IDN2 has a role downstream of siRNA formation in RNA-directed DNA methylation

Abstract

In plants, a particular class of short interfering (si)RNAs can serve as a signal to induce cytosine methylation at homologous genomic regions. If the targeted DNA has promoter function, this RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing (TGS). RNA-directed transcriptional gene silencing (RdTGS) of transgenes provides a versatile system for the study of epigenetic gene regulation. We used transcription of a nopaline synthase promoter (ProNOS)-inverted repeat (IR) to provide a RNA signal that triggers de novo cytosine methylation and TGS of a homologous ProNOS copy in trans. Utilizing a ProNOS-NPTII reporter gene showing high sensitivity to silencing in this two component system, a forward genetic screen for EMS-induced no rna-directed transcriptional silencing (nrd) mutations was performed in Arabidopsis thaliana. Three nrd mutant lines were found to contain one novel loss-of-function allele of idn2/rdm12 and two of nrpd2a/nrpe2a. IDN2/RDM12 encodes a XH/XS domain protein that is able to bind double-stranded RNA with 5′ overhangs, while NRPD2a/NRPE2a encodes the common second-largest subunit of the plant specific DNA-dependent RNA polymerases IV and V involved in silencing processes. Both idn2/rdm12 and nrpd2a/nrpe2a release target transgene expression and reduce CHH context methylation at transgenic as well as endogenous RdDM target regions to similar extents. Nevertheless, accumulation of IR-derived siRNA is not affected, allowing us to present a refined model for the pathway of RdDM and RdTGS that positions function of IDN2 downstream of siRNA formation and points to an important role for its XH domain.

Distinct and concurrent pathways of Pol II‐ and Pol IV‐dependent siRNA biogenesis at a repetitive trans‐silencer locus in Arabidopsis thaliana

“…The hairpin RNA is processed redundantly by several DCL enzymes, including DCL3, into siRNAs of 21-24 nt (Dunoyer et al, 2005;Kanno et al, 2008;Daxinger et al, 2009). Transgene IRs are frequently used to silence genes at the transcriptional (Eamens et al, 2008;Kanno et al, 2008;Finke et al, 2012) and post-transcriptional levels (Dunoyer et al, 2005;Fusaro et al, 2006;Molnar et al, 2010). In Arabidopsis thaliana (Arabidopsis), several endogenous IRs that give rise to hairpin-derived siRNAs have been described (Henderson et al, 2006;Zhang et al, 2007;Dunoyer et al, 2010).…”

Interplay among RNA polymerases II, IV and V in RNA-directed DNA methylation at a low copy transgene locus in Arabidopsis thaliana

“…A number of studies have shown that 21–24-nt hairpin-derived siRNAs, which depend on Pol II transcription of inverted DNA repeats, can act in trans to trigger DNA methylation of unlinked homologous target sequences (Kanno et al 2005, 2008; Eamens et al 2008; Finke et al 2012; this study). It has not been clear, however, whether Pol IV/RDR2-dependent 24-nt siRNAs, which can induce methylation in cis at the site where they are generated (Lister et al 2008; Daxinger et al 2009), can similarly elicit RdDM in trans .…”

DNA methylation maintenance consolidates RNA‐directed DNA methylation and transcriptional gene silencing over generations in Arabidopsis thaliana

“…We performed a screen for ethyl methanesulfonate‐induced mutants ( no rna‐directed transcriptional silencing , nrd ) in which expression of a silenced reporter transgene is reactivated (Finke et al ., ). The line K/K ; H/H that was subjected to mutagenesis was doubly homozygous for a SILENCER ( H ) transgene on chromosome 4 containing an inverted repeat (IR) of the NOPALIN SYNTHASE promoter ( ProNOS ) sequence under the control of the CaMV 35S promoter ( Pro35 ) (Aufsatz et al ., ) and a TARGET transgene ( K ) ( K chr1–10 in Fischer et al ., ) on chromosome 1 containing a NEOMYCIN PHOSPHOTRANSFERASE II ( NPTII ) reporter gene conferring resistance to kanamycin and controlled by a ProNOS sequence (Figure a).…”