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Abstract Imprinted expression is an essential process for seed viability affecting hundreds of genes in Zea mays endosperm; however, most studies have examined just one time point for analysis. The focus on single time points can limit our ability to identify imprinted genes and our ability to draw conclusions for the role of imprinting in endosperm. In this study, we examine imprinted expression across 4 time points ranging from the transition to endoreduplication from mitotic division through the beginning of programmed cell death. Additionally, we assessed imprinting variation across 8 diverse maize lines, 6 of which have never before been assessed for imprinting. Through this analysis, we identify over 700 imprinted genes with varying consistency across time points including 255 genes imprinted at every time point and 105 genes displaying transient imprinting. We find a correlation between high consistency of imprinting across time and high conservation of parental bias across 8 diverse maize lines reciprocally crossed with B73. Additionally, we identify evidence of imprinting for 3 zein genes that are critical for nutrient accumulation in the endosperm, suggesting that imprinting may play a more important role in seed composition than previously thought. Taken together, this study provides a more holistic view of imprinting variation across time and across genotypes in maize and enables us to more thoroughly investigate the complex imprinting landscape. 

Abstract DNA methylation in plants is depleted from cis-regulatory elements in and near genes but is present in some gene bodies, including exons. Methylation in exons solely in the CG context is called gene body methylation (gbM). Methylation in exons in both CG and non-CG contexts is called TE-like methylation (teM). Assigning functions to both forms of methylation in genes has proven to be challenging. Toward that end, we utilized recent genome assemblies, gene annotations, transcription data, and methylome data to quantify common patterns of gene methylation and their relations to gene expression in maize. We found that gbM genes exist in a continuum of CG methylation levels without a clear demarcation between unmethylated genes and gbM genes. Analysis of expression levels across diverse maize stocks and tissues revealed a weak but highly significant positive correlation between gbM and gene expression except in endosperm. gbM epialleles were associated with an approximately 3% increase in steady-state expression level relative to unmethylated epialleles. In contrast to gbM genes, which were conserved and were broadly expressed across tissues, we found that teM genes, which make up about 12% of genes, are mainly silent, are poorly conserved, and exhibit evidence of annotation errors. We used these data to flag teM genes in the 26 NAM founder genome assemblies. While some teM genes are likely functional, these data suggest that the majority are not, and their inclusion can confound the interpretation of whole-genome studies. 

Abstract Demethylation of transposons can activate the expression of nearby genes and cause imprinted gene expression in the endosperm; this demethylation is hypothesized to lead to expression of transposon small interfering RNAs (siRNAs) that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maize (Zea mays) maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis thaliana DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting is its main function in endosperm. 

Targeted demethylation by DNA glycosylases (DNGs) results in differential methylation between parental alleles in the endosperm, which drives imprinted expression. Here, we performed RNA sequencing on endosperm derived from DNG mutant mdr1 and wild-type (WT) endosperm. Consistent with the role of DNA methylation in gene silencing, we find 108 genes and 96 TEs differentially expressed (DE) transcripts that lost expression in the hypermethylated mdr1 mutant. Compared with other endosperm transcripts, the mdr1 targets are enriched for TEs (particularly Helitrons), and DE genes are depleted for both core genes and GO term assignments, suggesting that the majority of DE transcripts are TEs and pseudo-genes. By comparing DE genes to imprinting calls from prior studies, we find that the majority of DE genes have maternally biased expression, and approximately half of all maternally expressed genes (MEGs) are DE in this study. In contrast, no paternally expressed genes (PEGs) are DE. DNG-dependent imprinted genes are distinguished by maternal demethylation and expression primarily in the endosperm, so we also performed Enzymatic Methyl-seq on hybrids to identify maternal demethylation and utilized a W22 gene expression atlas to identify genes expressed primarily in the endosperm. Overall, approximately ⅔ of all MEGs show evidence of regulation by DNGs. Taken together, this study solidifies the role of MDR1 in the regulation of maternally expressed, imprinted genes and TEs and identifies subsets of genes with DNG-independent imprinting regulation. 

Paramutation is the transfer of mitotically and meiotically heritable silencing information between two alleles. With paramutation at the maize (Zea mays) booster1 (b1) locus, the low-expressed B′ epiallele heritably changes the high-expressed B-I epiallele into B′ with 100% frequency. This requires specific tandem repeats and multiple components of the RNA-directed DNA methylation pathway, including the RNA-dependent RNA polymerase (encoded by mediator of paramutation1, mop1), the second-largest subunit of RNA polymerase IV and V (NRP(D/E)2a, encoded by mop2), and the largest subunit of RNA Polymerase IV (NRPD1, encoded by mop3). Mutations in mop genes prevent paramutation and release silencing at the B′ epiallele. In this study, we investigated the effect of mutations in mop1, mop2, and mop3 on chromatin structure and DNA methylation at the B′ epiallele, and especially the regulatory hepta-repeat 100 kb upstream of the b1 gene. Mutations in mop1 and mop3 resulted in decreased repressive histone modifications H3K9me2 and H3K27me2 at the hepta-repeat. Associated with this decrease were partial activation of the hepta-repeat enhancer function, formation of a multi-loop structure, and elevated b1 expression. In mop2 mutants, which do not show elevated b1 expression, H3K9me2, H3K27me2 and a single-loop structure like in wild-type B′ were retained. Surprisingly, high CG and CHG methylation levels at the B′ hepta-repeat remained in all three mutants, and CHH methylation was low in both wild type and mutants. Our results raise the possibility of MOP factors mediating RNA-directed histone methylation rather than RNA-directed DNA methylation at the b1 locus.