The evolution and function of transposons in epigenetic regulation in response to the environment.

Transposable elements (TEs) make up a major proportion of plant genomes. Despite their prevalence genome-wide, TEs are often tossed aside as "junk DNA" since they rarely cause phenotypes, and epigenetic mechanisms silence TEs to prevent them from causing deleterious mutations through movement. While this bleak picture of TEs in genomes is true on average, a growing number of examples across many plant species point to TEs as drivers of phenotypic diversity and novel stress responses. Examples of TE-influenced phenotypes illustrate the many ways that novel transposition events can alter local gene expression and how this relates to potential variation in plant responses to environmental stress. Since TE families and insertions at the locus level lack evolutionary conservation, advancements in the field will require TE experts across diverse species to identify and utilize TE variation in their own systems as a means of crop improvement.

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.

The genomic ecosystem of transposable elements in maize.

Transposable elements (TEs) constitute the majority of flowering plant DNA, reflecting their tremendous success in subverting, avoiding, and surviving the defenses of their host genomes to ensure their selfish replication. More than 85% of the sequence of the maize genome can be ascribed to past transposition, providing a major contribution to the structure of the genome. Evidence from individual loci has informed our understanding of how transposition has shaped the genome, and a number of individual TE insertions have been causally linked to dramatic phenotypic changes. Genome-wide analyses in maize and other taxa have frequently represented TEs as a relatively homogeneous class of fragmentary relics of past transposition, obscuring their evolutionary history and interaction with their host genome. Using an updated annotation of structurally intact TEs in the maize reference genome, we investigate the family-level dynamics of TEs in maize. Integrating a variety of data, from descriptors of individual TEs like coding capacity, expression, and methylation, as well as similar features of the sequence they inserted into, we model the relationship between attributes of the genomic environment and the survival of TE copies and families. In contrast to the wholesale relegation of all TEs to a single category of junk DNA, these differences reveal a diversity of survival strategies of TE families. Together these generate a rich ecology of the genome, with each TE family representing the evolution of a distinct ecological niche. We conclude that while the impact of transposition is highly family- and context-dependent, a family-level understanding of the ecology of TEs in the genome can refine our ability to predict the role of TEs in generating genetic and phenotypic diversity.

Whole-genome variation of transposable element insertions in a maize diversity panel.

Intact transposable elements (TEs) account for 65% of the maize genome and can impact gene function and regulation. Although TEs comprise the majority of the maize genome and affect important phenotypes, genome-wide patterns of TE polymorphisms in maize have only been studied in a handful of maize genotypes, due to the challenging nature of assessing highly repetitive sequences. We implemented a method to use short-read sequencing data from 509 diverse inbred lines to classify the presence/absence of 445,418 nonredundant TEs that were previously annotated in four genome assemblies including B73, Mo17, PH207, and W22. Different orders of TEs (i.e., LTRs, Helitrons, and TIRs) had different frequency distributions within the population. LTRs with lower LTR similarity were generally more frequent in the population than LTRs with higher LTR similarity, though high-frequency insertions with very high LTR similarity were observed. LTR similarity and frequency estimates of nested elements and the outer elements in which they insert revealed that most nesting events occurred very near the timing of the outer element insertion. TEs within genes were at higher frequency than those that were outside of genes and this is particularly true for those not inserted into introns. Many TE insertional polymorphisms observed in this population were tagged by SNP markers. However, there were also 19.9% of the TE polymorphisms that were not well tagged by SNPs (R2 < 0.5) that potentially represent information that has not been well captured in previous SNP-based marker-trait association studies. This study provides a population scale genome-wide assessment of TE variation in maize and provides valuable insight on variation in TEs in maize and factors that contribute to this variation.

Widespread imprinting of transposable elements and variable genes in the maize endosperm.

Fertilization and seed development is a critical time in the plant life cycle, and coordinated development of the embryo and endosperm are required to produce a viable seed. In the endosperm, some genes show imprinted expression where transcripts are derived primarily from one parental genome. Imprinted gene expression has been observed across many flowering plant species, though only a small proportion of genes are imprinted. Understanding how imprinted expression arises has been complicated by the reliance on single nucleotide polymorphisms between alleles to enable testing for imprinting. Here, we develop a method to use whole genome assemblies of multiple genotypes to assess for imprinting of both shared and variable portions of the genome using data from reciprocal crosses. This reveals widespread maternal expression of genes and transposable elements with presence-absence variation within maize and across species. Most maternally expressed features are expressed primarily in the endosperm, suggesting that maternal de-repression in the central cell facilitates expression. Furthermore, maternally expressed TEs are enriched for maternal expression of the nearest gene, and read alignments over maternal TE-gene pairs indicate that these are fused rather than independent transcripts.

