An official website of the United States government
Abstract Cis-regulatory elements (CREs) are essential for regulating gene expression, yet their evolutionary dynamics in plants remain elusive. Here we constructed a single-cell chromatin accessibility atlas forOryza sativafrom 103,911 nuclei representing 126 cell states across nine organs. Comparative genomics betweenO. sativaand 57,552 nuclei from four additional grass species (Zea mays,Sorghum bicolor,Panicum miliaceumandUrochloa fusca) revealed that chromatin accessibility conservation varies with cell-type specificity. Epidermal accessible chromatin regions in the leaf were less conserved compared to other cell types, indicating accelerated regulatory evolution in the L1-derived epidermal layer ofO. sativarelative to other species. Conserved accessible chromatin regions overlapping the repressive histone modification H3K27me3 were identified as potentially silencer-like CREs, as deleting these regions led to up-regulation of gene expression. This study provides a comprehensive epigenomic resource for the rice community, demonstrating the utility of a comparative genomics approach that highlights the dynamics of plant cell-type-specific CRE evolution.
more »
« less
Exploring plant cis‐regulatory elements at single‐cell resolution: overcoming biological and computational challenges to advance plant research
SUMMARY Cis‐regulatory elements (CREs) are important sequences for gene expression and for plant biological processes such as development, evolution, domestication, and stress response. However, studying CREs in plant genomes has been challenging. The totipotent nature of plant cells, coupled with the inability to maintain plant cell types in culture and the inherent technical challenges posed by the cell wall has limited our understanding of how plant cell types acquire and maintain their identities and respond to the environment via CRE usage. Advances in single‐cell epigenomics have revolutionized the field of identifying cell‐type‐specific CREs. These new technologies have the potential to significantly advance our understanding of plant CRE biology, and shed light on how the regulatory genome gives rise to diverse plant phenomena. However, there are significant biological and computational challenges associated with analyzing single‐cell epigenomic datasets. In this review, we discuss the historical and foundational underpinnings of plant single‐cell research, challenges, and common pitfalls in the analysis of plant single‐cell epigenomic data, and highlight biological challenges unique to plants. Additionally, we discuss how the application of single‐cell epigenomic data in various contexts stands to transform our understanding of the importance of CREs in plant genomes.
more »
« less
- PAR ID:
- 10520978
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 115
- Issue:
- 6
- ISSN:
- 0960-7412
- Page Range / eLocation ID:
- 1486 to 1499
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Understanding the intimate interactions between plants and their microbiota at the cellular level is essential for unlocking the full potential of plant holobionts in agricultural systems. Traditional bulk and microbial community-level sequencing approaches reveal broad community patterns but fail to resolve how distinct plant cell types interact with or regulate microbial colonization, as well as the diverse antagonistic and synergistic interactions and responses existing between various microbial populations. Recent advances in single-cell and spatial multi-omics have transformed our understanding of plant cell identities as well as gene regulatory programs and their dynamic regulation in response to environmental stresses and plant development. In this review, we highlight the single-cell discoveries that uncover the plant cell-type-specific microbial perception, immune activation, and symbiotic differentiation, particularly in roots, nodules, and leaves. We further discuss how integrating transcriptomic, epigenomic, and spatial data can reconstruct multilayered interaction networks that connect plant cell-type-specific regulatory states with microbial spatial niches and inter-kingdom signaling (e.g., ligand–receptor and metabolite exchange), providing a foundation for developing new strategies to engineer crop–microbiome interactions to support sustainable agriculture. We conclude by outlining key methodological challenges and future research priorities that point toward building a fully integrated cellular interactome of the plant holobiont.more » « less
-
Abstract 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.more » « less
-
