RNA-directed DNA methylation mutants reduce histone methylation at the paramutated maize booster1 enhancer.

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.

The maize striate leaves2 (sr2) gene encodes a conserved DUF3732 domain and is homologous to the rice yss1 gene.

Maize striate leaves2 (sr2) is a mutant that causes white stripes on leaves that has been used in mapping studies for decades though the underlying gene has not been identified. The sr2 locus has been previously mapped to small regions of normal chromosome 10 (N10) and a rearranged variant called abnormal chromosome 10 (Ab10). A comparison of assembled genomes carrying N10 and Ab10 revealed only five candidate sr2 genes. Analysis of a stock carrying the sr2 reference allele (sr2-ref) showed that one of the five genes has a transposon insertion that disrupts its protein sequence and has a severe reduction in mRNA. An independent Mutator transposon insertion in the gene (sr2-Mu) failed to complement the sr2-ref mutation, and plants homozygous for sr2-Mu showed white striped leaf margins. The sr2 gene encodes a DUF3732 protein with strong homology to a rice gene with a similar mutant phenotype called young seedling stripe1 (yss1). These and other published data suggest that sr2 may have a function in plastid gene expression.

Potent pollen gene regulation by DNA glycosylases in maize.

Although DNA methylation primarily represses transposable elements (TEs) in plants, it also represses select endosperm and pollen genes. These genes, or their cis-regulatory elements, are methylated in plant body tissues but are demethylated by DNA glycosylases (DNGs) in endosperm and pollen, enabling their transcription. Activity of either one of two DNGs, MDR1 or DNG102, is essential for pollen viability in maize. Using single-pollen mRNA sequencing on pollen segregating mutations in both genes, we identified 58 candidate DNG target genes, whose expression is strongly decreased in double mutant pollen (124-fold decrease on average). These genes account for 11.1% of the wild-type pollen polyadenylated transcriptome, but they are silent or barely detectable in the plant body. They are unusual in their tendency to lack introns but even more so in their having TE-like methylation in their coding DNA sequence. Moreover, they are strongly enriched for predicted functions in cell wall modification. While some may support development of the pollen grain cell wall, expansins and pectinases in this set of genes suggest a function in cell wall loosening to support the rapid tip growth characteristic of pollen tubes as they carry the sperm cells through maternal apoplast and extracellular matrix of the pistil. These results suggest a critical role for DNA methylation and demethylation in regulating maize genes with potential for extremely high expression in pollen but constitutive silencing elsewhere.

Natural methylation epialleles correlate with gene expression in maize.

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.

Synthetic maize centromeres transmit chromosomes across generations.

Centromeres are long, often repetitive regions of genomes that bind kinetochore proteins and ensure normal chromosome segregation. Engineering centromeres that function in vivo has proven to be difficult. Here we describe a tethering approach that activates functional maize centromeres at synthetic sequence arrays. A LexA-CENH3 fusion protein was used to recruit native Centromeric Histone H3 (CENH3) to long arrays of LexO repeats on a chromosome arm. Newly recruited CENH3 was sufficient to organize functional kinetochores that caused chromosome breakage, releasing chromosome fragments that were passed through meiosis and into progeny. Several fragments formed independent neochromosomes with centromeres localized over the LexO repeat arrays. The new centromeres were self-sustaining and transmitted neochromosomes to subsequent generations in the absence of the LexA-CENH3 activator. Our results demonstrate the feasibility of using synthetic centromeres for karyotype engineering applications.

The maize gene maternal derepression of r1 encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in the endosperm.

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.

Resetting of the 24-nt siRNA landscape in rice zygotes.

The zygote, a totipotent stem cell, is crucial to the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells-the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Owing to technical challenges, the epigenetic changes that accompany the transition from differentiated gametes to totipotent zygote are poorly understood. Because siRNAs serve as both regulators and outputs of the epigenome, we characterized small RNA transcriptomes of zygotes from rice. Zygote small RNAs exhibit extensive maternal carryover and an apparent lack of paternal contribution, indicated by absence of sperm signature siRNAs. Zygote formation is accompanied by widespread redistribution of 24-nt siRNAs relative to gametes, such that ∼70% of the zygote siRNA loci do not overlap any egg cell siRNA loci. Newly detected siRNA loci in zygote are gene-proximal and not associated with centromeric heterochromatin, similar to canonical siRNAs, in sharp contrast to gametic siRNA loci that are gene-distal and heterochromatic. In addition, zygote but not egg siRNA loci are associated with high DNA methylation in the mature embryo. Thus, the zygote begins transitioning before the first embryonic division to an siRNA profile that is associated with future RdDM in embryogenesis. These findings indicate that, in addition to changes in gene expression, the transition to totipotency in the plant zygote is accompanied by resetting of the epigenetic reprogramming that occurred during gamete formation.

