essential for Parental Imprinting. Plant Cell 18:1360-72. Jullien P.E., Katz A., Oliva M., Ohad N., Berger F. (2006b) Polycomb Group Complexes Self-Regulate Imprinting of the Polycomb Group Gene MEDEA in Arabidopsis. Curr Biol 16:486-92. Jullien P.E., Mosquna A., Ingouff M., Sakata T., Ohad N., Berger F. (2008) Retinoblastoma and Its Binding Partner MSI1 Control Imprinting in Arabidopsis. PLoS Biol 6:e194. Kaneda M., Okano M., Hata K., Sado T., Tsujimoto N., Li E., Sasaki H. (2004) Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 429:900-3. Kanno T., Huettel B., Mette M.F., Aufsatz W., Jaligot E., Daxinger L., Kreil D.P., Matzke M., Matzke A.J. (2005) Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nat Genet 37:761-5. 108 Kasschau K.D., Fahlgren N., Chapman E.J., Sullivan C.M., Cumbie J.S., Givan S.A., Carrington J.C. (2007) Genome-Wide Profiling and Analysis of Arabidopsis siRNAs. PLoS Biol 5:e57. Katz A., Oliva M., Mosquna A., Hakim O., Ohad N. (2004) FIE and CURLY LEAF polycomb proteins interact in the regulation of homeobox gene expression during sporophyte development. Plant J 37:707-19. Kawabe A., Fujimoto R., Charlesworth D. (2007) High diversity due to balancing selection in the promoter region of the Medea gene in Arabidopsis lyrata. Curr Biol 17:1885-9. Kermicle J.L. (1970) Dependence of the R-Mottled Aleurone Phenotype in Maize on Mode of sexual tramission. Genetics 66:69 - 85. Kermicle J.L., Allemand, M. (1990) Gametic imprinting in maize in relation to the angiosperm life cycle. Development (Suppl) 1:9-14. Kinoshita T., Ikeda Y., Ishikawa R. (2008) Genomic imprinting: a balance between antagonistic roles of parental chromosomes. Semin Cell Dev Biol 19:574-9. Kinoshita T., Yadegari R., Harada J.J., Goldberg R.B., Fischer R.L. (1999) Imprinting of the MEDEA polycomb gene in the Arabidopsis endosperm. Plant Cell 11:1945-1952. Kinoshita T., Miura A., Choi Y., Kinoshita Y., Cao X., Jacobsen S.E., Fischer R.L., Kakutani T. (2004) One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation. Science 303:521-3. Kinoshita Y., Saze H., Kinoshita T., Miura A., Soppe W.J., Koornneef M., Kakutani T. (2007) Control of FWA gene silencing in Arabidopsis thaliana by SINErelated direct repeats. Plant J 49:38-45. Kiyosue T., Ohad N., Yadegari R., Hannon M., Dinneny J., Wells D., Katz A., Margossian L., Harada J.J., Goldberg R.B., Fischer R.L. (1999) Control of fertilization-independent endosperm development by the MEDEA polycomb gene in Arabidopsis. Proc. Natl. Acad. Sci. USA 96:4186-91. Kohler C., Weinhofer-Molisch I. (2010) Mechanisms and evolution of genomic imprinting in plants. Heredity 105:57-63. Kohler C., Page D.R., Gagliardini V., Grossniklaus U. (2005) The Arabidopsis thaliana MEDEA Polycomb group protein controls expression of PHERES1 by parental imprinting. Nat Genet 37:28-30. 109 Kohler C., Hennig L., Spillane C., Pien S., Gruissem W., Grossniklaus U. (2003a) The Polycomb-group protein MEDEA regulates seed development by controlling expression of the MADS-box gene PHERES1. Genes and development 17:1540-53. Kohler C., Hennig L., Bouveret R., Gheyselinck J., Grossniklaus U., Gruissem W. (2003b) Arabidopsis MSI1 is a component of the MEA/FIE Polycomb group complex and required for seed development. Embo J 22:4804-4814. Kouzarides T. (2007) Chromatin modifications and their function. Cell 128:693-705. Law J.A., Jacobsen S.E. (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11:204-220. Le Trionnaire G., Twell D. (2010) Small RNAs in angiosperm gametophytes: from epigenetics to gamete development. Genes & Development 24:1081-1085. DOI: 10.1101/gad.1936110. Lin B.