Investigating the cis-regulatory basis of C3 and C4 photosynthesis in grasses at single-cell resolution.

While considerable knowledge exists about the enzymes pivotal for C4 photosynthesis, much less is known about the cis-regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C4 enzymes for five different grass species. This study spans four C4 species, covering three distinct photosynthetic subtypes: Zea mays and Sorghum bicolor (NADP-dependent malic enzyme), Panicum miliaceum (NAD-dependent malic enzyme), Urochloa fusca (phosphoenolpyruvate carboxykinase), along with the C3 outgroup Oryza sativa. We studied the cis-regulatory landscape of enzymes essential across all C4 species and those unique to C4 subtypes, measuring cell-type-specific biases for C4 enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C4 evolution. Besides promoter proximal ACRs, we found that, on average, C4 genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C4 evolution. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution of cis-regulation at these C4 loci. This study illuminates the dynamic and complex nature of cis-regulatory elements evolution in C4 photosynthesis, particularly highlighting the intricate cis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C3 crop performance under changing climatic conditions.

GenomicLinks: deep learning predictions of 3D chromatin interactions in the maize genome.

Gene regulation in eukaryotes is partly shaped by the 3D organization of chromatin within the cell nucleus. Distal interactions between cis-regulatory elements and their target genes are widespread, and many causal loci underlying heritable agricultural traits have been mapped to distal non-coding elements. The biology underlying chromatin loop formation in plants is poorly understood. Dissecting the sequence features that mediate distal interactions is an important step toward identifying putative molecular mechanisms. Here, we trained GenomicLinks, a deep learning model, to identify DNA sequence features predictive of 3D chromatin interactions in maize. We found that the presence of binding motifs of specific transcription factor classes, especially bHLH, is predictive of chromatin interaction specificities. Using an in silico mutagenesis approach we show the removal of these motifs from loop anchors leads to reduced interaction probabilities. We were able to validate these predictions with single-cell co-accessibility data from different maize genotypes that harbor natural substitutions in these TF binding motifs. GenomicLinks is currently implemented as an open-source web tool, which should facilitate its wider use in the plant research community.

A spatially resolved multiomic single-cell atlas of soybean development.

Cis-regulatory elements (CREs) precisely control spatiotemporal gene expression in cells. Using a spatially resolved single-cell atlas of gene expression with chromatin accessibility across ten soybean tissues, we identified 103 distinct cell types and 303,199 accessible chromatin regions (ACRs). Nearly 40% of the ACRs showed cell-type-specific patterns and were enriched for transcription factor (TF) motifs defining diverse cell identities. We identified de novo enriched TF motifs and explored conservation of gene regulatory networks underpinning legume symbiotic nitrogen fixation. With comprehensive developmental trajectories for endosperm and embryo, we uncovered the functional transition of the three sub-cell types of endosperm, identified 13 sucrose transporters sharing the DOF11 motif that were co-up-regulated in late peripheral endosperm and identified key embryo cell-type specification regulators during embryogenesis, including a homeobox TF that promotes cotyledon parenchyma identity. This resource provides a valuable foundation for analyzing gene regulatory programs in soybean cell types across tissues and life stages.

scifi-ATAC-seq: massive-scale single-cell chromatin accessibility sequencing using combinatorial fluidic indexing.

Single-cell ATAC-seq has emerged as a powerful approach for revealing candidate cis-regulatory elements genome-wide at cell-type resolution. However, current single-cell methods suffer from limited throughput and high costs. Here, we present a novel technique called scifi-ATAC-seq, single-cell combinatorial fluidic indexing ATAC-sequencing, which combines a barcoded Tn5 pre-indexing step with droplet-based single-cell ATAC-seq using the 10X Genomics platform. With scifi-ATAC-seq, up to 200,000 nuclei across multiple samples can be indexed in a single emulsion reaction, representing an approximately 20-fold increase in throughput compared to the standard 10X Genomics workflow.

Investigating the cis-Regulatory Basis of C3 and C4 Photosynthesis in Grasses at Single-Cell Resolution.

While considerable knowledge exists about the enzymes pivotal for C4 photosynthesis, much less is known about the cis-regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C4 enzymes for five different grass species. This study spans four C4 species, covering three distinct photosynthetic subtypes: Zea mays and Sorghum bicolor (NADP-ME), Panicum miliaceum (NAD-ME), Urochloa fusca (PEPCK), along with the C3 outgroup Oryza sativa. We studied the cis-regulatory landscape of enzymes essential across all C4 species and those unique to C4 subtypes, measuring cell-type-specific biases for C4 enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C4 evolution. Besides promoter proximal ACRs, we found that, on average, C4 genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C4 evolution. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution of cis-regulation at these C4 loci. This study illuminates the dynamic and complex nature of CRE evolution in C4 photosynthesis, particularly highlighting the intricate cis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C3 crop performance under changing climatic conditions.