De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes.

We report de novo genome assemblies, transcriptomes, annotations, and methylomes for the 26 inbreds that serve as the founders for the maize nested association mapping population. The number of pan-genes in these diverse genomes exceeds 103,000, with approximately a third found across all genotypes. The results demonstrate that the ancient tetraploid character of maize continues to degrade by fractionation to the present day. Excellent contiguity over repeat arrays and complete annotation of centromeres revealed additional variation in major cytological landmarks. We show that combining structural variation with single-nucleotide polymorphisms can improve the power of quantitative mapping studies. We also document variation at the level of DNA methylation and demonstrate that unmethylated regions are enriched for cis-regulatory elements that contribute to phenotypic variation.

Maize centromeric chromatin scales with changes in genome size.

Centromeres are defined by the location of Centromeric Histone H3 (CENP-A/CENH3) which interacts with DNA to define the locations and sizes of functional centromeres. An analysis of 26 maize genomes including 110 fully assembled centromeric regions revealed positive relationships between centromere size and genome size. These effects are independent of variation in the amounts of the major centromeric satellite sequence CentC. We also backcrossed known centromeres into two different lines with larger genomes and observed consistent increases in functional centromere sizes for multiple centromeres. Although changes in centromere size involve changes in bound CENH3, we could not mimic the effect by overexpressing CENH3 by threefold. Literature from other fields demonstrate that changes in genome size affect protein levels, organelle size and cell size. Our data demonstrate that centromere size is among these scalable features, and that multiple limiting factors together contribute to a stable centromere size equilibrium.

Haploid induction by a maize cenh3 null mutant.

The production of haploids is an important first step in creating many new plant varieties. One approach used in Arabidopsis involves crossing plants expressing different forms of centromeric histone H3 (CENP-A/CENH3) and subsequent loss of genome with weaker centromeres. However, the method has been ineffective in crop plants. Here, we describe a greatly simplified method based on crossing maize lines that are heterozygous for a cenh3 null mutation. Crossing +/cenh3 to wild-type plants in both directions yielded haploid progeny. Genome elimination was determined by the cenh3 genotype of the gametophyte, suggesting that centromere failure is caused by CENH3 dilution during the postmeiotic cell divisions that precede gamete formation. The cenh3 haploid inducer works as a vigorous hybrid and can be transferred to other lines in a single cross, making it versatile for a variety of applications.

Distinct kinesin motors drive two types of maize neocentromeres.

A maize chromosome variant called abnormal chromosome 10 (Ab10) converts knobs on chromosome arms into neocentromeres, causing their preferential segregation to egg cells in a process known as meiotic drive. We previously demonstrated that the gene Kinesin driver (Kindr) on Ab10 encodes a kinesin-14 required to mobilize neocentromeres made up of the major tandem repeat knob180. Here we describe a second kinesin-14 gene, TR-1 kinesin (Trkin), that is required to mobilize neocentromeres made up of the minor tandem repeat TR-1. Trkin lies in a 4-Mb region of Ab10 that is not syntenic with any other region of the maize genome and shows extraordinary sequence divergence from Kindr and other kinesins in plants. Despite its unusual structure, Trkin encodes a functional minus end-directed kinesin that specifically colocalizes with TR-1 in meiosis, forming long drawn out neocentromeres. TRKIN contains a nuclear localization signal and localizes to knobs earlier in prophase than KINDR. The fact that TR-1 repeats often co-occur with knob180 repeats suggests that the current role of the TRKIN/TR-1 system is to facilitate the meiotic drive of the KINDR/knob180 system.

Gapless assembly of maize chromosomes using long-read technologies.

Creating gapless telomere-to-telomere assemblies of complex genomes is one of the ultimate challenges in genomics. We use two independent assemblies and an optical map-based merging pipeline to produce a maize genome (B73-Ab10) composed of 63 contigs and a contig N50 of 162 Mb. This genome includes gapless assemblies of chromosome 3 (236 Mb) and chromosome 9 (162 Mb), and 53 Mb of the Ab10 meiotic drive haplotype. The data also reveal the internal structure of seven centromeres and five heterochromatic knobs, showing that the major tandem repeat arrays (CentC, knob180, and TR-1) are discontinuous and frequently interspersed with retroelements.

Genome-wide redistribution of 24-nt siRNAs in rice gametes.

