Arabidopsis RPT2a, 19S Proteasome Subunit, Regulates Gene Silencing via DNA Methylation

The ubiquitin/proteasome pathway plays a crucial role in many biological processes. Here we report a novel role for the Arabidopsis 19S proteasome subunit RPT2a in regulating gene activity at the transcriptional level via DNA methylation. Knockout mutation of the RPT2a gene did not alter global protein levels; however, the transcriptional activities of reporter transgenes were severely reduced compared to those in the wild type. This transcriptional gene silencing (TGS) was observed for transgenes under control of either the constitutive CaMV 35S promoter or the cold-inducible RD29A promoter. Bisulfite sequencing analysis revealed that both the transgene and endogenous RD29A promoter regions were hypermethylated at CG and non-CG contexts in the rpt2a mutant. Moreover, the TGS of transgenes driven by the CaMV 35S promoters was released by treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine, but not by application of the inhibitor of histone deacetylase Trichostatin A. Genetic crosses with the DNA methyltransferase met1 single or drm1drm2cmt3 triple mutants also resulted in a release of CaMV 35S transgene TGS in the rpt2a mutant background. Increased methylation was also found at transposon sequences, suggesting that the 19S proteasome containing AtRPT2a negatively regulates TGS at transgenes and at specific endogenous genes through DNA methylation.