A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

Julie A. Law; Israel Ausín; Lianna M. Johnson; Ajay A Amar Vashisht; Jian-Kang Zhu; James A A. Wohlschlegel; Steven E. Jacobsen

doi:10.1016/j.cub.2010.03.062

A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

Julie A. Law, Israel Ausín, Lianna M. Johnson, Ajay A Amar Vashisht, Jian-Kang Zhu, James A A. Wohlschlegel, Steven E. Jacobsen

Biological, Environmental Sciences and Engineering

Research output: Contribution to journal › Article › peer-review

184 Scopus citations

Abstract

DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

Original language	English (US)
Pages (from-to)	951-956
Number of pages	6
Journal	Current Biology
Volume	20
Issue number	10
DOIs	https://doi.org/10.1016/j.cub.2010.03.062
State	Published - Apr 23 2010

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: We thank T. LaGrange for providing the NRPE1 antibody and members of the Jacobsen laboratory for helpful discussion. Jacobsen lab research was supported by U.S. National Institutes of Health (NIH) grant GM60398. I.A. was supported by a postdoctoral fellowship from the Ministerio de Educacion y Ciencia. J.A.L. was supported the NIH National Research Service Award 5F32GM820453. Wohlschlegel lab research was supported by University of California, Los Angeles Jonsson Cancer Center. S.E.J. is an investigator of the Howard Hughes Medical Institute. S.E.J., JAW., J.A.L., and I.A designed the experiments; J.A.L, I.A., L.M.J, and A.A.V. performed the experiments; J.K.Z. provided the ros1-1 rdm1-1 mutant allele and the RDM1 antibody; J.A.L. wrote the paper.

ASJC Scopus subject areas

General Agricultural and Biological Sciences
General Biochemistry, Genetics and Molecular Biology

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10.1016/j.cub.2010.03.062

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@article{0406e7cf97f14a31b79497e226e85f63,

title = "A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis",

abstract = "DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. {\textcopyright} 2010 Elsevier Ltd. All rights reserved.",

author = "Law, {Julie A.} and Israel Aus{\'i}n and Johnson, {Lianna M.} and Vashisht, {Ajay A Amar} and Jian-Kang Zhu and Wohlschlegel, {James A A.} and Jacobsen, {Steven E.}",

note = "KAUST Repository Item: Exported on 2020-10-01 Acknowledgements: We thank T. LaGrange for providing the NRPE1 antibody and members of the Jacobsen laboratory for helpful discussion. Jacobsen lab research was supported by U.S. National Institutes of Health (NIH) grant GM60398. I.A. was supported by a postdoctoral fellowship from the Ministerio de Educacion y Ciencia. J.A.L. was supported the NIH National Research Service Award 5F32GM820453. Wohlschlegel lab research was supported by University of California, Los Angeles Jonsson Cancer Center. S.E.J. is an investigator of the Howard Hughes Medical Institute. S.E.J., JAW., J.A.L., and I.A designed the experiments; J.A.L, I.A., L.M.J, and A.A.V. performed the experiments; J.K.Z. provided the ros1-1 rdm1-1 mutant allele and the RDM1 antibody; J.A.L. wrote the paper.",

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T1 - A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

AU - Law, Julie A.

AU - Ausín, Israel

AU - Johnson, Lianna M.

AU - Vashisht, Ajay A Amar

AU - Zhu, Jian-Kang

AU - Wohlschlegel, James A A.

AU - Jacobsen, Steven E.

N1 - KAUST Repository Item: Exported on 2020-10-01 Acknowledgements: We thank T. LaGrange for providing the NRPE1 antibody and members of the Jacobsen laboratory for helpful discussion. Jacobsen lab research was supported by U.S. National Institutes of Health (NIH) grant GM60398. I.A. was supported by a postdoctoral fellowship from the Ministerio de Educacion y Ciencia. J.A.L. was supported the NIH National Research Service Award 5F32GM820453. Wohlschlegel lab research was supported by University of California, Los Angeles Jonsson Cancer Center. S.E.J. is an investigator of the Howard Hughes Medical Institute. S.E.J., JAW., J.A.L., and I.A designed the experiments; J.A.L, I.A., L.M.J, and A.A.V. performed the experiments; J.K.Z. provided the ros1-1 rdm1-1 mutant allele and the RDM1 antibody; J.A.L. wrote the paper.

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N2 - DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

AB - DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

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A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

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