Prioritizing Metabolic Gene Regulators through Multi-Omic Network Integration in Maize.

Elucidating gene regulatory networks (GRNs) is a major area of study within plant systems biology. Phenotypic traits are intricately linked to specific gene expression profiles. These expression patterns arise primarily from regulatory connections between sets of transcription factors (TFs) and their target genes. In this study, we integrated publicly available co-expression networks derived from more than 6,000 RNA-seq samples, 283 protein-DNA interaction assays, and 16 million of SNPs used to identify expression quantitative loci (eQTL), to construct TF-target networks. In total, we analyzed ~4.6M interactions to generate four distinct types of TF-target networks: co-expression, protein-DNA interaction (PDI), trans-expression quantitative loci (trans-eQTL), and cis-eQTL combined with PDIs. To improve the functional annotation of TFs based on its target genes, we implemented three different strategies to integrate these four types of networks. We subsequently evaluated the effectiveness of our method through loss-of function mutant and random networks. The multi-network integration allowed us to identify transcriptional regulators of hormone-, metabolic- and development-related processes. Finally, using the topological properties of the fully integrated network, we identified potentially functional redundant TF paralogs. Our findings retrieved functions previously documented for numerous TFs and revealed novel functions that are crucial for informing the design of future experiments. The approach here-described lays the foundation for the integration of multi-omic datasets in maize and other plant systems.

Mutator transposon insertions within maize genes often provide a novel outward reading promoter.

The highly active family of Mutator (Mu) DNA transposons has been widely used for forward and reverse genetics in maize. There are examples of Mu-suppressible alleles that result in conditional phenotypic effects based on the activity of Mu. Phenotypes from these Mu-suppressible mutations are observed in Mu-active genetic backgrounds, but absent when Mu activity is lost. For some Mu-suppressible alleles, phenotypic suppression likely results from an outward-reading promoter within Mu that is only active when the autonomous Mu element is silenced or lost. We isolated 35 Mu alleles from the UniformMu population that represent insertions in 24 different genes. Most of these mutant alleles are due to insertions within gene coding sequences, but several 5' UTR and intron insertions were included. RNA-seq and de novo transcript assembly were utilized to document the transcripts produced from 33 of these Mu insertion alleles. For 20 of the 33 alleles, there was evidence of transcripts initiating within the Mu sequence reading through the gene. This outward-reading promoter activity was detected in multiple types of Mu elements and does not depend on the orientation of Mu. Expression analyses of Mu-initiated transcripts revealed the Mu promoter often provides gene expression levels and patterns that are similar to the wild-type gene. These results suggest the Mu promoter may represent a minimal promoter that can respond to gene cis-regulatory elements. Findings from this study have implications for maize researchers using the UniformMu population, and more broadly highlight a strategy for transposons to co-exist with their host.

A novel active transposon creates allelic variation through altered translation rate to influence protein abundance.

Protein translation is tightly and precisely controlled by multiple mechanisms including upstream open reading frames (uORFs), but the origins of uORFs and their role in maize are largely unexplored. In this study, an active transposition event was identified during the propagation of maize inbred line B73. The transposon, which was named BTA for 'B73 active transposable element hAT', creates a novel dosage-dependent hypomorphic allele of the hexose transporter gene ZmSWEET4c through insertion within the coding sequence in the first exon, and results in reduced kernel size. The BTA insertion does not affect transcript abundance but reduces protein abundance of ZmSWEET4c, probably through the introduction of a uORF. Furthermore, the introduction of BTA sequence in the exon of other genes can regulate translation efficiency without affecting their mRNA levels. A transposon capture assay revealed 79 novel insertions for BTA and BTA-like elements. These insertion sites have typical euchromatin features, including low levels of DNA methylation and high levels of H3K27ac. A putative autonomous element that mobilizes BTA and BTA-like elements was identified. Together, our results suggest a transposon-based origin of uORFs and document a new role for transposable elements to influence protein abundance and phenotypic diversity by affecting the translation rate.

cis-Regulatory Elements in Plant Development, Adaptation, and Evolution.

