A single-cell rice atlas integrates multi-species data to reveal cis-regulatory evolution

Cis-regulatory elements (CREs) are critical in regulating gene expression, and yet understanding of CRE evolution remains challenging. Here, we constructed a comprehensive single-cell atlas of chromatin accessibility in Oryza sativa, integrating data from 103,911 nuclei representing 126 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-derived 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 silencer-like CREs maintained by evolution. Collectively, this comparative genomics approach highlights the dynamics of plant cell-type-specific CRE evolution.

Decision letter: The single-cell chromatin accessibility landscape in mouse perinatal testis development

Decision letter: CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo

A number of deep learning models have been developed to predict epigenetic features such as chromatin accessibility from DNA sequence. Model evaluations commonly report performance genome-wide; however, cis regulatory elements (CREs), which play critical roles in gene regulation, make up only a small fraction of the genome. Furthermore, cell type specific CREs contain a large proportion of complex disease heritability. We evaluate genomic deep learning models in chromatin accessibility regions with varying degrees of cell type specificity. We assess two modeling directions in the field: general purpose models trained across thousands of outputs (cell types and epigenetic marks), and models tailored to specific tissues and tasks. We find that the accuracy of genomic deep learning models, including two state-of-the-art general purpose models - Enformer and Sei - varies across the genome and is reduced in cell type specific accessible regions. Using accessibility models trained on cell types from specific tissues, we find that increasing model capacity to learn cell type specific regulatory syntax - through single-task learning or high capacity multi-task models - can improve performance in cell type specific accessible regions. We also observe that improving reference sequence predictions does not consistently improve variant effect predictions, indicating that novel strategies are needed to improve performance on variants. Our results provide a new perspective on the performance of genomic deep learning models, showing that performance varies across the genome and is particularly reduced in cell type specific accessible regions. We also identify strategies to maximize performance in cell type specific accessible regions.

BackgroundA number of deep learning models have been developed to predict epigenetic features such as chromatin accessibility from DNA sequence. Model evaluations commonly report performance genome-wide; however, cis regulatory elements (CREs), which play critical roles in gene regulation, make up only a small fraction of the genome. Furthermore, cell type-specific CREs contain a large proportion of complex disease heritability.ResultsWe evaluate genomic deep learning models in chromatin accessibility regions with varying degrees of cell type specificity. We assess two modeling directions in the field: general purpose models trained across thousands of outputs (cell types and epigenetic marks) and models tailored to specific tissues and tasks. We find that the accuracy of genomic deep learning models, including two state-of-the-art general purpose models―Enformer and Sei―varies across the genome and is reduced in cell type-specific accessible regions. Using accessibility models trained on cell types from specific tissues, we find that increasing model capacity to learn cell type-specific regulatory syntax―through single-task learning or high capacity multi-task models―can improve performance in cell type-specific accessible regions. We also observe that improving reference sequence predictions does not consistently improve variant effect predictions, indicating that novel strategies are needed to improve performance on variants.ConclusionsOur results provide a new perspective on the performance of genomic deep learning models, showing that performance varies across the genome and is particularly reduced in cell type-specific accessible regions. We also identify strategies to maximize performance in cell type-specific accessible regions.

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.

Runx2 regulates chromatin accessibility to direct the osteoblast program at neonatal stages.

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.