-Y. (1984) Ploidy barrier to endosperm development in maize. Genetics 107:103-115. Lindroth A.M., Cao X., Jackson J.P., Zilberman D., McCallum C.M., Henikoff S., Jacobsen S.E. (2001) Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science 292:2077-80. Lippman Z., Gendrel A.-V., Black M., Vaughn M.W., Dedhia N., Richard McCombie W., Lavine K., Mittal V., May B., Kasschau K.D., Carrington J.C., Doerge R.W., Colot V., Martienssen R. (2004) Role of transposable elements in heterochromatin and epigenetic control. Nature 430:471-476. Luo M., Bilodeau P., Dennis E.S., Peacock W.J., Chaudhury A. (2000) Expression and parent-of-origin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds. Proc. Natl. Acad. Sci. USA 97:10637-10642. Luo M., Dennis E.S., Berger F., Peacock W.J., Chaudhury A. (2005) MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a leucine-rich repeat (LRR) KINASE gene, are regulators of seed size in Arabidopsis. Proc Natl Acad Sci U S A 102:17531-6. Luo M., Platten D., Chaudhury A., Peacock W.J., Dennis E.S. (2009) Expression, imprinting, and evolution of rice homologs of the polycomb group genes. Mol Plant 2:711-23. 110 Luo M., Bilodeau P., Koltunow A., Dennis E.S., Peacock W.J., Chaudhury A.M. (1999) Genes controlling fertilization-independent seed development in Arabidopsis thaliana. Proc Natl Acad Sci U S A 96:296-301. Makarevich G., Villar C.B., Erilova A., Kohler C. (2008) Mechanism of PHERES1 imprinting in Arabidopsis. J Cell Sci 121:906-12. Makarevich G., Leroy O., Akinci U., Schubert D., Clarenz O., Goodrich J., Grossniklaus U., Kohler C. (2006) Different Polycomb group complexes regulate common target genes in Arabidopsis. EMBO Rep 7:947-52. Martienssen R.A., Kloc A., Slotkin R.K., Tanurdzic M. (2008) Epigenetic inheritance and reprogramming in plants and fission yeast. Cold Spring Harb Symp Quant Biol 73:265-71. Matzke M., Kanno T., Daxinger L., Huettel B., Matzke A.J. (2009) RNA-mediated chromatin-based silencing in plants. Curr Opin Cell Biol 21:367-76. McCormick S. (2004) Control of male gametophyte development. Plant Cell 16 Suppl:S142-53. Miyake T., Takebayashi N., Wolf D.E. (2009) Possible Diversifying Selection in the Imprinted Gene, MEDEA, in Arabidopsis. Mol Biol Evol 26:843-857. DOI: 10.1093/molbev/msp001. Mosher R.A., Schwach F., Studholme D., Baulcombe D.C. (2008) PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis. Proc Natl Acad Sci U S A 105:3145-50. Mosher R.A., Melnyk C.W., Kelly K.A., Dunn R.M., Studholme D.J., Baulcombe D.C. (2009) Uniparental expression of PolIV-dependent siRNAs in developing endosperm of Arabidopsis. Nature 460:283-6. Numa H., Kim J.-M., Matsui A., Kurihara Y., Morosawa T., Ishida J., Mochizuki Y., Kimura H., Shinozaki K., Toyoda T., Seki M., Yoshikawa M., Habu Y. (2010) Transduction of RNA-directed DNA methylation signals to repressive histone marks in Arabidopsis thaliana. Ohad N., Yadegari R., Margossian L., Hannon M., Michaeli D., Harada J.J., Goldberg R.B., Fischer R.L. (1999) Mutations in FIE, a WD polycomb group gene, allow endosperm development without fertilization. Plant Cell 11:407-16. Olmedo-Monfil V., Duran-Figueroa N., Arteaga-Vazquez M., Demesa-Arevalo E., Autran D., Grimanelli D., Slotkin R.K., Martienssen R.A., Vielle-Calzada J.