Paramutation at the maize pl1 locus is associated with RdDM activity at distal tandem repeats.

Exceptions to Mendelian inheritance often highlight novel chromosomal behaviors. The maize Pl1-Rhoades allele conferring plant pigmentation can display inheritance patterns deviating from Mendelian expectations in a behavior known as paramutation. However, the chromosome features mediating such exceptions remain unknown. Here we show that small RNA production reflecting RNA polymerase IV function within a distal downstream set of five tandem repeats is coincident with meiotically-heritable repression of the Pl1-Rhoades transcription unit. A related pl1 haplotype with three, but not one with two, repeat units also displays the trans-homolog silencing typifying paramutations. 4C interactions, CHD3a-dependent small RNA profiles, nuclease sensitivity, and polyadenylated RNA levels highlight a repeat subregion having regulatory potential. Our comparative and mutant analyses show that transcriptional repression of Pl1-Rhoades correlates with 24-nucleotide RNA production and cytosine methylation at this subregion indicating the action of a specific DNA-dependent RNA polymerase complex. These findings support a working model in which pl1 paramutation depends on trans-chromosomal RNA-directed DNA methylation operating at a discrete cis-linked and copy-number-dependent transcriptional regulatory element.

Paramutation and development.

Paramutation describes a heritable change of gene expression that is brought about through interactions between homologous chromosomes. Genetic analyses in plants and, more recently, in mouse indicate that genomic sequences related to transcriptional control and molecules related to small RNA biology are necessary for specific examples of paramutation. Some of the molecules identified in maize are also required for normal plant development. These observations indicate a functional relationship between the nuclear mechanisms responsible for paramutation and modes of developmental gene control.

Paramutation and related phenomena in diverse species.

Paramutation describes a process that results in heritable epigenetic changes of gene regulation and trans-homologue interactions. Recent discoveries in model organisms have highlighted roles for the respective nuclear systems that regulate transposons via small RNA molecules both for paramutation and for defining transgenerational inheritance. Differences between plants and animals may influence specific transmission behaviours but the involvement of small RNA-based mechanisms identifies a unifying eukaryotic theme. These mechanisms that specify heritable epigenetic information represent genetic systems adjunct to DNA sequences that contribute to phenotypic diversity.

Locus-specific paramutation in Zea mays is maintained by a PICKLE-like chromodomain helicase DNA-binding 3 protein controlling development and male gametophyte function.

Paramutations represent directed and meiotically-heritable changes in gene regulation leading to apparent violations of Mendelian inheritance. Although the mechanism and evolutionary importance of paramutation behaviors remain largely unknown, genetic screens in maize (Zea mays) identify five components affecting 24 nucleotide RNA biogenesis as required to maintain repression of a paramutant purple plant1 (pl1) allele. Currently, the RNA polymerase IV largest subunit represents the only component also specifying proper development. Here we identify a chromodomain helicase DNA-binding 3 (CHD3) protein orthologous to Arabidopsis (Arabidopsis thaliana) PICKLE as another component maintaining both pl1 paramutation and normal somatic development but without affecting overall small RNA biogenesis. In addition, genetic tests show this protein contributes to proper male gametophyte function. The similar mutant phenotypes documented in Arabidopsis and maize implicate some evolutionarily-conserved gene regulation while developmental defects associated with the two paramutation mutants are largely distinct. Our results show that a CHD3 protein responsible for normal plant ontogeny and sperm transmission also helps maintain meiotically-heritable epigenetic regulatory variation for specific alleles. This finding implicates an intersection of RNA polymerase IV function and nucleosome positioning in the paramutation process.

Paramutation in maize and related behaviors in metazoans.

Paramutation refers to both the process and results of trans-homolog interactions causing heritable changes in both gene regulation and silencing abilities. Originally described in plants, paramutation-like behaviors have now been reported in model metazoans. Here we detail our current understanding of the paramutation mechanism as defined in Zea mays and compare this paradigm to these metazoan examples. Experimental results implicate functional roles of small RNAs in all these model organisms that highlight a diversity of mechanisms by which these molecules specify meiotically heritable regulatory information in the eukarya.

Maize RNA polymerase IV defines trans-generational epigenetic variation.