Cis-regulatory elements (CREs) are enriched in accessible chromatin regions (ACRs) of eukaryotes. Despite extensive research on genome-wide ACRs in various plant tissues, the global impact of these changes on developmental processes in maize seeds remains poorly understood. In this study, we employed the assay for transposase-accessible chromatin sequencing (ATAC-seq) to reveal the chromatin accessibility profile throughout the genome during the early stages of maize seed development. We identified a total of 37 952 to 59 887 high-quality ACRs in maize seeds at 0 to 8 days after pollination (DAP). Furthermore, we examined the correlation between the identified ACRs and gene expression. We observed a positive correlation between the open degree of promoter-ACRs and the expression of most genes. Moreover, we identified binding footprints of numerous transcription factors (TFs) within chromatin accessibility regions and revealed key TF families involved in different stages. Through the footprints of accessible chromatin regions, we predicted transcription factor regulatory networks during early maize embryo development. Additionally, we discovered that DNA sequence diversity was notably reduced at ACRs, yet trait-associated SNPs were more likely to be located within ACRs. We edited the ACR containing the trait-associated SNP of NKD1. Both NKD1pro-1 and NKD1pro-2 showed phenotypes corresponding to the trait-associated SNP. Our results suggest that alterations in chromatin accessibility play a crucial role in maize seed development and highlight the potential contribution of open chromatin regions to advancements in maize breeding.

The goal of the Cancer Dependency Map project (DepMap) is to identify the landscape of cancer vulnerabilities by systematically profiling hundreds of cell lines representing the heterogeneity of human cancer. Cancer cells are known to undergo significant alterations in the cancer genome including changes in the accessible chromatin regions (ACRs). These alterations could lead to novel cancer vulnerabilities but chromatin accessibility has not been systematically profiled in DepMap. Profiling hundreds of cell lines individually for ACRs would require large resources. Here we show that we can efficiently profile hundreds of cell lines using two single-cell approaches developed by our labs. SHARE-seq is a single-cell high-throughput combinatorial indexing method to measure both chromatin accessibility (scATAC-seq), and gene expression (scRNA-seq) in the same single cell. MIX-Seq is an approach that leverages the ability to pool hundreds of unique cancer cell lines and then apply single-cell genomics across cell populations from each cell line, resolving its identity using single nucleotide polymorphism (SNP) profiles. Here, we used a combination of MIX-seq (pooling cell lines) and SHARE-seq (scATAC-seq and RNA-seq) to simultaneously profile 500 unique cell lines covering 110 lineages within 22 cancer types at the single-cell level. Encouragingly, in a UMAP representation of the data, cancer cell lines cluster by lineage. Interestingly, we identified hundreds of thousands of peaks within cis-regulatory elements (CREs), 1/3 of which are unique to cancer cell lines and not present in normal cell types. Using published tools, we calculated an ATAC-seq gene activity prediction score per cell line. To identify cancer vulnerabilities associated with ACRs changes, we combined the ATAC-seq gene-activity predictions with RNAseq-based features to construct machine-learning models of cancer dependency scores. We found that the prediction accuracy for multiple dependencies improved with the addition of the ATAC-seq-based scores, indicating that chromatin accessibility provides information beyond gene expression. Additional analyses are underway to create other features. Overall, we have generated a large high-quality dataset to simultaneously map the chromatin accessibility in hundreds of cancer cell lines. Importantly, the chromatin accessibility adds to other omics profiling to identify biomarkers of response and understand how changes in chromatin can lead to new vulnerabilities. Citation Format: Patricia Borck, Kirsty Wienand, Fabiana Duarte, Alvin Qin, Ruochi Zhang, Juliana Babu, Simone Zhang, Samuel Maffa, Max Horlbeck, Rojesh Shrestha, Joshua Dempster, Jennifer Roth, Catarina Campbell, Jason Buenrostro, Francisca Vazquez. Identifying cancer vulnerabilities associated with changes in chromatin accessibility by simultaneously profiling hundreds of cancer cell lines with ATAC-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB234.