Evolution of plant cell-type-specific cis -regulatory elements.

Cis -regulatory elements (CREs) are critical in regulating gene expression, and yet our understanding of CRE evolution remains a challenge. Here, we constructed a comprehensive single-cell atlas of chromatin accessibility in Oryza sativa , integrating data from 104,029 nuclei representing 128 discrete cell states across nine distinct organs. We used comparative genomics to compare cell-type resolved chromatin accessibility between O. sativa and 57,552 nuclei from four additional grass species ( Zea mays, Sorghum bicolor, Panicum miliaceum , and Urochloa fusca ). Accessible chromatin regions (ACRs) had different levels of conservation depending on the degree of cell-type specificity. We found a complex relationship between ACRs with conserved noncoding sequences, cell-type specificity, conservation, and tissue-specific switching. Additionally, we found that epidermal ACRs were less conserved compared to other cell types, potentially indicating that more rapid regulatory evolution has occurred in the L1 epidermal layer of these species. Finally, we identified and characterized a conserved subset of ACRs that overlapped the repressive histone modification H3K27me3, implicating them as potentially critical silencer CREs maintained by evolution. Collectively, this comparative genomics approach highlights the dynamics of cell-type-specific CRE evolution in plants.

Massive-scale single-cell chromatin accessibility sequencing using combinatorial fluidic indexing.

Single-cell ATAC-seq has emerged as a powerful approach for revealing candidate cis-regulatory elements genome-wide at cell-type resolution. However, current single-cell methods suffer from limited throughput and high costs. Here, we present a novel technique called single-cell combinatorial fluidic indexing ATAC-sequencing ("scifi-ATAC-seq"), which combines a barcoded Tn5 pre-indexing step with droplet-based single-cell ATAC-seq using a widely commercialized microfluidics platform (10X Genomics). With scifi-ATAC-seq, up to 200,000 nuclei across multiple samples in a single emulsion reaction can be indexed, representing a ~20-fold increase in throughput compared to the standard 10X Genomics workflow.

Leveraging Single-Cell Populations to Uncover the Genetic Basis of Complex Traits.

The ease and throughput of single-cell genomics have steadily improved, and its current trajectory suggests that surveying single-cell populations will become routine. We discuss the merger of quantitative genetics with single-cell genomics and emphasize how this synergizes with advantages intrinsic to plants. Single-cell population genomics provides increased detection resolution when mapping variants that control molecular traits, including gene expression or chromatin accessibility. Additionally, single-cell population genomics reveals the cell types in which variants act and, when combined with organism-level phenotype measurements, unveils which cellular contexts impact higher-order traits. Emerging technologies, notably multiomics, can facilitate the measurement of both genetic changes and genomic traits in single cells, enabling single-cell genetic experiments. The implementation of single-cell genetics will advance the investigation of the genetic architecture of complex molecular traits and provide new experimental paradigms to study eukaryotic genetics.

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.

Computational Analysis of Maize Enhancer Regulatory Elements Using ATAC-STARR-seq.

The blueprints to development, response to the environment, and cellular function are largely the manifestation of distinct gene expression programs controlled by the spatiotemporal activity of cis-regulatory elements. Although biochemical methods for identifying accessible chromatin - a hallmark of active cis-regulatory elements - have been developed, approaches capable of measuring and quantifying cis-regulatory activity are only beginning to be realized. Massively Parallel Reporter Assays coupled to chromatin accessibility profiling present a high-throughput solution for testing the transcription-activating capacity of millions of putatively regulatory DNA sequences in parallel. However, clear computational pipelines for analyzing these high-throughput sequencing-based reporter assays are lacking. In this protocol, I layout and rationalize a computational framework for the processing and analysis of Assay for Transposase Accessible Chromatin profiling followed by Self-Transcribed Active Regulatory Region sequencing (ATAC-STARR-seq) data from a recent study in Zea mays. The approach described herein can be adapted to other sequencing-based reporter assays and is largely agnostic to the model organism with the appropriate input substitutions.

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.

A combinatorial indexing strategy for low-cost epigenomic profiling of plant single cells.

Understanding how cis-regulatory elements facilitate gene expression is a key question in biology. Recent advances in single-cell genomics have led to the discovery of cell-specific chromatin landscapes that underlie transcription programs in animal models. However, the high equipment and reagent costs of commercial systems limit their applications for many laboratories. In this study, we developed a combinatorial index and dual PCR barcode strategy to profile the Arabidopsis thaliana root single-cell epigenome without any specialized equipment. We generated chromatin accessibility profiles for 13 576 root nuclei with an average of 12 784 unique Tn5 integrations per cell. Integration of the single-cell assay for transposase-accessible chromatin sequencing and RNA sequencing data sets enabled the identification of 24 cell clusters with unique transcription, chromatin, and cis-regulatory signatures. Comparison with single-cell data generated using the commercial microfluidic platform from 10X Genomics revealed that this low-cost combinatorial index method is capable of unbiased identification of cell-type-specific chromatin accessibility. We anticipate that, by removing cost, instrumentation, and other technical obstacles, this method will be a valuable tool for routine investigation of single-cell epigenomes and provide new insights into plant growth and development and plant interactions with the environment.

