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

Abstract

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.

Tissue Culture-Induced Novel Epialleles of aMybTranscription Factor Encoded bypericarp color1in Maize

“…Since multiple P1-wr repeats are thought to be functional (Chopra et al 1998(Chopra et al , 2003Robbins et al 2008;Sekhon and Chopra 2009), common mutational mechanisms disrupting functionality of one of the repeats (e.g., mutagenesis with ethyl methanesulfonate) are unlikely to produce loss-of-function alleles. The unexpected frequent appearance of p1 mutant alleles (four of eight novel alleles analyzed for complementation) among this material indicates that (1) the P1-wr allele is hypersensitive to mutagenesis in culture, and (2) the mechanism of mutagenesis must affect multiple copies of the gene simultaneously.…”

“…Multiple alleles and epialleles of p1 are identified by accumulation of phlobaphenes in pericarp and cob glumes (Brink and Styles 1966;Chopra et al 1998); for example, P1-wr specifies white pericarp and red cob glumes, while p1-ww has white pericarp and white cob glumes. p1 is particularly sensitive to epigenetic modifications, and several examples of altered expression patterns due to epigenetic suppression have been reported (Das and Messing 1994;Cocciolone et al 2001;Sekhon et al 2007;Robbins et al 2008;Sekhon and Chopra 2009). Epigenetic silencing of the p1 alleles and epialleles can be relieved by the transacting factor Unstable factor for orange1 (Ufo1), resulting in gain of ectopic pigmentation (Chopra et al 2003;Sekhon et al 2007).…”

The Methylation of the PcMYB10 Promoter Is Associated with Green-Skinned Sport in Max Red Bartlett Pear

“…In region 21,218 to 21,649 bp of the PcMYB10 promoter, only CHH-type cytosine showed a higher methylation level in MRB-G fruit peel than in MRB. The type of DNA methylation that induced plant color mutation was largely CG/CHG cytosine methylation, such as the CG/CHG cytosine methylation that occurs in the promoter of the P1-wr gene, inducing color mutation of maize (Sekhon and Chopra, 2009). However, there are no previous reports showing that CHH cytosine hypermethylation and CG/CHG cytosine hypermethylation exist in different regions of a single promoter.…”

“…Hypermethylation of the phosphoribosylanthranilate isomerase1-4 (pai1-4) locus in Arabidopsis (Arabidopsis thaliana) is associated with transcriptional repression of PAI (Bender and Fink, 1995). Furthermore, hypermethylation in the promoter of the white pericarp/red cob (P1-wr) gene causes maize (Zea mays) color change, and hypermethylation in the 39 untranslated region of the allele gene of Pl (Pl-Bh) leads to the formation of red-blotting maize (Cocciolone and Cone, 1993;Sekhon and Chopra, 2009). Hypermethylation in the promoter of MdMYB10 causes a striped pigmentation on Honey Crisp apple fruit (Telias et al, 2011).…”

Flavonoid Phytoalexin-Dependent Resistance to Anthracnose Leaf Blight Requires a Functional yellow seed1 in Sorghum bicolor

“…DNA digests were fractionated on 0.8% agarose gels, and gels were blotted onto nylon membranes through standard protocol of capillary transfer (Sambrook and Russell 2001). DNA gel blots were hybridized using buffers and conditions described previously (Sekhon and Chopra 2009).…”