Cis-regulatory elements (CREs) are critical for modulating gene expression and phenotypic diversity in maize. While genome-wide association study (GWAS) hits and expression quantitative trait loci (eQTLs) are often enriched in CREs, their molecular mechanisms remain poorly understood. Characterizing CREs within accessible chromatin regions (ACRs) offers a powerful approach to link noncoding variants to chromatin structure alterations and phenotypic variation. Here, we generated ATAC-seq profiles from seedling leaves of 214 maize inbred lines, identifying 82 174 consensus ACRs. Notably, 39.55% of these ACRs exhibited significant population-wide chromatin accessibility variation. By mapping chromatin accessibility quantitative trait loci (caQTLs), we discovered 27 004 loci, including 1398 predicted to disrupt transcription factor (TF)-binding sites. Integration with multi-omics data revealed 7405 caACR-target gene pairs and linked 56 caACRs to GWAS signals for 51 agronomic traits, with significant enrichment in flowering-related pathways. Functional candidates such as ZmZIM30 - putatively regulated by caACRs - emerged as key regulators of flowering time. At the fad7 locus associated with linolenic acid content, allelic variants overlapping a caQTL showed differential chromatin accessibility. Our study provides a high-resolution cis-elements of maize leaves, deciphers the genetic basis of chromatin accessibility variation, and bridges noncoding caQTLs to molecular mechanisms underlying GWAS hits.

Single Nuclei Multiomics Analysis of Transcriptional and Chromatin Accessibility of Tumor Cells Uncovers Molecular Signatures and Regulatory Elements of Malignant Clonal Evolution in Multiple Myeloma

The utilization of rice heterosis is essential for ensuring global food security; however, its molecular mechanism remains unclear. In this study, comprehensive analyses of accessible chromatin regions (ACRs), DNA methylation, and gene expression in inter-subspecific hybrid and its parents were performed to determine the potential role of chromatin accessibility in rice heterosis. The hybrid exhibited abundant ACRs, in which the gene ACRs and proximal ACRs were directly related to transcriptional activation rather than the distal ACRs. Regarding the dynamic accessibility contribution of the parents, paternal ZHF1015 transmitted a greater number of ACRs to the hybrid. Accessible genotype-specific target genes were enriched with overrepresented transcription factors, indicating a unique regulatory network of genes in the hybrid. Compared with its parents, the differentially accessible chromatin regions with upregulated chromatin accessibility were much greater than those with downregulated chromatin accessibility, reflecting a stronger regulation in the hybrid. Furthermore, DNA methylation levels were negatively correlated with ACR intensity, and genes were strongly affected by CHH methylation in the hybrid. Chromatin accessibility positively regulated the overall expression level of each genotype. ACR-related genes with maternal Z04A-bias allele-specific expression tended to be enriched during carotenoid biosynthesis, whereas paternal ZHF1015-bias genes were more active in carbohydrate metabolism. Our findings provide a new perspective on the mechanism of heterosis based on chromatin accessibility in inter-subspecific hybrid rice.