- 111 P. (2010) Control of female gamete formation by a small RNA pathway in Arabidopsis. Onodera Y., Haag J.R., Ream T., Nunes P.C., Pontes O., Pikaard C.S. (2005) Plant nuclear RNA polymerase IV mediates siRNA and DNA methylationdependent heterochromatin formation. Cell 120:613-22. Pask A., Papenfuss A., Ager E., McColl K., Speed T., Renfree M. (2009) Analysis of the platypus genome suggests a transposon origin for mammalian imprinting. Genome Biology 10:R1. Pillot M., Baroux C., Vazquez M.A., Autran D., Leblanc O., Vielle-Calzada J.P., Grossniklaus U., Grimanelli D. (2010) Embryo and Endosperm Inherit Distinct Chromatin and Transcriptional States from the Female Gametes in Arabidopsis. Plant Cell 22:307-320. DOI: 10.1105/tpc.109.071647. Pontier D., Yahubyan G., Vega D., Bulski A., Saez-Vasquez J., Hakimi M.A., LerbsMache S., Colot V., Lagrange T. (2005) Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes Dev 19:2030-40. Randolph L. (1935) Cytogenetics of tetraploid maize. J Agr Res 50:591-595. Roudier F., Teixeira F.K., Colot V. (2009) Chromatin indexing in Arabidopsis: an epigenomic tale of tails and more. Trends Genet 25:511-7. Saze H., Scheid O.M., Paszkowski J. (2003) Maintenance of CpG methylation is essential for epigenetic inheritance during plant gametogenesis. Nat Genet 34:65-9. Schmittgen T.D., Livak K.J. (2008) Analyzing real-time PCR data by the comparative CT method. Nat. Protocols 3:1101-1108. Schuettengruber B., Chourrout D., Vervoort M., Leblanc B., Cavalli G. (2007) Genome regulation by polycomb and trithorax proteins. Cell 128:735-45. Scott R.J., Spielman M., Bailey J., Dickinson H.G. (1998) Parent-of-origin effects on seed development in Arabidopsis thaliana. Development 125:3329-41. Soppe W.J., Jacobsen S.E., Alonso-Blanco C., Jackson J.P., Kakutani T., Koornneef M., Peeters A.J. (2000) The late flowering phenotype of fwa mutants is caused by gain-of- function epigenetic alleles of a homeodomain gene. Mol Cell 6:791-802. 112 Sorensen M.B., Mayer U., Lukowitz W., Robert H., Chambrier P., Jurgens G., Somerville C., Lepiniec L., Berger F. (2002) Cellularisation in the endosperm of Arabidopsis thaliana is coupled to mitosis and shares multiple components with cytokinesis. Development 129:5567-76. Spielman M., Vinkenoog R., Dickinson H.G., Scott R.J. (2001) The epigenetic basis of gender in flowering plants and mammals. Trends Genet 17:705-11. Spillane C., Schmid K.J., Laoueille-Duprat S., Pien S., Escobar-Restrepo J.M., Baroux C., Gagliardini V., Page D.R., Wolfe K.H., Grossniklaus U. (2007) Positive darwinian selection at the imprinted MEDEA locus in plants. Nature 448:349-52. Suzuki S., Ono R., Narita T., Pask A.J., Shaw G., Wang C., Kohda T., Alsop A.E., Marshall Graves J.A., Kohara Y., Ishino F., Renfree M.B., Kaneko-Ishino T. (2007) Retrotransposon Silencing by DNA Methylation Can Drive Mammalian Genomic Imprinting. PLoS Genet 3:e55. Tiwari S., Schulz R., Ikeda Y., Dytham L., Bravo J., Mathers L., Spielman M., Guzman P., Oakey R.J., Kinoshita T., Scott R.J. (2008) MATERNALLY EXPRESSED PAB C-TERMINAL, a novel imprinted gene in Arabidopsis, encodes the conserved C-terminal domain of polyadenylate binding proteins. Plant Cell 20:2387-98. Vielle-Calzada J.P., Thomas J., Spillane C., Coluccio A., Hoeppner M.A., Grossniklaus U. (1999) Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity. Genes Dev. 13:2971-2982. Villar C.B.R., Erilova A., Makarevich G., Trosch R., Kohler C. (2009) Control of PHERES1 Imprinting in Arabidopsis by Direct Tandem Repeats. Mol Plant 2:654-660. DOI: 10.1093/mp/ssp014. Walia H., Josefsson C., Dilkes B., Kirkbride R., Harada J., Comai L. (2009) Dosagedependent deregulation of an AGAMOUS-LIKE gene cluster contributes to interspecific incompatibility. Curr Biol 19:1128-32. Wang A., Garcia D., Zhang H., Feng K., Chaudhury A., Berger F., Peacock W.J., Dennis E.S., Luo M. (2010) The VQ motif protein IKU1 regulates endosperm growth and seed size in Arabidopsis. The Plant Journal 63:670-679. DOI: 10.1111/j.1365-313X.2010.04271.x. Wang D., Tyson M.D., Jackson S.S., Yadegari R. (2006) Partially redundant functions of two SET-domain polycomb-group proteins in controlling initiation of seed development in Arabidopsis. Proc Natl Acad Sci U S A 103:13244-9. 