cis-Regulatory elements encode the genomic blueprints that ensure the proper spatiotemporal patterning of gene expression necessary for appropriate development and responses to the environment. Accumulating evidence implicates changes to gene expression as a major source of phenotypic novelty in eukaryotes, including acute phenotypes such as disease and cancer in mammals. Moreover, genetic and epigenetic variation affecting cis-regulatory sequences over longer evolutionary timescales has become a recurring theme in studies of morphological divergence and local adaptation. Here, we discuss the functions of and methods used to identify various classes of cis-regulatory elements, as well as their role in plant development and response to the environment. We highlight opportunities to exploit cis-regulatory variants underlying plant development and environmental responses for crop improvement efforts. Although a comprehensive understanding of cis-regulatory mechanisms in plants has lagged behind that in animals, we showcase several breakthrough findings that have profoundly influenced plant biology and shaped the overall understanding of transcriptional regulation in eukaryotes.

Mapping responsive genomic elements to heat stress in a maize diversity panel.

BACKGROUND: Many plant species exhibit genetic variation for coping with environmental stress. However, there are still limited approaches to effectively uncover the genomic region that regulates distinct responsive patterns of the gene across multiple varieties within the same species under abiotic stress. RESULTS: By analyzing the transcriptomes of more than 100 maize inbreds, we reveal many cis- and trans-acting eQTLs that influence the expression response to heat stress. The cis-acting eQTLs in response to heat stress are identified in genes with differential responses to heat stress between genotypes as well as genes that are only expressed under heat stress. The cis-acting variants for heat stress-responsive expression likely result from distinct promoter activities, and the differential heat responses of the alleles are confirmed for selected genes using transient expression assays. Global footprinting of transcription factor binding is performed in control and heat stress conditions to document regions with heat-enriched transcription factor binding occupancies. CONCLUSIONS: Footprints enriched near proximal regions of characterized heat-responsive genes in a large association panel can be utilized for prioritizing functional genomic regions that regulate genotype-specific responses under heat stress.

Modeling chromatin state from sequence across angiosperms using recurrent convolutional neural networks.

Accessible chromatin regions are critical components of gene regulation but modeling them directly from sequence remains challenging, especially within plants, whose mechanisms of chromatin remodeling are less understood than in animals. We trained an existing deep-learning architecture, DanQ, on data from 12 angiosperm species to predict the chromatin accessibility in leaf of sequence windows within and across species. We also trained DanQ on DNA methylation data from 10 angiosperms because unmethylated regions have been shown to overlap significantly with ACRs in some plants. The across-species models have comparable or even superior performance to a model trained within species, suggesting strong conservation of chromatin mechanisms across angiosperms. Testing a maize (Zea mays L.) held-out model on a multi-tissue chromatin accessibility panel revealed our models are best at predicting constitutively accessible chromatin regions, with diminishing performance as cell-type specificity increases. Using a combination of interpretation methods, we ranked JASPAR motifs by their importance to each model and saw that the TCP and AP2/ERF transcription factor (TF) families consistently ranked highly. We embedded the top three JASPAR motifs for each model at all possible positions on both strands in our sequence window and observed position- and strand-specific patterns in their importance to the model. With our publicly available across-species 'a2z' model it is now feasible to predict the chromatin accessibility and methylation landscape of any angiosperm genome.

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.

Epigenetic features drastically impact CRISPR-Cas9 efficacy in plants.

CRISPR-Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR-Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR-Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR-Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR-Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes.

Genome-wide loss of CHH methylation with limited transcriptome changes in Setaria viridis DOMAINS REARRANGED METHYLTRANSFERASE (DRM) mutants.

The DOMAINS REARRANGED METHYLTRANSFERASEs (DRMs) are crucial for RNA-directed DNA methylation (RdDM) in plant species. Setaria viridis is a model monocot species with a relatively compact genome that has limited transposable element (TE) content. CRISPR-based genome editing approaches were used to create loss-of-function alleles for the two putative functional DRM genes in S. viridis to probe the role of RdDM. Double mutant (drm1ab) plants exhibit some morphological abnormalities but are fully viable. Whole-genome methylation profiling provided evidence for the widespread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild-type plants. Evidence was also found for the locus-specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified genes with altered expression in the drm1ab mutants. However, the majority of genes with high levels of CHH methylation directly surrounding the transcription start site or in nearby promoter regions in wild-type plants do not have altered expression in the drm1ab mutant, even when this methylation is lost, suggesting limited regulation of gene expression by RdDM. Detailed analysis of the expression of TEs identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons are likely to require active RdDM for the maintenance of transcriptional repression.

