title: SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV.
abstract: DNA methylation is an evolutionarily conserved epigenetic modification that is critical for gene silencing and the maintenance of genome integrity. In Arabidopsis thaliana, the de novo DNA methyltransferase, domains rearranged methyltransferase 2 (DRM2), is targeted to specific genomic loci by 24 nt small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM). Biogenesis of the targeting siRNAs is thought to be initiated by the activity of the plant-specific RNA polymerase IV (Pol-IV). However, the mechanism through which Pol-IV is targeted to specific genomic loci and whether factors other than the core Pol-IV machinery are required for Pol-IV activity remain unknown. Through the affinity purification of nuclear RNA polymerase D1 (NRPD1), the largest subunit of the Pol-IV polymerase, we found that several previously identified RdDM components co-purify with Pol-IV, namely RNA-dependent RNA polymerase 2 (RDR2), CLASSY1 (CLSY1), and RNA-directed DNA methylation 4 (RDM4), suggesting that the upstream siRNA generating portion of the RdDM pathway may be more physically coupled than previously envisioned. A homeodomain protein, SAWADEE homeodomain homolog 1 (SHH1), was also found to co-purify with NRPD1; and we demonstrate that SHH1 is required for de novo and maintenance DNA methylation, as well as for the accumulation of siRNAs at specific loci, confirming it is a bonafide component of the RdDM pathway.
authors.affiliation: Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Law
authors.forename: Ajay A
authors.initials: AA
authors.lastname: Vashisht
authors.forename: James A
authors.initials: JA
authors.lastname: Wohlschlegel
authors.forename: Steven E
authors.initials: SE
authors.lastname: Jacobsen
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly / Q000235:genetics; D019175:DNA Methylation; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D030821:Plants, Genetically Modified; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D029681:Arabidopsis Proteins; C523533:CLASSY1 protein, Arabidopsis; C548147:DMS4 protein, Arabidopsis; D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; D021122:Protein Subunits; D034741:RNA, Small Interfering; C568760:SHH1 protein, Arabidopsis; C499181:RNA polymerase IV, Arabidopsis; C519655:RDR2 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; C568761:NRPD1A protein, Arabidopsis; D019951:DNA Polymerase beta