Gametes constitute a critical stage of the plant life cycle during which the genome undergoes reprogramming in preparation for embryogenesis. Here, we examined genome-wide distributions of small RNAs in the sperm and egg cells of rice. We found that 24-nt siRNAs, which are a hallmark of RNA-directed DNA methylation (RdDM) in plants, were depleted from heterochromatin boundaries in both gametes relative to vegetative tissues, reminiscent of siRNA patterns in DDM1-type nucleosome remodeler mutants. In sperm cells, 24-nt siRNAs were spread across heterochromatic regions, while in egg cells, 24-nt siRNAs were concentrated at a smaller number of heterochromatic loci throughout the genome, especially at loci which also produced siRNAs in other tissues. In both gametes, patterns of CHH methylation, typically a strong indicator of RdDM, were similar to vegetative tissues, although lower in magnitude. These findings indicate that the small RNA transcriptome undergoes large-scale redistribution in both male and female gametes, which is not correlated with recruitment of DNA methyltransferases in gametes and suggestive of unexplored regulatory activities of gamete small RNAs.

Loss of RNA-Directed DNA Methylation in Maize Chromomethylase and DDM1-Type Nucleosome Remodeler Mutants.

Plants make use of distinct types of DNA methylation characterized by their DNA methyltransferases and modes of regulation. One type, RNA-directed DNA methylation (RdDM), is guided by small interfering RNAs (siRNAs) to the edges of transposons that are close to genes, areas called mCHH islands in maize (Zea mays). Another type, chromomethylation, is guided by histone H3 lysine 9 methylation to heterochromatin across the genome. We examined DNA methylation and small RNA expression in plant tissues that were mutant for both copies of the genes encoding chromomethylases as well as mutants for both copies of the genes encoding DECREASED DNA METHYLATION1 (DDM1)-type nucleosome remodelers, which facilitate chromomethylation. Both sets of double mutants were nonviable but produced embryos and endosperm. RdDM was severely compromised in the double mutant embryos, both in terms of DNA methylation and siRNAs. Loss of 24-nucleotide siRNA from mCHH islands was coupled with a gain of 21-, 22-, and 24-nucleotide siRNAs in heterochromatin. These results reveal a requirement for both chromomethylation and DDM1-type nucleosome remodeling for RdDM in mCHH islands, which we hypothesize is due to dilution of RdDM components across the genome when heterochromatin is compromised.

A Kinesin-14 Motor Activates Neocentromeres to Promote Meiotic Drive in Maize.

Maize abnormal chromosome 10 (Ab10) encodes a classic example of true meiotic drive that converts heterochromatic regions called knobs into motile neocentromeres that are preferentially transmitted to egg cells. Here, we identify a cluster of eight genes on Ab10, called the Kinesin driver (Kindr) complex, that are required for both neocentromere motility and preferential transmission. Two meiotic drive mutants that lack neocentromere activity proved to be kindr epimutants with increased DNA methylation across the entire gene cluster. RNAi of Kindr induced a third epimutant and corresponding loss of meiotic drive. Kinesin gliding assays and immunolocalization revealed that KINDR is a functional minus-end-directed kinesin that localizes specifically to knobs containing 180 bp repeats. Sequence comparisons suggest that Kindr diverged from a Kinesin-14A ancestor ∼12 mya and has driven the accumulation of > 500 Mb of knob repeats and affected the segregation of thousands of genes linked to knobs on all 10 chromosomes.

Genome-wide mapping of transcriptional enhancer candidates using DNA and chromatin features in maize.

BACKGROUND: While most cells in multicellular organisms carry the same genetic information, in each cell type only a subset of genes is being transcribed. Such differentiation in gene expression depends, for a large part, on the activation and repression of regulatory sequences, including transcriptional enhancers. Transcriptional enhancers can be located tens of kilobases from their target genes, but display characteristic chromatin and DNA features, allowing their identification by genome-wide profiling. Here we show that integration of chromatin characteristics can be applied to predict distal enhancer candidates in Zea mays, thereby providing a basis for a better understanding of gene regulation in this important crop plant. RESULT: To predict transcriptional enhancers in the crop plant maize (Zea mays L. ssp. mays), we integrated available genome-wide DNA methylation data with newly generated maps for chromatin accessibility and histone 3 lysine 9 acetylation (H3K9ac) enrichment in young seedling and husk tissue. Approximately 1500 intergenic regions, displaying low DNA methylation, high chromatin accessibility and H3K9ac enrichment, were classified as enhancer candidates. Based on their chromatin profiles, candidate sequences can be classified into four subcategories. Tissue-specificity of enhancer candidates is defined based on the tissues in which they are identified and putative target genes are assigned based on tissue-specific expression patterns of flanking genes. CONCLUSIONS: Our method identifies three previously identified distal enhancers in maize, validating the new set of enhancer candidates and enlarging the toolbox for the functional characterization of gene regulation in the highly repetitive maize genome.

