Chromatin accessibility and transcriptome analyses reveal stress-responsive regulatory elements in Zygophyllum xanthoxylum.

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.

Lung cancer sequencing efforts have uncovered mutational signatures that are attributed to exposure to the cigarette smoke carcinogen benzo[a]pyrene. Benzo[a]pyrene metabolizes in cells to benzo[a]pyrene diol epoxide (BPDE) and reacts with guanine nucleotides to form bulky BPDE adducts. These DNA adducts block transcription and replication, compromising cell function and survival, and are repaired in human cells by the nucleotide excision repair pathway. Here, we applied high-resolution genomic assays to measure BPDE-induced damage formation and mutagenesis in human cells. We integrated the new damage and mutagenesis data with previous repair, DNA methylation, RNA expression, DNA replication, and chromatin component measurements in the same cell lines, along with lung cancer mutagenesis data. BPDE damage formation is significantly enhanced by DNA methylation and in accessible chromatin regions, including transcribed and early-replicating regions. Binding of transcription factors is associated primarily with reduced, but also enhanced damage formation, depending on the factor. While DNA methylation does not appear to influence repair efficiency, this repair was significantly elevated in accessible chromatin regions, which accumulated fewer mutations. Thus, when damage and repair drive mutagenesis in opposing directions, the final mutational patterns appear to be dictated by the efficiency of repair rather than the frequency of underlying damages.

Profiling of chromatin accessibility and regulatory elements toward understanding the mechanisms underlying growth regulation in the Pacific oyster (Crassostrea gigas)

Growing evidence indicates that transposons or transposable elements (TEs)-derived accessible chromatin regions (ACRs) play essential roles in multiple biological processes by interacting with trans-acting factors. However, the function of TE-derived ACRs in the regulation of gene expression in the rice genome has not been well characterized. In this study, we examined the chromatin dynamics in six types of rice tissues and found that ~8% of ACRs were derived from TEs and exhibited distinct levels of accessibility and conservation as compared to those without TEs. TEs exhibited a TE subtype-dependent impact on ACR formation, which can be mediated by changes in the underlying DNA methylation levels. Moreover, we found that tissue-specific TE-derived ACRs might function in the tissue development through the modulation of nearby gene expression. Interestingly, many genes in domestication sweeps were found to overlap with TE-derived ACRs, suggesting their potential functions in the rice domestication. In addition, we found that the expression divergence of 1070 duplicate gene pairs were associated with TE-derived ACRs and had distinct distributions of TEs and ACRs around the transcription start sites (TSSs), which may experience different selection pressures. Thus, our study provides some insights into the biological implications of TE-derived ACRs in the rice genome. Our results imply that these ACRs are likely involved in the regulation of tissue development, rice domestication and functional divergence of duplicated genes.

BackgroundThe pathological hallmarks of Alzheimer’s disease (AD) involve alterations in the expression of numerous genes associated with transcriptional levels, which are determined by chromatin accessibility. Here, the landscape of chromatin accessibility was studied to understand the outline of the transcription and expression of AD-associated metabolism genes in an AD mouse model.MethodsThe assay for transposase-accessible chromatin by sequencing (ATAC-seq) was used to investigate the AD-associated chromatin reshaping in the APPswe/PS1dE9 (APP/PS1) mouse model. ATAC-seq data in the hippocampus of 8-month-old APP/PS1 mice were generated, and the relationship between chromatin accessibility and gene expression was analyzed in combination with RNA sequencing. Gene ontology (GO) analysis was applied to elucidate biological processes and signaling pathways altered in APP/PS1 mice. Critical transcription factors were identified; alterations in chromatin accessibility were further confirmed using chromatin immunoprecipitation assays.ResultsWe identified 1690 increased AD-associated chromatin-accessible regions in the hippocampal tissues of APP/PS1 mice. These regions were enriched in genes related to diverse signaling pathways, including the PI3K-Akt, Hippo, TGF-β, and Jak-Stat signaling pathways, which play essential roles in regulating cell proliferation, apoptosis, and inflammatory responses. A total of 1003 decreased chromatin-accessible regions were considered to be related with declined AD-associated biological processes including cellular response to hyperoxia and insulin stimulus, synaptic transmission, and positive regulation of autophagy. In the APP/PS1 hippocampus, 1090 genes were found to be upregulated and 1081 downregulated. Interestingly, enhanced ATAC-seq signal was found in approximately 740 genes, with 43 exhibiting upregulated mRNA levels. Several genes involved in AD development were found to have a significantly increased expression in APP/PS1 mice compared to controls, including Sele, Clec7a, Cst7, and Ccr6. The signatures of numerous transcription factors, including Olig2, NeuroD1, TCF4, and NeuroG2, were found enriched in the AD-associated accessible chromatin regions. The transcription-activating marks of H3K4me3 and H3K27ac were also found increased in the promoters of these genes. These results indicate that the mechanism for the upregulation of genes could be attributed to the enrichment of open chromatin regions with transcription factors motifs and the histone marks H3K4me3 and H3K27ac.ConclusionOur study reveals that alterations in chromatin accessibility may be an initial mechanism in AD pathogenesis.

