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.     

Ac insertion site affects the frequency of transposon-induced homologous recombination at the maize p1 locus

The maize p1 gene regulates the production of a red pigment in the kernel pericarp, cob, and other maize floral tissues. Insertions of the transposable element Ac can induce recombination between two highly homologous 5.2-kb direct repeat sequences that flank the p1 gene-coding region. Here, we tested the effects of the Ac insertion site and orientation on the induction of recombination at the p1 locus. A collection of unique p1 gene alleles was used, which carry Ac insertions at different sites in and near the p1 locus, outside of the direct repeats, within the direct repeat sequences, and between the direct repeats, in both orientations. Recombination was scored by the numbers of colorless pericarp sectors (somatic frequency) and heritable mutations (germinal frequency). In both the somatic and germinal tests, the frequency of homologous recombination is significantly higher when Ac is inserted between the direct repeats than when Ac is inserted either within or outside the repeats. In contrast, Ac orientation had no significant effect on recombination frequency. We discuss these results in terms of the possible mechanisms of transposon-induced recombination.     

Ac Induces Homologous Recombination at the Maize P Locus

The maize P gene conditions red phlobaphene pigmentation to the pericarp and cob. Starting from two unstable P alleles which carry insertions of the transposable element Ac, we have derived 51 P null alleles; 47 of the 51 null alleles have a 17-kb deletion which removes the 4.5-kb Ac element and 12.5 kb of P sequences flanking both sides of Ac. The deletion endpoints lie within two 5.2-kb homologous direct repeats which flank the P gene. A P allele which contains the direct repeats, but does not have an Ac insertion between the direct repeats, shows very little sporophytic or gametophytic instability. The apparent frequency of sporophytic mutations was not increased when Ac was introduced in trans. Southern analysis of DNA prepared from the pericarp tissue demonstrates that the deletions can occur premeiotically, in the somatic cells during development of the pericarp. Evidence is presented that the deletions occurred by homologous recombination between the two direct repeats, and that the presence of an Ac element at the P locus is associated with the recombination/deletion. These results add another aspect to the spectrum of activities of Ac: the destabilization of flanking direct repeat sequences.     

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.     

Comparative Structural and Functional Characterization of Sorghum and Maize Duplications Containing Orthologous Myb Transcription Regulators of 3-Deoxyflavonoid Biosynthesis

Sequence characterization of the genomic region of sorghum yellow seed 1 shows the presence of two genes that are arranged in a head to tail orientation. The two duplicated gene copies, y1 and y2 are separated by a 9.084 kbp intergenic region, which is largely composed of highly repetitive sequences. The y1 is the functional copy, while the y2 may represent a pseudogene; there are several sequence indels and rearrangements within the putative coding region of y2. The y1 gene encodes a R2R3 type of Myb domain protein that regulates the expression of chalcone synthase, chalcone isomerase and dihydroflavonol reductase genes required for the biosynthesis of 3-deoxyflavonoids. Expression of y1 can be observed throughout the plant and it represents a combination of expression patterns produced by different alleles of the maize p1. Comparative sequence analysis within the coding regions and flanking sequences of y1, y2 and their maize and teosinte orthologs show local rearrangements and insertions that may have created modified regulatory regions. These micro-colinearity modifications possibly are responsible for differential patterns of expression in maize and sorghum floral and vegetative tissues. Phylogenetic analysis indicates that sorghum y1 and y2 sequences may have arisen by gene duplication mechanisms and represent an evolutionarily parallel event to the duplication of maize p2 and p1 genes.     

Transposon-induced inversions activate gene expression in the maize pericarp

Chromosomal inversions can have considerable biological and agronomic impacts including disrupted gene function, change in gene expression, and inhibited recombination. Here, we describe the molecular structure and functional impact of six inversions caused by Alternative Transpositions between p1 and p2 genes responsible for floral pigmentation in maize. In maize line p1-wwB54, the p1 gene is null and the p2 gene is expressed in anther and silk but not in pericarp, making the kernels white. By screening for kernels with red pericarp, we identified inversions in this region caused by transposition of Ac and fractured Ac (fAc) transposable elements. We hypothesize that these inversions place the p2 gene promoter near a p1 gene enhancer, thereby activating p2 expression in kernel pericarp. To our knowledge, this is the first report of multiple recurrent inversions that change the position of a gene promoter relative to an enhancer to induce ectopic expression in a eukaryote.     

Insertional Mutagenesis of the Maize P Gene by Intragenic Transposition of Ac

The P-rr allele of the maize P gene regulates the synthesis of pigments derived from flavan-4-ol in the pericarp, cob glumes and other floral organs. We characterized 21 P alleles derived by intragenic transposition of Ac from three known positions. Ac transpositions can occur in either direction in the P gene, and with no apparent minimum distance: in one case Ac transposed just 6 bp from its original insertion site. However, the distribution of transposed Ac elements was markedly nonrandom: of 19 transposed Ac elements derived from a single Ac donor, 15 were inserted in a 1.1-kb region at the 5' end of P, while none had inserted in an adjacent 3.2-kb intronic region. All of the Ac insertions affect both pericarp and cob glume pigmentation, providing further evidence that the P-rr allele contains a single gene required for both pericarp and cob glume pigmentation. The distribution of the inserted Ac elements and the phenotype conditioned by each allele suggests a structure of P-rr which is similar to that previously determined molecularly. Possible explanations for the nonrandom distribution of transposed Ac elements are discussed.