Assessing the regulatory potential of transposable elements using chromatin accessibility profiles of maize transposons.

Transposable elements (TEs) have the potential to create regulatory variation both through the disruption of existing DNA regulatory elements and through the creation of novel DNA regulatory elements. In a species with a large genome, such as maize, many TEs interspersed with genes create opportunities for significant allelic variation due to TE presence/absence polymorphisms among individuals. We used information on putative regulatory elements in combination with knowledge about TE polymorphisms in maize to identify TE insertions that interrupt existing accessible chromatin regions (ACRs) in B73 as well as examples of polymorphic TEs that contain ACRs among four inbred lines of maize including B73, Mo17, W22, and PH207. The TE insertions in three other assembled maize genomes (Mo17, W22, or PH207) that interrupt ACRs that are present in the B73 genome can trigger changes to the chromatin, suggesting the potential for both genetic and epigenetic influences of these insertions. Nearly 20% of the ACRs located over 2 kb from the nearest gene are located within an annotated TE. These are regions of unmethylated DNA that show evidence for functional importance similar to ACRs that are not present within TEs. Using a large panel of maize genotypes, we tested if there is an association between the presence of TE insertions that interrupt, or carry, an ACR and the expression of nearby genes. While most TE polymorphisms are not associated with expression for nearby genes, the TEs that carry ACRs exhibit enrichment for being associated with higher expression of nearby genes, suggesting that these TEs may contribute novel regulatory elements. These analyses highlight the potential for a subset of TEs to rewire transcriptional responses in eukaryotic genomes.

Genetic and epigenetic variation in transposable element expression responses to abiotic stress in maize.

Transposable elements (TEs) pervade most eukaryotic genomes. The repetitive nature of TEs complicates the analysis of their expression. Evaluation of the expression of both TE families (using unique and multi-mapping reads) and specific elements (using uniquely mapping reads) in leaf tissue of three maize (Zea mays) inbred lines subjected to heat or cold stress reveals no evidence for genome-wide activation of TEs; however, some specific TE families generate transcripts only in stress conditions. There is substantial variation for which TE families exhibit stress-responsive expression in the different genotypes. In order to understand the factors that drive expression of TEs, we focused on a subset of families in which we could monitor expression of individual elements. The stress-responsive activation of a TE family can often be attributed to a small number of elements in the family that contains regions lacking DNA methylation. Comparisons of the expression of TEs in different genotypes revealed both genetic and epigenetic variation. Many of the specific TEs that are activated in stress in one inbred are not present in the other inbred, explaining the lack of activation. Among the elements that are shared in both genomes but only expressed in one genotype, we found that many exhibit differences in DNA methylation such that the genotype without expression is fully methylated. This study provides insights into the regulation of expression of TEs in normal and stress conditions and highlights the role of chromatin variation between elements in a family or between genotypes for contributing to expression variation. The highly repetitive nature of many TEs complicates the analysis of their expression. Although most TEs are not expressed, some exhibits expression in certain tissues or conditions. We monitored the expression of both TE families (using unique and multi-mapping reads) and specific elements (using uniquely mapping reads) in leaf tissue of three maize (Zea mays) inbred lines subjected to heat or cold stress. While genome-wide activation of TEs did not occur, some TE families generated transcripts only in stress conditions with variation by genotype. To better understand the factors that drive expression of TEs, we focused on a subset of families in which we could monitor expression of individual elements. In most cases, stress-responsive activation of a TE family was attributed to a small number of elements in the family. The elements that contained small regions lacking DNA methylation regions showed enriched expression while fully methylated elements were rarely expressed in control or stress conditions. The cause of varied expression in the different genotypes was due to both genetic and epigenetic variation. Many specific TEs activated by stress in one inbred were not present in the other inbred. Among the elements shared in both genomes, full methylation inhibited expression in one of the genotypes. This study provides insights into the regulation of TE expression in normal and stress conditions and highlights the role of chromatin variation between elements in a family or between genotypes for contributing to expression.

Monitoring the interplay between transposable element families and DNA methylation in maize.

DNA methylation and epigenetic silencing play important roles in the regulation of transposable elements (TEs) in many eukaryotic genomes. A majority of the maize genome is derived from TEs that can be classified into different orders and families based on their mechanism of transposition and sequence similarity, respectively. TEs themselves are highly methylated and it can be tempting to view them as a single uniform group. However, the analysis of DNA methylation profiles in flanking regions provides evidence for distinct groups of chromatin properties at different TE families. These differences among TE families are reproducible in different tissues and different inbred lines. TE families with varying levels of DNA methylation in flanking regions also show distinct patterns of chromatin accessibility and modifications within the TEs. The differences in the patterns of DNA methylation flanking TE families arise from a combination of non-random insertion preferences of TE families, changes in DNA methylation triggered by the insertion of the TE and subsequent selection pressure. A set of nearly 70,000 TE polymorphisms among four assembled maize genomes were used to monitor the level of DNA methylation at haplotypes with and without the TE insertions. In many cases, TE families with high levels of DNA methylation in flanking sequence are enriched for insertions into highly methylated regions. The majority of the >2,500 TE insertions into unmethylated regions result in changes in DNA methylation in haplotypes with the TE, suggesting the widespread potential for TE insertions to condition altered methylation in conserved regions of the genome. This study highlights the interplay between TEs and the methylome of a major crop species.