The maize (Zea mays) RNA Polymerase IV (Pol IV) largest subunit, RNA Polymerase D1 (RPD1 or NRPD1), is required for facilitating paramutations, restricting expression patterns of genes required for normal development, and generating small interfering RNA (siRNAs). Despite this expanded role for maize Pol IV relative to Arabidopsis thaliana, neither the general characteristics of Pol IV-regulated haplotypes, nor their prevalence, are known. Here, we show that specific haplotypes of the purple plant1 locus, encoding an anthocyanin pigment regulator, acquire and retain an expanded expression domain following transmission from siRNA biogenesis mutants. This conditioned expression pattern is progressively enhanced over generations in Pol IV mutants and then remains heritable after restoration of Pol IV function. This unusual genetic behavior is associated with promoter-proximal transposon fragments but is independent of sequences required for paramutation. These results indicate that trans-generational Pol IV action defines the expression patterns of haplotypes using co-opted transposon-derived sequences as regulatory elements. Our results provide a molecular framework for the concept that induced changes to the heterochromatic component of the genome are coincident with heritable changes in gene regulation. Alterations of this Pol IV-based regulatory system can generate potentially desirable and adaptive traits for selection to act upon.

required to maintain repression2 is a novel protein that facilitates locus-specific paramutation in maize.

Meiotically heritable epigenetic changes in gene regulation known as paramutations are facilitated by poorly understood trans-homolog interactions. Mutations affecting paramutations in maize (Zea mays) identify components required for the accumulation of 24-nucleotide RNAs. Some of these components have Arabidopsis thaliana orthologs that are part of an RNA-directed DNA methylation (RdDM) pathway. It remains unclear if small RNAs actually mediate paramutations and whether the maize-specific molecules identified to date define a mechanism distinct from RdDM. Here, we identify a novel protein required for paramutation at the maize purple plant1 locus. This required to maintain repression2 (RMR2) protein represents the founding member of a plant-specific clade of predicted proteins. We show that RMR2 is required for transcriptional repression at the Pl1-Rhoades haplotype, for accumulation of 24-nucleotide RNA species, and for maintenance of a 5-methylcytosine pattern distinct from that maintained by RNA polymerase IV. Genetic tests indicate that RMR2 is not required for paramutation occurring at the red1 locus. These results distinguish the paramutation-type mechanisms operating at specific haplotypes. The RMR2 clade of proteins provides a new entry point for understanding the diversity of epigenomic control operating in higher plants.

Production and processing of siRNA precursor transcripts from the highly repetitive maize genome.

Mutations affecting the maintenance of heritable epigenetic states in maize identify multiple RNA-directed DNA methylation (RdDM) factors including RMR1, a novel member of a plant-specific clade of Snf2-related proteins. Here we show that RMR1 is necessary for the accumulation of a majority of 24 nt small RNAs, including those derived from Long-Terminal Repeat (LTR) retrotransposons, the most common repetitive feature in the maize genome. A genetic analysis of DNA transposon repression indicates that RMR1 acts upstream of the RNA-dependent RNA polymerase, RDR2 (MOP1). Surprisingly, we show that non-polyadenylated transcripts from a sampling of LTR retrotransposons are lost in both rmr1 and rdr2 mutants. In contrast, plants deficient for RNA Polymerase IV (Pol IV) function show an increase in polyadenylated LTR RNA transcripts. These findings support a model in which Pol IV functions independently of the small RNA accumulation facilitated by RMR1 and RDR2 and support that a loss of Pol IV leads to RNA Polymerase II-based transcription. Additionally, the lack of changes in general genome homeostasis in rmr1 mutants, despite the global loss of 24 nt small RNAs, challenges the perceived roles of siRNAs in maintaining functional heterochromatin in the genomes of outcrossing grass species.

Diversity of Pol IV function is defined by mutations at the maize rmr7 locus.

Mutations affecting the heritable maintenance of epigenetic states in maize identify multiple small RNA biogenesis factors including NRPD1, the largest subunit of the presumed maize Pol IV holoenzyme. Here we show that mutations defining the required to maintain repression7 locus identify a second RNA polymerase subunit related to Arabidopsis NRPD2a, the sole second largest subunit shared between Arabidopsis Pol IV and Pol V. A phylogenetic analysis shows that, in contrast to representative eudicots, grasses have retained duplicate loci capable of producing functional NRPD2-like proteins, which is indicative of increased RNA polymerase diversity in grasses relative to eudicots. Together with comparisons of rmr7 mutant plant phenotypes and their effects on the maintenance of epigenetic states with parallel analyses of NRPD1 defects, our results imply that maize utilizes multiple functional NRPD2-like proteins. Despite the observation that RMR7/NRPD2, like NRPD1, is required for the accumulation of most siRNAs, our data indicate that different Pol IV isoforms play distinct roles in the maintenance of meiotically-heritable epigenetic information in the grasses.

