Divergence among rice cultivars reveals roles for transposition and epimutation in ongoing evolution of genomic imprinting.

Parent-of-origin-dependent gene expression in mammals and flowering plants results from differing chromatin imprints (genomic imprinting) between maternally and paternally inherited alleles. Imprinted gene expression in the endosperm of seeds is associated with localized hypomethylation of maternally but not paternally inherited DNA, with certain small RNAs also displaying parent-of-origin-specific expression. To understand the evolution of imprinting mechanisms in Oryza sativa (rice), we analyzed imprinting divergence among four cultivars that span both japonica and indica subspecies: Nipponbare, Kitaake, 93-11, and IR64. Most imprinted genes are imprinted across cultivars and enriched for functions in chromatin and transcriptional regulation, development, and signaling. However, 4 to 11% of imprinted genes display divergent imprinting. Analyses of DNA methylation and small RNAs revealed that endosperm-specific 24-nt small RNA-producing loci show weak RNA-directed DNA methylation, frequently overlap genes, and are imprinted four times more often than genes. However, imprinting divergence most often correlated with local DNA methylation epimutations (9 of 17 assessable loci), which were largely stable within subspecies. Small insertion/deletion events and transposable element insertions accompanied 4 of the 9 locally epimutated loci and associated with imprinting divergence at another 4 of the remaining 8 loci. Correlating epigenetic and genetic variation occurred at key regulatory regions-the promoter and transcription start site of maternally biased genes, and the promoter and gene body of paternally biased genes. Our results reinforce models for the role of maternal-specific DNA hypomethylation in imprinting of both maternally and paternally biased genes, and highlight the role of transposition and epimutation in rice imprinting evolution.

Genomic analysis uncovers functional variation in the C-terminus of anthocyanin-activating MYB transcription factors.

MYB transcription factors regulate diverse aspects of plant development and secondary metabolism, often by partnering in transcriptional regulatory complexes. Here, we harness genomic resources to identify novel MYBs, thereby producing an updated eudicot MYB phylogeny with revised relationships among subgroups as well as new information on sequence variation in the disordered C-terminus of anthocyanin-activating MYBs. BLAST® and hidden Markov model scans of gene annotations identified a total of 714 MYB transcription factors across the genomes of four crops that span the eudicots: apple, grape, kiwifruit and tomato. Codon model-based phylogenetic inference identified novel members of previously defined subgroups, and the function of specific anthocyanin-activating subgroup 6 members was assayed transiently in tobacco leaves. Sequence conservation within subgroup 6 highlighted one previously described and two novel short linear motifs in the disordered C-terminal region. The novel motifs have a mix of hydrophobic and acidic residues and are predicted to be relatively ordered compared with flanking protein sequences. Comparison of motifs with the Eukaryotic Linear Motif database suggests roles in protein-protein interaction. Engineering of motifs and their flanking regions from strong anthocyanin activators into weak activators, and vice versa, affected function. We conclude that, although the MYB C-terminal sequence diverges greatly even within MYB clades, variation within the C-terminus at and near relatively ordered regions offers opportunities for exploring MYB function and developing superior alleles for plant breeding.

Evolution and function of genomic imprinting in plants.

Genomic imprinting, an inherently epigenetic phenomenon defined by parent of origin-dependent gene expression, is observed in mammals and flowering plants. Genome-scale surveys of imprinted expression and the underlying differential epigenetic marks have led to the discovery of hundreds of imprinted plant genes and confirmed DNA and histone methylation as key regulators of plant imprinting. However, the biological roles of the vast majority of imprinted plant genes are unknown, and the evolutionary forces shaping plant imprinting remain rather opaque. Here, we review the mechanisms of plant genomic imprinting and discuss theories of imprinting evolution and biological significance in light of recent findings.

Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm.

Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally inherited chromosomes. Demethylation mediates parent-of-origin-specific (imprinted) gene expression but is apparently unnecessary for the extensive accumulation of maternally biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin specific is unknown. Imprinted expression of sRNA also remains uninvestigated in monocot seeds. Here, we report high-coverage sequencing of the Kitaake rice cultivar that enabled us to show that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants.

Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes.

The Arabidopsis thaliana central cell, the companion cell of the egg, undergoes DNA demethylation before fertilization, but the targeting preferences, mechanism, and biological significance of this process remain unclear. Here, we show that active DNA demethylation mediated by the DEMETER DNA glycosylase accounts for all of the demethylation in the central cell and preferentially targets small, AT-rich, and nucleosome-depleted euchromatic transposable elements. The vegetative cell, the companion cell of sperm, also undergoes DEMETER-dependent demethylation of similar sequences, and lack of DEMETER in vegetative cells causes reduced small RNA-directed DNA methylation of transposons in sperm. Our results demonstrate that demethylation in companion cells reinforces transposon methylation in plant gametes and likely contributes to stable silencing of transposable elements across generations.