113 Wiley C.D., Matundan H.H., Duselis A.R., Isaacs A.T., Vrana P.B. (2008) Patterns of hybrid loss of imprinting reveal tissue- and cluster-specific regulation. PLoS One 3:e3572. Wilkins J.F., Haig D. (2003) What good is genomic imprinting: the function of parent-specific gene expression. Nat Rev Genet 4:359-68. Wuest S.E., Vijverberg K., Schmidt A., Weiss M., Gheyselinck J., Lohr M., Wellmer F., Rahnenführer J., von Mering C., Grossniklaus U. (2010a) Arabidopsis Female Gametophyte Gene Expression Map Reveals Similarities between Plant and Animal Gametes. Curr Biol 20:506-512. Xiao W., Brown R.C., Lemmon B.E., Harada J.J., Goldberg R.B., Fischer R.L. (2006) Regulation of seed size by hypomethylation of maternal and paternal genomes. Plant Physiol 142:1160-8. Xie Z., Johansen L.K., Gustafson A.M., Kasschau K.D., Lellis A.D., Zilberman D., Jacobsen S.E., Carrington J.C. (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2:E104. Yadegari R., Drews G.N. (2004) Female gametophyte development. Plant Cell 16 Suppl:S133-41. Yokthongwattana C., Bucher E., Caikovski M., Vaillant I., Nicolet J., Mittelsten Scheid O., Paszkowski J. (2010) MOM1 and Pol-IV/V interactions regulate the intensity and specificity of transcriptional gene silencing. Embo J 29:34051. Zhai J., Liu J., Liu B., Li P., Meyers B.C., Chen X., Cao X. (2008) Small RNADirected Epigenetic Natural Variation in Arabidopsis thaliana. PLoS Genet 4:e1000056. Zhang X., Henderson I.R., Lu C., Green P.J., Jacobsen S.E. (2007a) Role of RNA polymerase IV in plant small RNA metabolism. Proceedings of the National Academy of Sciences 104:4536-4541. DOI: 10.1073/pnas.0611456104. Zhang X., Bernatavichute Y.V., Cokus S., Pellegrini M., Jacobsen S.E. (2009) Genome-wide analysis of mono-, di- and trimethylation of histone H3 lysine in Arabidopsis thaliana. Genome Biol 10:R62. Zhang X., Germann S., Blus B.J., Khorasanizadeh S., Gaudin V., Jacobsen S.E. (2007b) The Arabidopsis LHP1 protein colocalizes with histone H3 Lys27 trimethylation. Nat Struct Mol Biol 14:869-71. 114 Zhang X., Yazaki J., Sundaresan A., Cokus S., Chan S.W., Chen H., Henderson I.R., Shinn P., Pellegrini M., Jacobsen S.E., Ecker J.R. (2006) Genome-wide highresolution mapping and functional analysis of DNA methylation in arabidopsis. Cell 126:1189-201. Zhou Y., Zhang X., Kang X., Zhao X., Zhang X., Ni M. (2009) SHORT HYPOCOTYL UNDER BLUE1 associates with MINISEED3 and HAIKU2 promoters in vivo to regulate Arabidopsis seed development. Plant Cell 21:106-17. Zilberman D., Cao X., Johansen L.K., Xie Z., Carrington J.C., Jacobsen S.E. (2004) Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. Curr Biol 14:1214-20. 