A novel Snf2 protein maintains trans-generational regulatory states established by paramutation in maize.

Paramutations represent heritable epigenetic alterations that cause departures from Mendelian inheritance. While the mechanism responsible is largely unknown, recent results in both mouse and maize suggest paramutations are correlated with RNA molecules capable of affecting changes in gene expression patterns. In maize, multiple required to maintain repression (rmr) loci stabilize these paramutant states. Here we show rmr1 encodes a novel Snf2 protein that affects both small RNA accumulation and cytosine methylation of a proximal transposon fragment at the Pl1-Rhoades allele. However, these cytosine methylation differences do not define the various epigenetic states associated with paramutations. Pedigree analyses also show RMR1 does not mediate the allelic interactions that typically establish paramutations. Strikingly, our mutant analyses show that Pl1-Rhoades RNA transcript levels are altered independently of transcription rates, implicating a post-transcriptional level of RMR1 action. These results suggest the RNA component of maize paramutation maintains small heterochromatic-like domains that can affect, via the activity of a Snf2 protein, the stability of nascent transcripts from adjacent genes by way of a cotranscriptional repression process. These findings highlight a mechanism by which alleles of endogenous loci can acquire novel expression patterns that are meiotically transmissible.

Sensing the epigenome.

Recent studies of plant development and environmental stress responses have converged on the roles of RNA and its metabolism as primary regulators of gene action. This RNA-based system appears to represent a versatile platform both for maintaining epigenetic memory and for reprogramming gene control in response to external signals. The fast-paced research reviewed here highlights exciting new trends in plant research relating to mechanisms and roles of the RNA-dependent epigenome in both development and evolution.

Multiple trans-sensing interactions affect meiotically heritable epigenetic states at the maize pl1 locus.

Interactions between specific maize purple plant1 (pl1) alleles result in heritable changes of gene regulation that are manifested as differences in anthocyanin pigmentation. Transcriptionally repressed states of Pl1-Rhoades alleles (termed Pl') are remarkably stable and invariably facilitate heritable changes of highly expressed states (termed Pl-Rh) in Pl'/Pl-Rh plants. However, Pl' can revert to Pl-Rh when hemizygous, when heterozygous with pl1 alleles other than Pl1-Rhoades, or in the absence of trans-acting factors required to maintain repressed states. Cis-linked features of Pl1-Rhoades responsible for these trans-sensing behaviors remain unknown. Here, genetic tests of a pl1 allelic series identify two potentially separate cis-linked features: one facilitating repression of Pl-Rh and another stabilizing Pl' in trans. Neither function is affected in ethyl-methanesulfonate-induced Pl1-Rhoades derivatives that produce truncated PL1 peptides, indicating that PL1 is unlikely to mediate trans interactions. Both functions, however, are impaired in a spontaneous Pl1-Rhoades derivative that fails to produce detectable pl1 RNA. Pl'-like states can also repress expression of a pl1-W22 allele, but this repression is not meiotically heritable. As the Pl' state is not associated with unique small RNA species representing the pl1-coding region, the available data suggest that interactions between elements required for transcription underlie Pl1-Rhoades epigenetic behaviors.

Rmr6 maintains meiotic inheritance of paramutant states in Zea mays.

