Genetic factors required to maintain repression of a paramutagenic maize pl1 allele

A genetic screen identified two novel gene functions required to maintain mitotically and meiotically heritable gene silencing associated with paramutation of the maize purple plant 1 (pl1) locus. Paramutation at pl1 leads to heritable alterations of pl1 gene regulation; the Pl-Rhoades (Pl-Rh) allele, which typically confers strong pigmentation to juvenile and adult plant structures, changes to a lower expression state termed Pl'-mahogany (Pl'). Paramutation spontaneously occurs at low frequencies in Pl-Rh homozygotes but always occurs when Pl-Rh is heterozygous with Pl'. We identified four mutations that caused increased Pl' pigment levels. Allelism tests revealed that three mutations identified two new maize loci, required to maintain repression 1 (rmr1) and rmr2 and that the other mutation represents a new allele of the previously described mediator of paramutation 1 (mop1) locus. RNA levels from Pl' are elevated in rmr mutants and genetic tests demonstrate that Pl' can heritably change back to Pl-Rh in rmr mutant individuals at variable frequencies. Pigment levels controlled by two pl1 alleles that do not participate in paramutation are unaffected in rmr mutants. These results suggest that RMR functions are intimately involved in maintaining the repressed expression state of paramutant Pl' alleles. Despite strong effects on Pl' repression, rmr mutant plants have no gross developmental abnormalities even after several generations of inbreeding, implying that RMR1 and RMR2 functions are not generally required for developmental homeostasis.     

Paramutation in maize

Paramutation is a heritable change in gene expression induced by allele interactions. This review summarizes key experiments on three maize loci, which undergo paramutation. Similarities and differences between the phenomenology at the three loci are described. In spite of many differences with respect to the stability of the reduced expression states at each locus or whether paramutation correlates with DNA methylation and repeated sequences within the loci, recent experiments are consistent with a common mechanism underlying paramutation at all three loci. Most strikingly, trans-acting mutants have been isolated that prevent paramutation at all three loci and lead to the activation of silenced Mutator transposable elements. Models consistent with the hypothesis that paramutation involves heritable changes in chromatin structure are presented. Several potential roles for paramutation are discussed. These include localizing recombination to low-copy sequences within the genome, establishing and maintaining chromatin domain boundaries, and providing a mechanism for plants to transmit an environmentally influenced expression state to progeny.     

Epigenetic inheritance of transcriptional states in S. cerevisiae

SIR1, one of several genes required for repression of yeast silent mating type loci, has a unique role in repression of the HML alpha locus. Single-cell assays revealed that cells with mutant alleles of SIR1, including presumptive null alleles, existed as populations of genetically identical cells whose members were in one of two different regulatory states. A minority of cells had a repressed HML alpha locus whereas the majority had a derepressed HML alpha locus. The two states were mitotically stable, although rare changes in state were observed during mitotic growth, possibly reflecting heritable changes to the HML alpha locus at or before replication. Analysis of changes in state suggests that SIR1 protein has a role in the establishment but not the maintenance of repression of silent mating type genes, whereas SIR2, SIR3, and SIR4 are required for maintenance.     

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.     

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.     

The maize b1 paramutation control region causes epigenetic silencing in Drosophila melanogaster

Paramutation is an epigenetic process in which a combination of alleles in a heterozygous organism results in a meiotically stable change in expression of one of the alleles. The mechanisms underlying paramutation are being actively investigated, and examples have been described in both plants and mammals, suggesting that it may utilize epigenetic mechanisms that are widespread and evolutionarily conserved. Paramutation at the well-studied maize b1 locus requires a control region consisting of seven 853 bp tandem repeats. To study the conservation of the epigenetic mechanisms underlying seemingly unique epigenetic processes such as paramutation, we created transgenic Drosophila melanogaster carrying the maize b1 control region adjacent to the Drosophila white reporter gene. We show that the b1 tandem repeats cause silencing of the white reporter in Drosophila. A single copy of the tandem repeat sequence is sufficient to cause silencing, and silencing strength increases as the number of tandem repeats increases. Additionally, transgenic lines with the full seven tandem repeats demonstrate evidence of either pairing-sensitive silencing and silencing in trans, or epigenetic activation in trans. These trans-interactions are dependent on repeat number, similar to maize b1 paramutation. Also, as in maize, the tandem repeats are bidirectionally transcribed in Drosophila. These results indicate that the maize b1 tandem repeats function as an epigenetic silencer and mediate trans-interactions in Drosophila, and support the hypothesis that the mechanisms underlying such epigenetic processes are conserved.