Stable centromere positioning in diverse sequence contexts of complex and satellite centromeres of maize and wild relatives.

BACKGROUND: Paradoxically, centromeres are known both for their characteristic repeat sequences (satellite DNA) and for being epigenetically defined. Maize (Zea mays mays) is an attractive model for studying centromere positioning because many of its large (~2 Mb) centromeres are not dominated by satellite DNA. These centromeres, which we call complex centromeres, allow for both assembly into reference genomes and for mapping short reads from ChIP-seq with antibodies to centromeric histone H3 (cenH3). RESULTS: We found frequent complex centromeres in maize and its wild relatives Z. mays parviglumis, Z. mays mexicana, and particularly Z. mays huehuetenangensis. Analysis of individual plants reveals minor variation in the positions of complex centromeres among siblings. However, such positional shifts are stochastic and not heritable, consistent with prior findings that centromere positioning is stable at the population level. Centromeres are also stable in multiple F1 hybrid contexts. Analysis of repeats in Z. mays and other species (Zea diploperennis, Zea luxurians, and Tripsacum dactyloides) reveals tenfold differences in abundance of the major satellite CentC, but similar high levels of sequence polymorphism in individual CentC copies. Deviation from the CentC consensus has little or no effect on binding of cenH3. CONCLUSIONS: These data indicate that complex centromeres are neither a peculiarity of cultivation nor inbreeding in Z. mays. While extensive arrays of CentC may be the norm for other Zea and Tripsacum species, these data also reveal that a wide diversity of DNA sequences and multiple types of genetic elements in and near centromeres support centromere function and constrain centromere positions.

Gene Expression and Chromatin Modifications Associated with Maize Centromeres.

Centromeres are defined by the presence of CENH3, a variant of histone H3. Centromeres in most plant species contain exclusively highly repetitive DNA sequences, which has hindered research on structure and function of centromeric chromatin. Several maize centromeres have been nearly completely sequenced, providing a sequence-based platform for genomic and epigenomic research of plant centromeres. Here we report a high resolution map of CENH3 nucleosomes in the maize genome. Although CENH3 nucleosomes are spaced ∼190 bp on average, CENH3 nucleosomes that occupied CentC, a 156-bp centromeric satellite repeat, showed clear positioning aligning with CentC monomers. Maize centromeres contain alternating CENH3-enriched and CENH3-depleted subdomains, which account for 87% and 13% of the centromeres, respectively. A number of annotated genes were identified in the centromeres, including 11 active genes that were located exclusively in CENH3-depleted subdomains. The euchromatic histone modification marks, including H3K4me3, H3K36me3 and H3K9ac, detected in maize centromeres were associated mainly with the active genes. Interestingly, maize centromeres also have lower levels of the heterochromatin histone modification mark H3K27me2 relative to pericentromeric regions. We conclude that neither H3K27me2 nor the three euchromatic histone modifications are likely to serve as functionally important epigenetic marks of centromere identity in maize.

RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome.

The maize genome is relatively large (∼ 2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (∼ 100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes.

Stable Patterns of CENH3 Occupancy Through Maize Lineages Containing Genetically Similar Centromeres.

While the approximate chromosomal position of centromeres has been identified in many species, little is known about the dynamics and diversity of centromere positions within species. Multiple lines of evidence indicate that DNA sequence has little or no impact in specifying centromeres in maize and in most multicellular organisms. Given that epigenetically defined boundaries are expected to be dynamic, we hypothesized that centromere positions would change rapidly over time, which would result in a diversity of centromere positions in isolated populations. To test this hypothesis, we used CENP-A/cenH3 (CENH3 in maize) chromatin immunoprecipitation to define centromeres in breeding pedigrees that included the B73 inbred as a common parent. While we found a diversity of CENH3 profiles for centromeres with divergent sequences that were not inherited from B73, the CENH3 profiles from centromeres that were inherited from B73 were indistinguishable from each other. We propose that specific genetic elements in centromeric regions favor or inhibit CENH3 accumulation, leading to reproducible patterns of CENH3 occupancy. These data also indicate that dramatic shifts in centromere position normally originate from accumulated or large-scale genetic changes rather than from epigenetic positional drift.