Previous studies showed that the pathological hallmarks of Alzheimer’s disease (AD) are characterized by β‐amyloid(Aβ) deposition, neurofibrillary tangles, the upregulation of inflammation and neuronal apoptosis. However, the molecular mechanisms of most genes participated in the pathways above remained unclear. Gene expression is associated with the transcriptional level which is decided by the chromatin accessibility. In this study, the landscape of chromatin accessibility was described to disclose the outline of the transcriptions of AD‐associated metabolism and gene expression in an AD mouse model.The assay for transposase‐accessible chromatin by sequencing (ATAC‐seq) was utilized to investigate the AD‐associated chromatin reshaping in APPswe/PS1dE9 AD mouse model. The ATAC‐seq data in the hippocampus of 8‐month‐old APP/PS1 mice was generated and the relationship between the chromatin accessibility and the gene expressions was analyzed in combination with RNA‐seq. Gene ontology (GO) analysis and KEGG were applied to facilitate elucidating the alteration of biological process and signaling pathways. In addition, critical transcription factors were identified and, the alteration of the chromatin accessibility was further confirmed using ChIP assays.The genomic location distribution of peaks showed that the peaks distributed on the promoter‐TSS regions of APP/PS1 mice is significantly more than WT mice and, 1690 AD‐associated chromatin accessible regions in the hippocampus tissues of APP/PS1 mice were identified. These regions were enriched in the signaling pathways included PI3K‐Akt signaling pathway, Hippo signaling pathway, TGF‐β signaling pathway and Jak‐Stat signaling pathway which play essential roles in regulating of cell proliferation, cell apoptosis and inflammatory. GO analysis showed that many AD‐associated biological processes declined including cellular response to hyperoxia and insulin stimulus, synaptic transmission, and positive regulation of autophagy. 1090 genes were found to be up‐regulated, and 1081 genes were found to be down‐regulated in the hippocampus of the AD model mice. Interestingly, enhanced ATAC‐seq signal were found around 740 genes, of which 43 genes exhibited up‐regulated mRNA level. Several most significantly increased genes were also identified, including Sele, Clec7a, Cst7 and Ccr6 which critically involved in the development of AD. In addition, a great deal of transcription factors, including Olig2, NeuroD1, TCF4, NeuroG2 were found to enrich in the AD‐associated accessible chromatin regions. Besides, the transcription‐activating marks of H3K4me3 and H3K27ac were increased in the promoters of Sele, Clec7a, Cst7 and Ccr6 gene. These results above indicates that the mechanism for the up‐regulation genes could attribute to the enrichment of open chromatin regions by transcription factors and histone marks H3K4me3 and H3K27ac.Altogether, our study reveals that the alterations of chromatin accessibility might be an initial mechanism in AD pathogenesis.Support or Funding InformationThis work was supported by grants from the National Natural Science Foundation of China (No.81430037, No.81871714) and the Beijing Postdoctoral Sustentation Fund of China (No.ZZ2019‐13).ATAC‐seq chromatin accessibility analysis in hippocampus of Alzheimer’s disease (AD) model mice. A. Distribution of chromatin‐accessible regions across the genome in AD and WT mice. B. Signaling pathway associated with chromatin accessibility in AD mice. C. GO analysis of biological process associated with accessible chromatin regions in AD mice. D. Venn diagram showing genes associated with the chromatin‐accessible regions in AD and differentially‐expressed genes.Figure 1Motif enrichment and Histone modifications at the accessible chromatin regions. A. The 15 motifs with the greatest enrichment. B. The fold changes of histone H3K4me3 and H3K27ac are determined by ChIP‐qPCR, in chromatin‐accessible regions of SELE, Ccr6, CD300lf, Clec7a and Cst7 genes. Data are shown as mean ± SD. *p<0.05 and **p<0.01.Figure 2

The adaptive evolution of xerophytes to abiotic stresses is intimately linked with evolutionarily conserved cis-regulatory elements (CREs). However, the key CREs and conserved non-coding sequences (CNSs) involved in stress responses remain largely unexplored. In this study, we investigated the epigenetic regulatory mechanisms of the xerophyte Zygophyllum xanthoxylum under abiotic stress using an integrated multi-omics approach. Using ATAC-seq, we mapped genome-wide chromatin accessibility profiles in shoots and roots under heat, salt, and drought treatments, identifying 2,423-77,497 high-quality accessible chromatin regions (ACRs). Zygophyllum xanthoxylum employs distinct chromatin remodeling strategies for different stresses: chromatin tends to open under salt and drought conditions but close under heat stress. By integrating RNA-seq data, we correlated ACR dynamics with gene expression changes and identified significant alterations in transcription factor binding profiles within these accessible regions. We validated a stress-responsive regulatory pathway where the ZxNF-YC10-ZxAPK1 module induces promoter-associated ACR opening, leading to gene expression upregulation. Comparative genomic analysis across five xerophyte species identified 165,896 phylogenetically conserved non-coding sequences containing critical transcription factor binding sites. These species-specific CNSs were significantly enriched within central regions of ACRs and exhibited higher overlap with root ACRs, suggesting they may represent crucial CREs that evolved during adaptation to adverse environments. Our study provides a comprehensive chromatin accessibility atlas for Z. xanthoxylum under diverse abiotic stresses and establishes cross-species CNS resources that may facilitate genetic improvement of stress-tolerant crops.