115 Annex: Maternal effect of mutation in RdDM pathway on seed development Rationale Apart from three canonical RNA polymerases I, II and II plants possess two distinct RNA polymerase defined as PolIV and PolV. Both of them were characterized in reversed genetics to identify the essential components for RNA-based silencing pathway. POLIV and POLV participate in gene silencing through the RNA-directed DNA methylation (RdDM) which targets transgenes, transposable elements, and repetitive elements (Herr et al., 2005; Mosher et al., 2008; Zhang et al., 2007a). In RdDM, POLIV is thought to transcribe repeat elements to produce single stranded RNAs which are processed into 24 nt siRNAs by a RDR2 and DCL3. Whereas the POLV acts in the later step as a component of AGO4-siRNA-POLV complex recruited to the target sequence. It is suggested that POLV transcribed the target DNA to generate the ssRNA molecules to which the AGO-siRNA complex can hybridize and DRM2 is able methylate its targets (Henderson and Jacobsen, 2007; Jia et al., 2009). However, the transcriptase activity of POLIV and POLV has not been demonstrated yet. The number of siRNAs produced by POLIV/POLV account for 90% of total siRNAs in Arabidopsis (Jia et al., 2009; Kasschau et al., 2007; Le Trionnaire and Twell, 2010; Zhang et al., 2007a). However, it is surprising that mutants impaired in POLIV/POLV complexes have no visible developmental phenotypes and are fertile. In this study, we characterized seed phenotype caused by a mutation for NRPD2a. The gene encodes 116 the second biggest subunit of both POLIV and POLV, therefore loss of NRPD2 theoretically leads to loss of POLIV and POLV functions. Preliminary Results Loss of NRPD2a caused abnormally developing seed NPRD2 has two homologs in Arabidopsis genome including NRPD2a and NRPD2b (Figure A1-A). It seems that only NRPD2a is expressed and has been shown to play an important role in transcription silencing through the RdDM pathway (Herr et al., 2005; Mosher et al., 2008). In contrast, NRPD2b coding sequence contains a stop codon in the first exon, therefore the gene is unlikely to produce a functional protein (Pontier et al., 2005). We obtained the double mutant nrpd2a-1/nrpd2a-1; nrpd2b1/nrpd2b-1 to completely remove any functional redundancy of NRPD2a and NRPD2b (Onodera et al., 2005). Interestingly, we observed that in selfed nrpd2a/nrpd2a mutant, 30% of seeds developed abnormally leading to seed abortion (Figure A1-B). Seeds were affected with a pleiotropic phenotype including early embryo arrest, embryo polarity defects and reduced endosperm growth. At DAP, we observed a significant difference in term of size between seeds of mutant plants and seed of wild type plants. More frequently we found that mutant seeds are smaller than wild type, while they displayed the same embryo size (Figure A2). More than 30% of mature seeds collected from viable seeds of mutant are smaller than wild type mature seeds 117 (Figure A3). This result suggested that endosperm development is affected in the nrpd2a, nrpd2b mutant background. In order to confirm the phenotype observed is associated with nrpd2a-1, the mutant line was backcrossed with the wild type plant for two times. The homozygous lines recovered from the back-crosses displayed the same phenotypes including the same percentage of aborted seeds and same percentage small seed size (data not shown). We observed the same phenotype in the mutation for NRPD2a (nrpd2a-1) but not in NRPD2b (nrpd2b-1) (Figure A1-C). We thus conclude that NRPD2a is required for normal seed development in Arabidopsis. 