Quality control and evaluation of plant epigenomics data.

Epigenomics is the study of molecular signatures associated with discrete regions within genomes, many of which are important for a wide range of nuclear processes. The ability to profile the epigenomic landscape associated with genes, repetitive regions, transposons, transcription, differential expression, cis-regulatory elements, and 3D chromatin interactions has vastly improved our understanding of plant genomes. However, many epigenomic and single-cell genomic assays are challenging to perform in plants, leading to a wide range of data quality issues; thus, the data require rigorous evaluation prior to downstream analyses and interpretation. In this commentary, we provide considerations for the evaluation of plant epigenomics and single-cell genomics data quality with the aim of improving the quality and utility of studies using those data across diverse plant species.

Single-cell analysis of cis-regulatory elements.

Plant tissues and organs are composed of functionally discrete cell types that are all defined by the same genome sequence. Cell-type variation in part arises from differential accessibility of cis-regulatory elements that encode the blueprints for transcriptional programs underlying cell identity and function. Owing to technical limitations, the role of cis-regulatory elements in cell identity maintenance, differentiation, and functional specialization has remained relatively unexplored in plant systems. Single-cell profiling has emerged as a powerful tool to circumvent these past obstacles by enabling unbiased charting of transcriptional and cis-regulatory states at the resolution of individual cells. Here, we review state-of-the-art single-cell approaches and analytical frameworks that have paved the way for establishing the link between cellular phenotypic variation and cis-regulatory mechanisms in plants.

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.

Leveraging histone modifications to improve genome annotations.

Accurate genome annotations are essential to modern biology; however, they remain challenging to produce. Variation in gene structure and expression across species, as well as within an organism, make correctly annotating genes arduous; an issue exacerbated by pitfalls in current in silico methods. These issues necessitate complementary approaches to add additional confidence and rectify potential misannotations. Integration of epigenomic data into genome annotation is one such approach. In this study, we utilized sets of histone modification data, which are precisely distributed at either gene bodies or promoters to evaluate the annotation of the Zea mays genome. We leveraged these data genome wide, allowing for identification of annotations discordant with empirical data. In total, 13,159 annotation discrepancies were found in Z. mays upon integrating data across three different tissues, which were corroborated using RNA-based approaches. Upon correction, genes were extended by an average of 2128 base pairs, and we identified 2529 novel genes. Application of this method to five additional plant genomes identified a series of misannotations, as well as identified novel genes, including 13,836 in Asparagus officinalis, 2724 in Setaria viridis, 2446 in Sorghum bicolor, 8631 in Glycine max, and 2585 in Phaseolous vulgaris. This study demonstrates that histone modification data can be leveraged to rapidly improve current genome annotations across diverse plant lineages.

Profiling single-cell chromatin accessibility in plants.

Coupling assay for transposase-accessible chromatin sequencing (ATAC-seq) with microfluidic separation and cellular barcoding has emerged as a powerful approach to investigate chromatin accessibility of individual cells. Here, we define a protocol for constructing single-cell ATAC-seq libraries from maize seedling nuclei and the preliminary computational steps for assessing data quality. This protocol can be readily adapted to other plant species or tissues with minor changes to reveal chromatin accessibility variation among individual cells. For complete details on the use and execution of this protocol, please refer to Marand et al. (2021).

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.

A cis-regulatory atlas in maize at single-cell resolution.

cis-regulatory elements (CREs) encode the genomic blueprints of spatiotemporal gene expression programs enabling highly specialized cell functions. Using single-cell genomics in six maize organs, we determined the cis- and trans-regulatory factors defining diverse cell identities and coordinating chromatin organization by profiling transcription factor (TF) combinatorics, identifying TFs with non-cell-autonomous activity, and uncovering TFs underlying higher-order chromatin interactions. Cell-type-specific CREs were enriched for enhancer activity and within unmethylated long terminal repeat retrotransposons. Moreover, we found cell-type-specific CREs are hotspots for phenotype-associated genetic variants and were targeted by selection during modern maize breeding, highlighting the biological implications of this CRE atlas. Through comparison of maize and Arabidopsis thaliana developmental trajectories, we identified TFs and CREs with conserved and divergent chromatin dynamics, showcasing extensive evolution of gene regulatory networks. In addition to this rich dataset, we developed single-cell analysis software, Socrates, which can be used to understand cis-regulatory variation in any species.

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.