Local DNA hypomethylation activates genes in rice endosperm.

Cytosine methylation silences transposable elements in plants, vertebrates, and fungi but also regulates gene expression. Plant methylation is catalyzed by three families of enzymes, each with a preferred sequence context: CG, CHG (H = A, C, or T), and CHH, with CHH methylation targeted by the RNAi pathway. Arabidopsis thaliana endosperm, a placenta-like tissue that nourishes the embryo, is globally hypomethylated in the CG context while retaining high non-CG methylation. Global methylation dynamics in seeds of cereal crops that provide the bulk of human nutrition remain unknown. Here, we show that rice endosperm DNA is hypomethylated in all sequence contexts. Non-CG methylation is reduced evenly across the genome, whereas CG hypomethylation is localized. CHH methylation of small transposable elements is increased in embryos, suggesting that endosperm demethylation enhances transposon silencing. Genes preferentially expressed in endosperm, including those coding for major storage proteins and starch synthesizing enzymes, are frequently hypomethylated in endosperm, indicating that DNA methylation is a crucial regulator of rice endosperm biogenesis. Our data show that genome-wide reshaping of seed DNA methylation is conserved among angiosperms and has a profound effect on gene expression in cereal crops.

New Zealand Ginger mouse: novel model that associates the tyrp1b pigmentation gene locus with regulation of lean body mass.

The study of spontaneous mutations in mice over the last century has been fundamental to our understanding of normal physiology and mechanisms of disease. Here we studied the phenotype and genotype of a novel mouse model we have called the New Zealand Ginger (NZG/Kgm) mouse. NZG/Kgm mice are very large, rapidly growing, ginger-colored mice with pink eyes. Breeding NZG/Kgm mice with CAST/Ei or C57BL/6J mice showed that the ginger coat colour is a recessive trait, while the excessive body weight and large body size exhibit a semidominant pattern of inheritance. Backcrossing F1 (NZG/Kgm x CAST/Ei) to NZG/Kgm mice to produce the N2 generation determined that the NZG/Kgm mouse has two recessive pigmentation variant genes (oca2(p) and tyrp-1(b)) and that the tyrp-1(b) gene locus associates with large body size. Three coat colors appeared in the N2 generation; ginger, brown, and dark. Strikingly, N2 male coat colour associated with body weight; the brown-colored mice weighed the most followed by ginger and then dark. The male brown coat-colored offspring reached adult body weights indistinguishable from NZG/Kgm males. The large NZG/Kgm mouse body size is a result of excessive lean body mass since these mice are not obese or diabetic. NZG/Kgm mice exhibit an unusual pattern of fat distribution; compared with other mouse strains they have disproportionately higher amounts of subcutaneous and gonadal fat. These mice are susceptible to high-fat diet-induced obesity but are resistant to high-fat diet-induced diabetes. We propose NZG/Kgm mice as a novel model to delineate gene(s) that regulate 1) growth and metabolism, 2) resistance to Type 2 diabetes, and 3) preferential fat deposition in the subcutaneous and gonadal areas.

Newly weaned nonobese diabetic mice show heightened early diabetes sensitivity to multiple low doses of streptozotocin than nondiabetes-prone CD-1 mice: initial beta-cell damage a key trigger for type 1 diabetes?

OBJECTIVES: We determined if newly weaned female nonobese diabetic (NOD) mice show greater diabetes sensitivity to dose-adjusted regimens of multiple low doses of streptozotocin (Stz) than nondiabetes-prone CD-1 mice. METHODS: Female NOD mice received 5 daily doses of Stz from day 21 (0, 5, 10, 15, 20, 30, and 40 mg/kg body weight) and CD-1 mice 20, 30, and 40 mg. RESULTS: : Streptozotocin, at the 15-, 20-, 30-, and 40-mg dose, induced rapid diabetes in NOD mice. By day 100, 90% to 95% of NOD mice became diabetic after the 40- and 30-mg dose and 33% to 40% with the 15- and 20-mg dose. In comparison, only about 50% and 33% of CD-1 mice developed diabetes with the 40- and 30-mg dose, respectively, and 5.5% with the 20-mg dose. In NOD mice, the 20-mg dose also partially suppressed spontaneous diabetes. All diabetic mice displayed insulitis, variable immunostaining for insulin, and redistribution of glucagon and somatostatin cells. Glucose transporter-2 was markedly attenuated in selective beta cells. CONCLUSIONS: Newly weaned female NOD mice show heightened early sensitivity to low doses of Stz than CD-1 mice. At diabetes, several beta cells remain and show variable immunostaining for insulin and an attenuated expression for glucose transporter-2. Specific low doses of Stz may also suppress spontaneous diabetes.