DNA demethylation affects imprinted gene expression in maize endosperm.

BACKGROUND: DNA demethylation occurs in many species and is involved in diverse biological processes. However, the occurrence and role of DNA demethylation in maize remain unknown. RESULTS: We analyze loss-of-function mutants of two major genes encoding DNA demethylases. No significant change in DNA methylation has been detected in these mutants. However, we detect increased DNA methylation levels in the mutants around genes and some transposons. The increase in DNA methylation is accompanied by alteration in gene expression, with a tendency to show downregulation, especially for the genes that are preferentially expressed in endosperm. Imprinted expression of both maternally and paternally expressed genes changes in F1 hybrid with the mutant as female and the wild-type as male parental line, but not in the reciprocal hybrid. This alteration in gene expression is accompanied by allele-specific DNA methylation differences, suggesting that removal of DNA methylation of the maternal allele is required for the proper expression of these imprinted genes. Finally, we demonstrate that hypermethylation in the double mutant is associated with reduced binding of transcription factor to its target, and altered gene expression. CONCLUSIONS: Our results suggest that active removal of DNA methylation is important for transcription factor binding and proper gene expression in maize endosperm.

Prediction of conserved and variable heat and cold stress response in maize using cis-regulatory information.

Changes in gene expression are important for responses to abiotic stress. Transcriptome profiling of heat- or cold-stressed maize genotypes identifies many changes in transcript abundance. We used comparisons of expression responses in multiple genotypes to identify alleles with variable responses to heat or cold stress and to distinguish examples of cis- or trans-regulatory variation for stress-responsive expression changes. We used motifs enriched near the transcription start sites (TSSs) for thermal stress-responsive genes to develop predictive models of gene expression responses. Prediction accuracies can be improved by focusing only on motifs within unmethylated regions near the TSS and vary for genes with different dynamic responses to stress. Models trained on expression responses in a single genotype and promoter sequences provided lower performance when applied to other genotypes but this could be improved by using models trained on data from all three genotypes tested. The analysis of genes with cis-regulatory variation provides evidence for structural variants that result in presence/absence of transcription factor binding sites in creating variable responses. This study provides insights into cis-regulatory motifs for heat- and cold-responsive gene expression and defines a framework for developing models to predict expression responses across multiple genotypes.

Stability of DNA methylation and chromatin accessibility in structurally diverse maize genomes.

Accessible chromatin and unmethylated DNA are associated with many genes and cis-regulatory elements. Attempts to understand natural variation for accessible chromatin regions (ACRs) and unmethylated regions (UMRs) often rely upon alignments to a single reference genome. This limits the ability to assess regions that are absent in the reference genome assembly and monitor how nearby structural variants influence variation in chromatin state. In this study, de novo genome assemblies for four maize inbreds (B73, Mo17, Oh43, and W22) are utilized to assess chromatin accessibility and DNA methylation patterns in a pan-genome context. A more complete set of UMRs and ACRs can be identified when chromatin data are aligned to the matched genome rather than a single reference genome. While there are UMRs and ACRs present within genomic regions that are not shared between genotypes, these features are 6- to 12-fold enriched within regions between genomes. Characterization of UMRs present within shared genomic regions reveals that most UMRs maintain the unmethylated state in other genotypes with only ∼5% being polymorphic between genotypes. However, the majority (71%) of UMRs that are shared between genotypes only exhibit partial overlaps suggesting that the boundaries between methylated and unmethylated DNA are dynamic. This instability is not solely due to sequence variation as these partially overlapping UMRs are frequently found within genomic regions that lack sequence variation. The ability to compare chromatin properties among individuals with structural variation enables pan-epigenome analyses to study the sources of variation for accessible chromatin and unmethylated DNA.