Dynamic Patterns of Transcript Abundance of Transposable Element Families in Maize.

Transposable Elements (TEs) are mobile elements that contribute the majority of DNA sequences in the maize genome. Due to their repetitive nature, genomic studies of TEs are complicated by the difficulty of properly attributing multi-mapped short reads to specific genomic loci. Here, we utilize a method to attribute RNA-seq reads to TE families rather than particular loci in order to characterize transcript abundance for TE families in the maize genome. We applied this method to assess per-family expression of transposable elements in >800 published RNA-seq libraries representing a range of maize development, genotypes, and hybrids. While a relatively small proportion of TE families are transcribed, expression is highly dynamic with most families exhibiting tissue-specific expression. A large number of TE families were specifically detected in pollen and endosperm, consistent with reproductive dynamics that maintain silencing of TEs in the germ line. We find that B73 transcript abundance is a poor predictor of TE expression in other genotypes and that transcript levels can differ even for shared TEs. Finally, by assessing recombinant inbred line and hybrid transcriptomes, complex patterns of TE transcript abundance across genotypes emerged. Taken together, this study reveals a dynamic contribution of TEs to maize transcriptomes.

Transposable elements contribute to dynamic genome content in maize.

Transposable elements (TEs) are ubiquitous components of eukaryotic genomes and can create variation in genome organization and content. Most maize genomes are composed of TEs. We developed an approach to define shared and variable TE insertions across genome assemblies and applied this method to four maize genomes (B73, W22, Mo17 and PH207) with uniform structural annotations of TEs. Among these genomes we identified approximately 400 000 TEs that are polymorphic, encompassing 1.6 Gb of variable TE sequence. These polymorphic TEs include a combination of recent transposition events as well as deletions of older TEs. There are examples of polymorphic TEs within each of the superfamilies of TEs and they are found distributed across the genome, including in regions of recent shared ancestry among individuals. There are many examples of polymorphic TEs within or near maize genes. In addition, there are 2380 gene annotations in the B73 genome that are located within variable TEs, providing evidence for the role of TEs in contributing to the substantial differences in annotated gene content among these genotypes. TEs are highly variable in our survey of four temperate maize genomes, highlighting the major contribution of TEs in driving variation in genome organization and gene content. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://github.com/SNAnderson/maizeTE_variation; https://mcstitzer.github.io/maize_TEs.

Hybrid Decay: A Transgenerational Epigenetic Decline in Vigor and Viability Triggered in Backcross Populations of Teosinte with Maize.

In the course of generating populations of maize with teosinte chromosomal introgressions, an unusual sickly plant phenotype was noted in individuals from crosses with two teosinte accessions collected near Valle de Bravo, Mexico. The plants of these Bravo teosinte accessions appear phenotypically normal themselves and the F1 plants appear similar to typical maize × teosinte F1s. However, upon backcrossing to maize, the BC1 and subsequent generations display a number of detrimental characteristics including shorter stature, reduced seed set, and abnormal floral structures. This phenomenon is observed in all BC individuals and there is no chromosomal segment linked to the sickly plant phenotype in advanced backcross generations. Once the sickly phenotype appears in a lineage, normal plants are never again recovered by continued backcrossing to the normal maize parent. Whole-genome shotgun sequencing reveals a small number of genomic sequences, some with homology to transposable elements, that have increased in copy number in the backcross populations. Transcriptome analysis of seedlings, which do not have striking phenotypic abnormalities, identified segments of 18 maize genes that exhibit increased expression in sickly plants. A de novo assembly of transcripts present in plants exhibiting the sickly phenotype identified a set of 59 upregulated novel transcripts. These transcripts include some examples with sequence similarity to transposable elements and other sequences present in the recurrent maize parent (W22) genome as well as novel sequences not present in the W22 genome. Genome-wide profiles of gene expression, DNA methylation, and small RNAs are similar between sickly plants and normal controls, although a few upregulated transcripts and transposable elements are associated with altered small RNA or methylation profiles. This study documents hybrid incompatibility and genome instability triggered by the backcrossing of Bravo teosinte with maize. We name this phenomenon "hybrid decay" and present ideas on the mechanism that may underlie it.

The maize W22 genome provides a foundation for functional genomics and transposon biology.