118 Figure A1. Seed phenotype caused by loss of NRPD2 function A. Nrpd2a mutant line: nrpd2a-1 is SALK_095689 located at the last exon encoding the 3’ CTD domain which is found to play an important role for polymerase activity in the canonical RNA polymerase I, II and III. B. Seed phenotype in the double mutant nrpd2a-1, nrpd2b-1 C. Seed phenotype in the single mutant nrpd2a-1 Figure A2. Defect in seed development in nrpd2 mutant Seeds from DAP siliques were cleared and observed with differential interference contrast optics. A population of nrpd2 seeds is significant smaller than WT seed. The mutant seed (nrpd2a, #1) was observed with more frequencies, in which the seed size is smaller than WT but the embryo size is the same in both mutant and WT. The nrpd2a, #2 seeds showed the smaller size with smaller embryo size and short suspensor compared to WT seeds. 119 Figure A3. Seed size comparison between col-0 wild type and nrpd2 mutant (A) Size of viable seeds collected from the selfed nrpd2a-1, nrpd2b-1 was compared to Col-0 WT seed. 38% of mutant seeds are significant smaller than wild type seeds. (B)Wild type and mutant nrpd2a-1 plants were cultured in the same environmental condition. Seeds are produced by manual pollination. Size of the viable seeds was measured using ImageJ ver 1.43. The data was plotted by using Sigma Plot v10.0. 120 Maternal effect of nrpd2a mutant on seed development In order to know whether the phenotype of mutant seed is gametophytic recessive origin or sporophytic recessive origin. We phenotyped the selfed nrpd2a-1/+, nrpd2b1/+, however we did not observe abnormal seed phenotypes. The similar result was observed when heterozygous nrpd2a-1/+, nrpd2b-1/+ was backcrossed with wild type plant either as male or female. These results ruled out the sporophytic recessive origin and the gametophytic recessive origin of the nrpd2 mutant phenotypes. However, the phenotype associated with nrpd2 was observed in backcrosses involving nrpd2 homozygous plants as female but not as male (Figure A4). The origin of the nrpd2 sporophytic maternal effect remains unknown though it is possible that it affects meiosis and chromosome segregation. In conclusion, we described for the first time that loss of POLIV/POLV caused a maternal sporophytic effect on seed development which. Our results suggested that an additional important role of PolIV/PolV in plant life. 121 Figure A4. Maternal effect of nrpd2 on seed viability and seed size A) Seed abortion was only observed when nrpd2 was used as a mother to cross with a WT father. B) More than 30% of viable seeds from the crosses nrpd2a mother x wild type father were smaller than viable seeds came from the crosses WT mother x nrpd2 father. 122 [...]... discovery of imprinting 1.1.1 First reports of imprinting in plants The term imprinting was originally adopted to qualify the differential elimination of paternal chromosomes in the mealybug Sciara (Crouse, 1960) The first example of imprinted expression of a gene was identified in the study of pigmentation of the outer layers of the endosperm in maize (Kermicle, 1970) Irregular anthocyanin pigmentation... contains two doses of the maternal genome and one dose of the paternal genome This specific parental genomic dosage attracted interest in early studies of plant reproduction, which led to the discovery of imprinting in plants After an historical account, we will review in this section the identity and function of imprinted genes in plants 3 Figure 1-1 Gametogenesis in flowering plants In flowering plants,... requirement of paternal genome expression suggests the existence of yet unidentified paternally expressed imprinted genes Imprinting affects genes encoding members of a conserved PcG complex, which plays a key role in the control of several aspects of endosperm development including polarity, growth, and temporal