Plant height heterosis is quantitatively associated with expression levels of plastid ribosomal proteins.

The use of hybrids is widespread in agriculture, yet the molecular basis for hybrid vigor (heterosis) remains obscure. To identify molecular components that may contribute to trait heterosis, we analyzed paired proteomic and transcriptomic data from seedling leaf and mature leaf blade tissues of maize hybrids and their inbred parents. Nuclear- and plastid-encoded subunits of complexes required for protein synthesis in the chloroplast and for the light reactions of photosynthesis were expressed above midparent and high-parent levels, respectively. Consistent with previous reports in Arabidopsis, ethylene biosynthetic enzymes were expressed below midparent levels in the hybrids, suggesting a conserved mechanism for heterosis between monocots and dicots. The ethylene biosynthesis mutant, acs2/acs6, largely phenocopied the hybrid proteome, indicating that a reduction in ethylene biosynthesis may mediate the differences between inbreds and their hybrids. To rank the relevance of expression differences to trait heterosis, we compared seedling leaf protein levels to the adult plant height of 15 hybrids. Hybrid/midparent expression ratios were most positively correlated with hybrid/midparent plant height ratios for the chloroplast ribosomal proteins. Our results show that increased expression of chloroplast ribosomal proteins in hybrid seedling leaves is mediated by reduced expression of ethylene biosynthetic enzymes and that the degree of their overexpression in seedlings can quantitatively predict adult trait heterosis.

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.

A capture-based assay for detection and characterization of transposon polymorphisms in maize.

Transposons can create allelic diversity that affects gene expression and phenotypic diversity. The detection of transposon polymorphisms at a genome-wide scale across a large population is difficult. Here, we developed a targeted sequencing approach to monitor transposon polymorphisms of interest. This approach can interrogate the presence or absence of transposons reliably across various genotypes. Using this approach, we genotyped a set of 965 transposon-related presence/absence polymorphisms in a diverse panel of 16 maize (Zea mays L.) inbred lines that are representative of the major maize breeding groups. About 70% of the selected regions can be effectively assayed in each genotype. The consistency between the capture-based assay and PCR-based assay are 98.6% based on analysis of 24 randomly selected transposon polymorphisms. By integrating the transposon polymorphisms data with gene expression data, ∼18% of the assayed transposon polymorphisms were found to be associated with variable gene expression levels. A detailed analysis of 18 polymorphisms in a larger association panel confirmed the effects of 10 polymorphisms, with one of them having a stronger association with expression than nearby SNP markers. The effects of seven polymorphisms were tested using a luciferase-based expression assay, and one was confirmed. Together, this study demonstrates that the targeted sequencing assay is an effective way to explore transposon function in a high-throughput manner.

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.

Maize decrease in DNA methylation 1 targets RNA-directed DNA methylation on active chromatin.

DNA methylation plays vital roles in repressing transposable element activity and regulating gene expression. The chromatin-remodeling factor Decrease in DNA methylation 1 (DDM1) is crucial for maintaining DNA methylation across diverse plant species, and is required for RNA-directed DNA methylation (RdDM) to maintain mCHH islands in maize (Zea mays). However, the mechanisms by which DDM1 is involved in RdDM are not well understood. In this work, we used chromatin immunoprecipitation coupled with high-throughput sequencing to ascertain the genome-wide occupancy of ZmDDM1 in the maize genome. The results revealed that ZmDDM1 recognized an 8-bp-long GC-rich degenerate DNA sequence motif, which is enriched in transcription start sites and other euchromatic regions. Meanwhile, 24-nucleotide siRNAs and CHH methylation were delineated at the edge of ZmDDM1-occupied sites. ZmDDM1 co-purified with Argonaute 4 (ZmAGO4) proteins, providing further evidence that ZmDDM1 is a component of RdDM complexes in planta. Consistent with this, the vast majority of ZmDDM1-targeted regions co-localized with ZmAGO4-bound genomic sites. Overall, our results suggest a model that ZmDDM1 may be recruited to euchromatic regions via recognition of a GC-rich motif, thereby remodeling chromatin to provide access for RdDM activities in maize.

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.