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.

Subtle Perturbations of the Maize Methylome Reveal Genes and Transposons Silenced by Chromomethylase or RNA-Directed DNA Methylation Pathways.

DNA methylation is a chromatin modification that can provide epigenetic regulation of gene and transposon expression. Plants utilize several pathways to establish and maintain DNA methylation in specific sequence contexts. The chromomethylase (CMT) genes maintain CHG (where H = A, C or T) methylation. The RNA-directed DNA methylation (RdDM) pathway is important for CHH methylation. Transcriptome analysis was performed in a collection of Zea mays lines carrying mutant alleles for CMT or RdDM-associated genes. While the majority of the transcriptome was not affected, we identified sets of genes and transposon families sensitive to context-specific decreases in DNA methylation in mutant lines. Many of the genes that are up-regulated in CMT mutant lines have high levels of CHG methylation, while genes that are differentially expressed in RdDM mutants are enriched for having nearby mCHH islands, implicating context-specific DNA methylation in the regulation of expression for a small number of genes. Many genes regulated by CMTs exhibit natural variation for DNA methylation and transcript abundance in a panel of diverse inbred lines. Transposon families with differential expression in the mutant genotypes show few defining features, though several families up-regulated in RdDM mutants show enriched expression in endosperm tissue, highlighting the potential importance for this pathway during reproduction. Taken together, our findings suggest that while the number of genes and transposon families whose expression is reproducibly affected by mild perturbations in context-specific methylation is small, there are distinct patterns for loci impacted by RdDM and CMT mutants.

Potential roles for transposable elements in creating imprinted expression.

Changes in gene expression can have profound effects on phenotype. Nature has provided many complex patterns of gene regulation such as imprinting. Imprinted genes exhibit differences in the expression of the maternal and paternal alleles, even though they reside in the same nucleus with access to the same trans-acting factors. Significant attention has been focused on the potential reasons that imprinted expression could be beneficial and stabilized by selection. However, less attention has focused on understanding how imprinted expression might arise or decay. We discuss the evidence for frequent turnover of imprinted expression based on evolutionary analyses in plants and the potential role for transposable elements (TEs) in creating imprinted expression patterns.

The Zygotic Transition Is Initiated in Unicellular Plant Zygotes with Asymmetric Activation of Parental Genomes.

The zygotic transition, from a fertilized egg to an embryo, is central to animal and plant reproduction. Animal embryos depend upon maternally provided factors until zygotic genome activation (ZGA). In plants, the timing and parental genome contributions to ZGA are unresolved. Here, we use the flowering plant Oryza sativa (rice) to characterize transcriptomes of time-staged isogenic and hybrid zygotes following fertilization. Large-scale transcriptomic changes were observed in unicellular zygotes, including upregulation of S-phase genes, a characteristic of ZGA. The parental contributions to ZGA were highly asymmetric. Zygotic transcription was primarily from the maternal genome and included genes for basic cellular processes. Transcription of the paternal genome was highly restricted but unexpectedly included genes encoding putative pluripotency factors expressed at the onset of ZGA. Thus, distinct transcriptional activities are exhibited by the parental genomes during the initiation of embryogenesis, which presumptively derive from divergent pre-zygotic transcriptional states established in the gametes.

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.

Transcriptomes of isolated Oryza sativa gametes characterized by deep sequencing: evidence for distinct sex-dependent chromatin and epigenetic states before fertilization.

The formation of a zygote by the fusion of egg and sperm involves the two gametic transcriptomes. In flowering plants, the embryo sac embedded within the ovule contains the egg cell, whereas the pollen grain contains two sperm cells inside a supporting vegetative cell. The difficulties of collecting isolated gametes and consequent low recovery of RNA have restricted in-depth analysis of gametic transcriptomes in flowering plants. We isolated living egg cells, sperm cells and pollen vegetative cells from Oryza sativa (rice), and identified transcripts for approximately 36 000 genes by deep sequencing. The three transcriptomes are highly divergent, with about three-quarters of those genes differentially expressed in the different cell types. Distinctive expression profiles were observed for genes involved in chromatin conformation, including an unexpected expression in the sperm cell of genes associated with active chromatin. Furthermore, both the sperm cell and the pollen vegetative cell were deficient in expression of key RNAi components. Differences in gene expression were also observed for genes for hormonal signaling and cell cycle regulation. The egg cell and sperm cell transcriptomes reveal major differences in gene expression to be resolved in the zygote, including pathways affecting chromatin configuration, hormones and cell cycle. The sex-specific differences in the expression of RNAi components suggest that epigenetic silencing in the zygote might act predominantly through female-dependent pathways. More generally, this study provides a detailed gene expression landscape for flowering plant gametes, enabling the identification of specific gametic functions, and their contributions to zygote and seed development.