aspects Studies using interploid crosses suggest that the overall function of imprinting related... prefigure the imprinted expression after fertilization in the endosperm Figure 1-5 Establishment of DNA methylation- dependent imprinted genes throughout the plant life cycle DNA methylation dependent imprinted genes are silenced by DNA methyltransferase MET1 in the vegetative tissue Maintain of DNA methylation is also required for silencing of imprinted genes in pollen Whereas DNA methylation was actively... regulation of MEA imprinting, disruption of PcG function provided by the Mez1 maternal allele causes expression of the Mez1 paternal allele (Haun et al., 2009), suggesting a conservation of the mechanisms that regulate imprinting of MEA and its homolog Mez1 PHERES1 (PHE1) is a paternally expressed imprinted gene (Kohler et al., 2005) The silencing of the maternal allele of PHE1 is mediated by the maternal... by removal of chromatin modification and remains active after fertilization while the other allele remains silenced, leading to imprinted gene expression Imprinting mechanisms are conserved across plant species and to a certain extent there is evidence of convergent evolution of imprinting mechanisms between plants and mammals The physiological significance and evolutionary origin of imprinting are still... to the control of endosperm growth and seed size However, a comprehensive picture of the total number of imprinted genes is still unknown It is likely that the development of deep sequencing technologies coupled with the use of polymorphisms will enable rapid discovery of new imprinted genes in various species 2 Molecular mechanisms controlling imprinting Parental genomic imprinting originates from epigenetic... imprinting is contained in a 400bp domain of the AtFH5 promoter; but the detailed mechanism causing imprinting of AtFH5 is not known Although histone methylation by Pc-G is involved in imprinting in mammals (Feil and Berger, 2007), it does not appear to act as the essential repressor of the silenced alleles of imprinted genes as shown for certain imprinted genes in plants A major challenge is to understand... cells MET1 maintains DNA methylation silencing marks (grey triangles) on the paternal allele of imprinted genes During the second part of female gametogenesis, MET1 expression is repressed by the Retinoblastoma (pRB) pathway This repression causes passive removal of DNA methylation on the maternal allele This mode of demethylation is not sufficient to cause expression of the target gene Only in the mature... mechanism causing such a removal remains unknown Figure 1-7 Polycomb Repressive Complex 2 (PRC2) dependent mechanisms leading to imprinting of maternally expressed genes in Arabidopsis PRC2 maintains H3K27 methylation silencing marks on the parental alleles of MEA (green spheres) The two sperm cells fertilize the egg cell and the central cell During male gametogenesis, H3K27 methylation is maintained while . mechanisms controlling imprinting 15 2.1 Imprinting by DNA methylation 16 2.1.1 Maintenance of DNA methylation on the silent alleles 16 2.1.2 Two-step removal of DNA methylation in the central. origin of imprinting are still unclear but in plants, imprinting may be the consequence of global epigenetic reprogramming during sexual reproduction. 3 1. Introduction of imprinting In. 25 2.4 Genomic imprinting and RNA- directed DNA methylation 27 2.5 Imprinting, a by-product of the global reprogramming? 30 iv 3. Biological significance and evolution of imprinting 31