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.     

The mop1 (mediator of paramutation1) mutant progressively reactivates one of the two genes encoded by the MuDR transposon in maize

Transposons make up a sizable portion of most genomes, and most organisms have evolved mechanisms to silence them. In maize, silencing of the Mutator family of transposons is associated with methylation of the terminal inverted repeats (TIRs) surrounding the autonomous element and loss of mudrA expression (the transposase) as well as mudrB (a gene involved in insertional activity). We have previously reported that a mutation that suppresses paramutation in maize, mop1, also hypomethylates Mu1 elements and restores somatic activity to silenced MuDR elements. Here, we describe the progressive reactivation of silenced mudrA after several generations in a mop1 background. In mop1 mutants, the TIRA becomes hypomethylated immediately, but mudrA expression and significant somatic reactivation is not observed until silenced MuDR has been exposed to mop1 for several generations. In subsequent generations, individuals that are heterozygous or wild type for the Mop1 allele continue to exhibit hypomethylation at Mu1 and mudrA TIRs as well as somatic activity and high levels of mudrA expression. Thus, mudrA silencing can be progressively and heritably reversed. Conversely, mudrB expression is never restored, its TIR remains methylated, and new insertions of Mu elements are not observed. These data suggest that mudrA and mudrB silencing may be maintained via distinct mechanisms.     

Tissue-specific patterns of a maize Myb transcription factor are epigenetically regulated

The maize p1 gene encodes a Myb-homologous regulator of red pigment biosynthesis. To investigate the tissue-specific regulation of the p1 gene, maize plants were transformed with constructs combining promoter and cDNA sequences of two alleles which differ in pigmentation patterns: P1-wr (white pericarp/red cob) and P1-rr (red pericarp/red cob). Surprisingly, all promoter/cDNA combinations produced transgenic plants with red pericarp and red cob (RR pattern), indicating that the P1-wr promoter and encoded protein can function in pericarp. Some of the RR patterned transgenic plants produced progeny plants with white pericarp and red cob (WR pattern), and this switch in tissue-specificity correlated with increased transgene methylation. A similar inverse correlation between pericarp pigmentation and DNA methylation was observed for certain natural p1 alleles, which have a gene structure characteristic of standard P1-wr alleles, but which confer red pericarp pigmentation and are consistently less methylated than standard P1-wr alleles. Although we cannot rule out the possible existence of tissue-specific regulatory elements within the p1 non-coding sequences or flanking regions, the data from transgenic and natural alleles suggest that the tissue-specific pigmentation pattern characteristic of the P1-wr phenotype is epigenetically controlled.     

A Mutator transposon insertion is associated with ectopic expression of a tandemly repeated multicopy Myb gene pericarp color1 of maize

The molecular basis of tissue-specific pigmentation of maize carrying a tandemly repeated multicopy allele of pericarp color1 (p1) was examined using Mutator (Mu) transposon-mediated mutagenesis. The P1-wr allele conditions a white or colorless pericarp and a red cob glumes phenotype. However, a Mu-insertion allele, designated as P1-wr-mum6, displayed an altered phenotype that was first noted as occasional red stripes on pericarp tissue. This gain-of-pericarp-pigmentation phenotype was heritable, yielding families that displayed variable penetrance and expressivity. In one fully penetrant family, deep red pericarp pigmentation was observed. Several reports on Mu suppressible alleles have shown that Mu transposons can affect gene expression by mechanisms that depend on transposase activity. Conversely, the P1-wr-mum6 phenotype is not affected by transposase activity. The increased pigmentation was associated with elevated mRNA expression of P1-wr-mum6 copy (or copies) that was uninterrupted by the transposons. Genomic bisulfite sequencing analysis showed that the elevated expression was associated with hypomethylation of a floral-specific enhancer that is approximately 4.7 kb upstream of the Mu1 insertion site and may be proximal to an adjacent repeated copy. We propose that the Mu1 insertion interferes with the DNA methylation and related chromatin packaging of P1-wr, thereby inducing expression from gene copy (or copies) that is otherwise suppressed.     

A natural mutation in rc reverts white-rice-pericarp to red and results in a new, dominant, wild-type allele: Rc-g

The Rc locus regulates pigmentation of the rice bran layer, and selection for the rc allele (white pericarp) occurred during domestication of the crop. White bran is now ubiquitous among cultivated varieties throughout rice growing regions of the world. We identified a new allele that arose by natural mutation within the rc pseudogene of the cultivar 'Wells'. The mutation restored the reading frame of the gene, and reverted the bran layer pigmentation to red (wild-type). By sequencing the Rc locus in plants derived from red seeds, and linkage analysis in a segregating population, we were able to demonstrate that mutation within rc resulted in the new, dominant, wild-type allele Rc-g.     

Progressive Loss of DNA Methylation Releases Epigenetic Gene Silencing From a Tandemly Repeated Maize Myb Gene

Maize pericarp color1 (p1) gene, which regulates phlobaphene biosynthesis in kernel pericarp and cob glumes, offers an excellent genetic system to study tissue-specific gene regulation. A multicopy p1 allele, P1-wr (white pericarp/red cob) is epigenetically regulated. Hypomethylation of P1-wr in the presence of Unstable factor for orange1 (Ufo1), leads to ectopic pigmentation of pericarp and other organs. The Ufo1-induced phenotypes show incomplete penetrance and poor expressivity: gain of pigmentation is observed only in a subset of plants carrying Ufo1 mutation, and the extent of pigmentation is highly variable. We show that Ufo1 induces progressive hypomethylation of P1-wr repeats over generations. After five generations of exposure to Ufo1, a 30–40% decrease in CG and CNG methylation was observed in an upstream enhancer and an intron region of P1-wr. Interestingly, such hypomethylation correlated with an increase in penetrance of the Ufo1-induced pigmentation phenotype from ~27 to 61%. Expressivity of the Ufo1-induced phenotype also improved markedly as indicated by increased uniformity of pericarp pigmentation in the later generations. Furthermore, the poor expressivity of the Uo1 is associated with mosaic methylation patterns of the P1-wr upstream enhancer in individual cells and distinct P1-wr gene copies. Finally, comparison of methylation among different tissues indicated that Ufo1 induces rapid CG and CNG hypomethylation of P1-wr repeats during plant development. Together, these data indicate that the poor penetrance and expressivity of Ufo1-induced phenotypes is caused by mosaicism of methylation, and progressive mitotic hypomethylation leads to improved meiotic heritability of the mutant phenotype. In duplicated genomes like maize, loss of an epigenetic regulator may produce mosaic patterns due to redundancy of epigenetic regulators and their target sequences. We show here that multiple developmental cycles may be required for complete disruption of suppressed epigenetic states and appearance of heritable phenotypes.