Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis

Abstract

REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylation enzyme that was previously identified during a genetic screen for the silencing of both RD29A-LUC and 35S-NPTII transgenes on a T-DNA construct. Here we performed a genetic screen to identify additional mutants in which the 35S-NPTII transgene is silenced. We identified several alleles of ros1 and of the following components of the RNA-directed DNA methylation (RdDM) pathway: NRPD1 (the largest subunit of polymerase IV), RDR2, NRPE1 (the largest subunit of polymerase V), NRPD2, AGO4, and DMS3. Our results show that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants. We also identified a putative histone acetyltransferase (ROS4) from the genetic screen. The acetyltransferase contains a PHD-finger domain that binds to unmethylated histone H3K4. The mutation in ROS4 led to reduction of H3K18 and H3K23 acetylation levels. We show that the silencing of 35S-NPTII and some transposable element genes was released by the ddm1 mutation but that this also required ROS4. Our study identifies a unique antisilencing factor, and reveals that the RdDM pathway has an antisilencing function due to its role in maintaining ROS1 expression.transcriptional gene silencing | epigenetic | gene silencing | histone modification D NA cytosine methylation is an important epigenetic modification in plants and animals. The molecular mechanism for RNA-directed DNA methylation (RdDM) has been well-established by both genetic and biochemical studies in Arabidopsis (1-4). Small RNAs (∼24 bp) and long noncoding RNAs produced by a complex transcriptional machinery composed of polymerase (Pol) IV and Pol V, together with other proteins, target specific sequences for de novo DNA methylation (1-4). RdDM mainly occurs in transposons, retrotransposons, rDNA arrays, and centromeric repeat regions, and accounts for about 30% of the DNA methylation in the genome of Arabidopsis (5, 6).DNA methylation can be removed passively during DNA replication or actively by the DNA glycosylase REPRESSOR OF SILENCING 1 (ROS1) and its homologs DEMETER (DME), DEMETER-LIKE 2 (DML2), and DML3 (1, 2, 7, 8). ROS1 was identified in a genetic screen in which the originally active RD29A-LUC was silenced in ros1 mutants (9). ROS1 is a key DNA demethylation component in vegetative tissues (9), and its transcript level is regulated by MET1 and several RdDM components (10). Screening for second-site suppressors of ros1 mutants by using silenced RD29A-LUC has enabled researchers to identify the main components of the RdDM pathway and has further indicated that RdDM and ROS1 antagonistically regulate DNA methylation (11)(12)(13)(14).ros1 mutations also cause the silencing of another transgene, 35S-NPTII, which is in the same T-DNA construct as RD29A-LUC (9). RD29A-LUC has been used to identify ROS1 and ROS3 genes (9, 15). By screening for second-site suppressors of ros1 using silenced 35S-NPTII, we previously identified genes that are involved in DNA replication and maint...

High-resolution mapping of H4K16 and H3K23 acetylation reveals conserved and unique distribution patterns inArabidopsisand rice

“…14,22,23,[55][56][57][58] This distribution pattern is also in agreement with an anticorrelation of H3K23ac with DNA methylation. 18,59 Surprisingly, we found that H4K16ac and H3K23ac are also enriched across the gene-body of many lowly expressed genes in rice (Fig. 5C and 5D).…”

DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV

“…Although genome-wide analysis of the relationship between H3K9me1/2 and RdDM targets has not been reported, multiple RdDM target loci were shown to associate with H3K9me2 (31,32). Based on these observations we propose a model that DTF1 assists in recruiting Pol IV to genomic regions associated with H3K9me1/2, possibly through the interaction with CLSY1.…”

The second subunit of DNA-polymerase delta is required for genomic stability and epigenetic regulation

“…dms3-4 was identified during a genetic screen for mutants that silence Pro35S:NPTII in a transgenic Arabidopsis line carrying expressed ProRD29A:LUC and Pro35S:NPTII (C24 accession, which was used as the wild type in this study; Li et al, 2012). We performed a forward genetic screen using an ethyl methanesulfonate (EMS)-mutagenized dms3-4 population to isolate the mutants that release the silenced Pro35S:NPTII in dms3-4.…”

“…In a previous study, we performed a genetic screen for additional ROS genes by using a transgenic Arabidopsis line that carries a T-DNA insertion expressing both the ProRD29A:LUC (firefly luciferase reporter driven by the stress-responsive RD29A promoter) gene and the Pro35S:NPTII (neomycin phosphotransferase II driven by the CaMV 35S promoter) gene. Using this screen, we identified several genes in the RNA-directed DNA methylation (RdDM) pathway whose mutations silence Pro35S:NPTII because of the reduced expression of ROS1, indicating that the RdDM pathway positively modulates ROS1 expression (Huettel et al, 2006;Li et al, 2012). One of these mutants isolated from the screen was defective in meristem silencing 3 (dms3-4; Li et al, 2012).…”

“…Using this screen, we identified several genes in the RNA-directed DNA methylation (RdDM) pathway whose mutations silence Pro35S:NPTII because of the reduced expression of ROS1, indicating that the RdDM pathway positively modulates ROS1 expression (Huettel et al, 2006;Li et al, 2012). One of these mutants isolated from the screen was defective in meristem silencing 3 (dms3-4; Li et al, 2012). DMS3 is a silencing factor in RdDM that coordinates the formation of the DDR complex by defective in RNA-directed DNA methylation 1 and RNA-directed DNA methylation 1 and that facilitates Pol V transcript (Law and Jacobsen, 2010).…”