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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. 

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.

 

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.

 

Core Ideas Cross‐species models of chromatin state from sequence are comparable or superior to within‐species models. Model performance is highest on accessible regions open in many tissues. Transcription factor motifs can be ranked by importance to each species and chromatin state. 

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.

By analyzing the transcriptomes of more than 100 maize inbreds, we reveal manycis- andtrans-acting eQTLs that influence the expression response to heat stress. Thecis-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. Thecis-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.

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.

 

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.

 

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.

 

Abstract 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.