Author Summary How an individual’s genes are activated or silenced is an essential question impacting all fields of biology. Usually gene expression patterns, i.e., which genes are on and which are off in different tissues and during development, are highly reproducible; and those patterns are efficiently reset in the next generation of progeny. Paramutation represents an exception to these genetic rules, in that for certain genes the silencing that is established in an individual is efficiently transmitted to their progeny. Importantly, in these subsequent generations, the silenced gene continues to silence active versions of that gene. Prior work has demonstrated that these heritable gene expression changes are not accompanied by changes in DNA sequence: they are epigenetic. Understanding mechanisms for heritable changes in gene expression has major implications for researchers studying complex traits, including diseases. In this manuscript we demon- strate that a subunit of a RNA polymerase is required for paramutation in maize and other gene silencing processes that also involve RNA–mediated chromatin changes. We show that the multiple, closely related, plant-specific RNA polymerases mediating gene silencing have diverged functions in maize. Results from our experiments suggest testable models for the role of these polymerases in multiple gene-silencing processes. and inverted repeats and the strength of paramutation correlates with repeat number [14]; paramutable alleles have inverted repeats [15–18]. Thus, while there are no sequence similarities between the regions that mediate paramutation at these distinct loci, a common theme is the presence of direct or inverted repeat sequences. Mutations that alter paramutation have been isolated using screens with either the b1 or pl1 paramutation systems [19–21]. Several of the genes identified in these paramutation screens have been cloned and to date all share homology with genes in Arabidopsis that mediate RNAi transcriptional silencing of transgenes or endogenous genes. The first cloned gene required for paramutation was mediator of paramutation 1 (mop1), which encodes a RNA dependent RNA polymerase most similar to Arabidopsis RDR2 [22] that mediates heterochromatic silencing of repeats through 24 nt siRNAs [23]. In addition to preventing paramutation at multiple loci and increasing the transcription of paramutated alleles [20], mop1 mutations also reactivate Mutator transposons [24–26] and transcriptionally silenced transgenes [27]. The second gene cloned, required to maintain repression1 (rmr1), encodes a SNF2-like ATPase [28], a factor similar to, but distinct from Arabidopsis DRD1 (Defective in RNA Directed DNA methylation1) involved in RNAi-mediated transcriptional silencing [29] and CLSY1 (CLASSY1) involved in RNA signal spreading [30]. The rmr1 mutation increases the expression of previously silenced pl1 and b1 alleles, but does not prevent paramutation at pl1 [19,28] or b1 (V. Chandler, unpublished data), suggesting it is involved in maintaining the silenced epigenetic states. While rmr1 mutations can also reactivate transcriptionally silenced transgenes, these transgenes are efficiently resilenced upon the introduction of a wild type allele [27], in contrast to mop1 mutations in which the reactivated transgenes can remain heritably active even when the wild type allele is reintroduced [27]. The third gene cloned, rmr6, encodes the largest subunit of the plant specific DNA-dependent RNA polymerase most similar to Arabidopsis NRPD1 [31], the largest subunit of the Pol-IV complex required for primary siRNA biogenesis [32,33]. Mutations in rmr6 cause dramatic develop- mop2 Is Required for Multiple Silencing Processes mental phenotypes and prevent paramutation at pl1, b1, and r1 as well as relieve silencing of paramutant alleles [21]. The genes cloned to date, when mutated, show a dramatic reduction in 24 nt siRNAs normally associated with heterochromatic silencing of repeated sequences [31,34]. In Arabidopsis the related RNAi heterochromatic silencing pathway is often referred to as RdDM (RNA directed DNA Methylation); the transcriptional silencing requires small RNA biogenesis and targets homologous promoters with DNA methyl- ation and repressive histone modifications [35–37]. This pathway in Arabidopsis mediates transcriptional silencing by transgenes in which inverted repeats of promoters are transcribed to generate dsRNA (referred to a pIR transgenes) [38]. The dsRNA is processed to 24 nt siRNAs, leading to DNA and chromatin modifications and silencing of any endogenous gene or transgene sharing homologous promoter sequences [36–39]. This pathway also mediates de novo DNA methylation and silencing of several endogenous genes associated with tandem repeats [40]. The current model for the Arabidopsis RNAi heterochromatic silencing pathway involves the genes identified in maize discussed above as well as other factors. Pol-IV is required for siRNA biogenesis and is thought to mediate the synthesis of non-coding RNAs at multiple repetitive endogenous loci using either double- stranded DNA, or single- or double-stranded RNA as a template [for a review, see [37]]. The resulting single stranded RNA is postulated to be used by the RNA dependent RNA polymerase, encoded by RDR2 [23], to generate double stranded RNA molecules that are then diced into double stranded 24 nt siRNAs by an RNAseIII-like endonuclease, encoded by Dicer-like 3 (DCL3) [23]. Transgenes or endogenous genes that can produce dsRNA via strong Pol-II promoters, such as pIR transgenes, do not require Pol-IV or RDR2 for silencing [41]. Another plant-specific Pol-II related polymerase complex, Pol-V [42], associates with target DNA with the help of the SNF2-like ATP-dependent chromatin remodeler, encoded by DRD1 (RNA-directed DNA methylation1) [43], and a hinge protein, DMS3 [44]. Pol-V produces transcripts regardless of the presence or absence of the 24 nt siRNA signal [45,46]. Nascent Pol-V RNA transcripts associate with an RNA binding protein KTF1 (KOW domain containing transcription factor 1) [47] and recruit the ARGONAUTE4 (AGO4) protein [48], which is complexed with a guiding strand of complementary 24 nt siRNA. The AGO4 complex then recruits the de-novo DNA methylation enzyme DRM2 (domain rearranged methytrasfer- ase2) [45–47,49], and histone modification factors such as HDA6 (Histone deacetylase6), and a histone methyltransferase, KYP (Kryptonite), to establish and reinforce silencing at target loci [50,51]. The requirements of a RDR2-like RNA dependent RNA Polymerase encoded by mop1 [22], a NRPD1-like large subunit of Pol-IV encoded by rmr6 [31], and the SNF2-like factor encoded by rmr1 [28], coupled with the requirements for transcribed tandem repeats to mediate b1 paramutation [11,22], loss of siRNAs in several paramutation mutants [31,34], and associated chromatin differences between paramutagenic and paramutable b1 and p1 alleles [4,52,53], suggest that paramutation involves a mechanism similar to the RdDM pathway in Arabidopsis. However, paramutation has properties that are distinct from RdDM [8,40]. Most dramatically, the silencing associated with paramuta- tion is highly heritable after the paramutagenic allele is segregated away and the newly silenced allele itself becomes paramutagenic in subsequent generations. These characteristics do not occur with RdDM in Arabidopsis; in most instances expression of the targeted loci returns to normal after the inducing transgene (or locus) is segregated away. Even in the examples of heritable silencing at the PLoS Genetics | www.plosgenetics.org 2 November 2009 | Volume 5 | Issue 11 | e1000725