Paramutation generates heritable changes affecting regulation of specific alleles found at several Zea mays (maize) loci that encode transcriptional regulators of anthocyanin biosynthetic genes. Although the direction and extent of paramutation is influenced by poorly understood allelic interactions occurring in diploid sporophytes, two required to maintain repression loci (rmr1 and rmr2), as well as mediator of paramutation1 (mop1), affect this process at the purple plant1 (pl1) locus. Here we show that the rmr6 locus is required for faithful transmission of weakly expressed paramutant states previously established at both pl1 and red1 (r1) loci. Transcriptional repression occurring at both pl1 and booster1 (b1) loci as a result of paramutation also requires Rmr6 action. Reversions to highly expressed, nonparamutant states at both r1 and pl1 occur in plants homozygous for rmr6 mutations. Pedigree analysis of reverted pl1 alleles reveals variable latent susceptibilities to spontaneous paramutation in future generations, suggesting a quantitative nature of Rmr6-based alterations. Genetic tests demonstrate that Rmr6 encodes a common component required for establishing paramutations at diverse maize loci. Our analyses at pl1 and r1 suggest that this establishment requires Rmr6-dependent somatic maintenance of meiotically heritable epigenetic marks.

Nascent transcription affected by RNA polymerase IV in Zea mays.

All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3'-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance.

Paramutation: a trans-homolog interaction affecting heritable gene regulation.

Paramutation describes both the process and results of trans-sensing between chromosomes that causes specific heritable changes in gene regulation. RNA molecules are implicated in mediating similar events in maize, mouse, and Drosophila. Changes in both small RNA profiles and cytosine methylation patterns in Arabidopsis hybrids represent a potential molecular equivalent to the interactions responsible for paramutations. Despite a seemingly unifying feature of RNA-directed changes, both recent and historical works show that paramutations in maize require plant-specific proteins and lack expected hallmarks of a trans-effect mediated solely by RNAs. Recent examples of nearby transposons affecting RNA polymerase II functions lead to an opinion that paramutations represent an emergent property of the transcriptional dynamics ongoing in plant genomes between repetitious features and nearby genes.

Paramutation: a process for acquiring trans-generational regulatory states.

Basic tenets of Mendelian inheritance are violated by paramutations in which trans-homolog interactions lead to heritable changes in gene regulation and phenotype. First described in plants, similar behaviors have now been noted in diverse eukaryotes. Genetic and molecular studies of paramutations occurring in maize indicate that components of a small interfering RNA (siRNA) biogenesis pathway are required for the maintenance of meiotically heritable regulatory states. Although these findings lead to a hypothesis that siRNAs themselves mediate paramutation interactions, an assessment of existing data supports the opinion that siRNAs alone are insufficient. Recent evidence implies that transcription of paramutation-associated repeats and siRNA-facilitated chromatin changes at affected loci are involved in directing and maintaining the heritable changes in gene regulation that typify paramutations.

RNA polymerase IV functions in paramutation in Zea mays.

Plants have distinct RNA polymerase complexes (Pol IV and Pol V) with largely unknown roles in maintaining small RNA-associated gene silencing. Curiously, the eudicot Arabidopsis thaliana is not affected when either function is lost. By use of mutation selection and positional cloning, we showed that the largest subunit of the presumed maize Pol IV is involved in paramutation, an inherited epigenetic change facilitated by an interaction between two alleles, as well as normal maize development. Bioinformatics analyses and nuclear run-on transcription assays indicate that Pol IV does not engage in the efficient RNA synthesis typical of the three major eukaryotic DNA-dependent RNA polymerases. These results indicate that Pol IV employs abnormal RNA polymerase activities to achieve genome-wide silencing and that its absence affects both maize development and heritable epigenetic changes.

Maize sex determination and abaxial leaf fates are canalized by a factor that maintains repressed epigenetic states.

In maize (Zea mays ssp. mays), the meiotically heritable maintenance of specific transcriptionally repressed epigenetic states is facilitated by a putative RNA-dependent RNA polymerase encoded by mediator of paramutation1 (mop1) and an unknown factor encoded by the required to maintain repression6 (rmr6) locus. These so-called "paramutant" states occur at certain alleles of loci encoding regulators of anthocyanin pigment biosynthesis. Here we show Rmr6 acts to canalize leaf and inflorescence development by prohibiting the ectopic action of key developmental regulators. Phenotypic and genetic analyses suggest that Rmr6 ensures proper adaxial-abaxial polarity of the leaf sheath by limiting the expression domain of a putative adaxializing factor. Similar tests indicate that Rmr6 maintains maize's monoecious pattern of sex determination by restricting the function of the pistil-protecting factor, silkless1, from the apical inflorescence. Phenotypic similarities with mop1 mutant plants together with current models of heterochromatin maintenance and leaf polarity imply Rmr6 functions to maintain epigenetic repression established by non-coding small RNA molecules.