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.

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.

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.

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes.

Base J, ß-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.

Stable unmethylated DNA demarcates expressed genes and their cis-regulatory space in plant genomes.

The genomic sequences of crops continue to be produced at a frenetic pace. It remains challenging to develop complete annotations of functional genes and regulatory elements in these genomes. Chromatin accessibility assays enable discovery of functional elements; however, to uncover the full portfolio of cis-elements would require profiling of many combinations of cell types, tissues, developmental stages, and environments. Here, we explore the potential to use DNA methylation profiles to develop more complete annotations. Using leaf tissue in maize, we define ∼100,000 unmethylated regions (UMRs) that account for 5.8% of the genome; 33,375 UMRs are found greater than 2 kb from genes. UMRs are highly stable in multiple vegetative tissues, and they capture the vast majority of accessible chromatin regions from leaf tissue. However, many UMRs are not accessible in leaf, and these represent regions with potential to become accessible in specific cell types or developmental stages. These UMRs often occur near genes that are expressed in other tissues and are enriched for binding sites of transcription factors. The leaf-inaccessible UMRs exhibit unique chromatin modification patterns and are enriched for chromatin interactions with nearby genes. The total UMR space in four additional monocots ranges from 80 to 120 megabases, which is remarkably similar considering the range in genome size of 271 megabases to 4.8 gigabases. In summary, based on the profile from a single tissue, DNA methylation signatures provide powerful filters to distill large genomes down to the small fraction of putative functional genes and regulatory elements.

Historical Meiotic Crossover Hotspots Fueled Patterns of Evolutionary Divergence in Rice.

Recombination plays an integral role in the creation of novel genetic variation in sexually reproducing species. Despite this important role, the determinants and evolution of crossover hotspots have remained poorly understood in plants. Here, we present a comparative analysis of two rice (Oryza sativa) historical recombination maps from two subspecies (indica and japonica) using 150 resequenced genomes. Fine-scale recombination rates and crossover hotspots were validated by comparison with a consensus genetic map and empirically derived crossovers, respectively. Strikingly, nearly 80% of crossover hotspots were unique to each subspecies, despite their relatively recent divergence and broad-scale correlated recombination rates. Crossover hotspots were enriched with Stowaway and P instability factor (PIF)/Harbinger transposons and overlapped accessible chromatin regions. Increased nucleotide diversity and signatures of population differentiation augmented by Stowaway and PIF/Harbinger transposons were prevalent at subspecies-specific crossover hotspots. Motifs derived from lineage-specific indica and japonica crossover hotspots were nearly identical in the two subspecies, implicating a core set of crossover motifs in rice. Finally, Stowaway and PIF/Harbinger transposons were associated with stabilized G/C bias within highly active hotspots, suggesting that hotspot activity can be fueled by de novo variation. These results provide evolutionary insight into historical crossover hotspots as potentially powerful drivers of sequence and subspecies evolution in plants.

Genome sequence of M6, a diploid inbred clone of the high-glycoalkaloid-producing tuber-bearing potato species Solanum chacoense, reveals residual heterozygosity.

Cultivated potato (Solanum tuberosum L.) is a highly heterozygous autotetraploid that presents challenges in genome analyses and breeding. Wild potato species serve as a resource for the introgression of important agronomic traits into cultivated potato. One key species is Solanum chacoense and the diploid, inbred clone M6, which is self-compatible and has desirable tuber market quality and disease resistance traits. Sequencing and assembly of the genome of the M6 clone of S. chacoense generated an assembly of 825 767 562 bp in 8260 scaffolds with an N50 scaffold size of 713 602 bp. Pseudomolecule construction anchored 508 Mb of the genome assembly into 12 chromosomes. Genome annotation yielded 49 124 high-confidence gene models representing 37 740 genes. Comparative analyses of the M6 genome with six other Solanaceae species revealed a core set of 158 367 Solanaceae genes and 1897 genes unique to three potato species. Analysis of single nucleotide polymorphisms across the M6 genome revealed enhanced residual heterozygosity on chromosomes 4, 8 and 9 relative to the other chromosomes. Access to the M6 genome provides a resource for identification of key genes for important agronomic traits and aids in genome-enabled development of inbred diploid potatoes with the potential to accelerate potato breeding.

Proliferation of Regulatory DNA Elements Derived from Transposable Elements in the Maize Genome.

Genomic regions free of nucleosomes, which are hypersensitive to DNase I digestion, are known as DNase I hypersensitive sites (DHSs) and frequently contain cis-regulatory DNA elements. To investigate their prevalence and characteristics in maize (Zea mays), we developed high-resolution genome-wide DHS maps using a modified DNase-seq technique. Maize DHSs exhibit depletion of nucleosomes and low levels of DNA methylation and are enriched with conserved noncoding sequences (CNSs). We developed a protoplast-based transient transformation assay to assess the potential gene expression enhancer and/or promoter functions associated with DHSs, which showed that more than 80% of DHSs overlapping with CNSs showed an enhancer function. Strikingly, nearly 25% of maize DHSs were derived from transposable elements (TEs), including both class I and class II transposons. Interestingly, TE-derived DHSs (teDHSs) homologous to retrotransposons were enriched with sequences related to the intrinsic cis-regulatory elements within the long terminal repeats of retrotransposons. We demonstrate that more than 80% of teDHSs can drive transcription of a reporter gene in protoplast assays. These results reveal the widespread occurrence of TE-derived cis-regulatory sequences and suggest that teDHSs play a major role in transcriptional regulation in maize.

PlantDHS: a database for DNase I hypersensitive sites in plants.

Gene expression is regulated by orchestrated binding of regulatory proteins to promoters and other cis-regulatory DNA elements (CREs). Several plant databases have been developed for mapping promoters or DNA motifs associated with promoters. However, there is a lack of databases that allow investigation for all CREs. Here we present PlantDHS (http://plantdhs.org), a plant DNase I hypersensitive site (DHS) database that integrates histone modification, RNA sequencing, nucleosome positioning/occupancy, transcription factor binding sites, and genomic sequence within an easily navigated user interface. DHSs are indicative of all CREs, including promoters, enhancers, silencers, insulators and transcription factor binding sites; all of which play immense roles in global gene expression regulation. PlantDHS provides a platform to predict all CREs associated with individual genes from three model plant species, including Arabidopsis thaliana, Brachypodium distachyon and rice (Oryza sativa). PlantDHS is especially valuable in the detection of distant CREs that are located away from promoters.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.