Abstract Cis-regulatory elements (CREs) control gene expression, orchestrating tissue identity, developmental timing and stimulus responses, which collectively define the thousands of unique cell types in the body1–3. While there is great potential for strategically incorporating CREs in therapeutic or biotechnology applications that require tissue specificity, there is no guarantee that an optimal CRE for these intended purposes has arisen naturally. Here we present a platform to engineer and validate synthetic CREs capable of driving gene expression with programmed cell-type specificity. We take advantage of innovations in deep neural network modelling of CRE activity across three cell types, efficient in silico optimization and massively parallel reporter assays to design and empirically test thousands of CREs4–8. Through large-scale in vitro validation, we show that synthetic sequences are more effective at driving cell-type-specific expression in three cell lines compared with natural sequences from the human genome and achieve specificity in analogous tissues when tested in vivo. Synthetic sequences exhibit distinct motif vocabulary associated with activity in the on-target cell type and a simultaneous reduction in the activity of off-target cells. Together, we provide a generalizable framework to prospectively engineer CREs from massively parallel reporter assay models and demonstrate the required literacy to write fit-for-purpose regulatory code.more » « less
-
INTRODUCTION Genome-wide association studies (GWASs) have identified thousands of human genetic variants associated with diverse diseases and traits, and most of these variants map to noncoding loci with unknown target genes and function. Current approaches to understand which GWAS loci harbor causal variants and to map these noncoding regulators to target genes suffer from low throughput. With newer multiancestry GWASs from individuals of diverse ancestries, there is a pressing and growing need to scale experimental assays to connect GWAS variants with molecular mechanisms. Here, we combined biobank-scale GWASs, massively parallel CRISPR screens, and single-cell sequencing to discover target genes of noncoding variants for blood trait loci with systematic targeting and inhibition of noncoding GWAS loci with single-cell sequencing (STING-seq). RATIONALE Blood traits are highly polygenic, and GWASs have identified thousands of noncoding loci that map to candidate cis -regulatory elements (CREs). By combining CRE-silencing CRISPR perturbations and single-cell readouts, we targeted hundreds of GWAS loci in a single assay, revealing target genes in cis and in trans . For select CREs that regulate target genes, we performed direct variant insertion. Although silencing the CRE can identify the target gene, direct variant insertion can identify magnitude and direction of effect on gene expression for the GWAS variant. In select cases in which the target gene was a transcription factor or microRNA, we also investigated the gene-regulatory networks altered upon CRE perturbation and how these networks differ across blood cell types. RESULTS We inhibited candidate CREs from fine-mapped blood trait GWAS variants (from ~750,000 individual of diverse ancestries) in human erythroid progenitors. In total, we targeted 543 variants (254 loci) mapping to candidate CREs, generating multimodal single-cell data including transcriptome, direct CRISPR gRNA capture, and cell surface proteins. We identified target genes in cis (within 500 kb) for 134 CREs. In most cases, we found that the target gene was the closest gene and that specific enhancer-associated biochemical hallmarks (H3K27ac and accessible chromatin) are essential for CRE function. Using multiple perturbations at the same locus, we were able to distinguished between causal variants from noncausal variants in linkage disequilibrium. For a subset of validated CREs, we also inserted specific GWAS variants using base-editing STING-seq (beeSTING-seq) and quantified the effect size and direction of GWAS variants on gene expression. Given our transcriptome-wide data, we examined dosage effects in cis and trans in cases in which the cis target is a transcription factor or microRNA. We found that trans target genes are also enriched for GWAS loci, and identified gene clusters within trans gene networks with distinct biological functions and expression patterns in primary human blood cells. CONCLUSION In this work, we investigated noncoding GWAS variants at scale, identifying target genes in single cells. These methods can help to address the variant-to-function challenges that are a barrier for translation of GWAS findings (e.g., drug targets for diseases with a genetic basis) and greatly expand our ability to understand mechanisms underlying GWAS loci. Identifying causal variants and their target genes with STING-seq. Uncovering causal variants and their target genes or function are a major challenge for GWASs. STING-seq combines perturbation of noncoding loci with multimodal single-cell sequencing to profile hundreds of GWAS loci in parallel. This approach can identify target genes in cis and trans , measure dosage effects, and decipher gene-regulatory networks.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1111/tpj.16351