The genomes of flowering plants consist largely of transposable elements (TEs), some of which modulate gene regulation and function. However, the repetitive nature of TEs and difficulty of mapping individual TEs by short-read sequencing have hindered our understanding of their regulatory potential. Here we show that long-read chromatin fibre sequencing (Fiber-seq) comprehensively identifies accessible chromatin regions (ACRs) and CpG methylation across the maize genome. We uncover stereotypical ACR patterns at young TEs that degenerate with evolutionary age, resulting in TE enhancers preferentially marked by a novel plant-specific epigenetic feature: simultaneous hyper-CpG methylation and chromatin accessibility. We show that TE ACRs are co-opted as gene promoters and that ACR-containing TEs can facilitate gene amplification. Lastly, we uncover a pervasive epigenetic signature-hypo-5mCpG methylation and diffuse chromatin accessibility-directing TEs to specific loci, including the loci that sparked McClintock's discovery of TEs.

Regulatory regions maintain nucleosome-depleted, open chromatin status but simultaneously require the presence of nucleosomes for specific histone modifications. It remains unclear how these can be achieved for proper regulatory function. Here we demonstrate that nucleosomes positioned within accessible chromatin regions near the boundaries provide platforms for histone modifications while preventing the occlusion of regulatory elements. These boundary nucleosomes were particularly enriched for active or poised regulatory marks in human, such as histone acetylations, H3K4 methylations, H3K9me3, H3K79me2, and H4K20me1. Additionally, we found that based on a genome-wide profiling of ∼100 recombinant yeast strains, the location of open chromatin borders tends to vary mostly within 150 bp upon genetic perturbation whereas this positional variation increases in proportion to the sequence preferences of the underlying DNA for nucleosome formation. More than 40% of the local boundary shifts were associated with genetic variation in cis- or trans-acting factors. A sizeable fraction of the identified genetic factors was also associated with nearby gene expression, which was correlated with the distance between the transcription start site (tss) and the boundary that faces the tss. Taken together, the variation in the width of accessible chromatin regions may arise in conjunction with the modulation of the boundary nucleosomes by post-translational modifications or by chromatin regulators and in association with the activity of nearby gene transcription.

JASPAR (http://jaspar.genereg.net) is an open-access database storing curated, non-redundant transcription factor (TF) binding profiles representing transcription factor binding preferences as position frequency matrices for multiple species in six taxonomic groups. For this 2016 release, we expanded the JASPAR CORE collection with 494 new TF binding profiles (315 in vertebrates, 11 in nematodes, 3 in insects, 1 in fungi and 164 in plants) and updated 59 profiles (58 in vertebrates and 1 in fungi). The introduced profiles represent an 83% expansion and 10% update when compared to the previous release. We updated the structural annotation of the TF DNA binding domains (DBDs) following a published hierarchical structural classification. In addition, we introduced 130 transcription factor flexible models trained on ChIP-seq data for vertebrates, which capture dinucleotide dependencies within TF binding sites. This new JASPAR release is accompanied by a new web tool to infer JASPAR TF binding profiles recognized by a given TF protein sequence. Moreover, we provide the users with a Ruby module complementing the JASPAR API to ease programmatic access and use of the JASPAR collection of profiles. Finally, we provide the JASPAR2016 R/Bioconductor data package with the data of this release.

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.

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.

Whole human genome is packed into chromatin, which is dynamically remodeled. Chromatin structure has been extensively studied with cell lines, but information about chromatin structure in tissue context is lacking. We present genome-wide chromatin accessibility analysis of clinical tissue samples using transposase-accessible chromatin sequencing (ATAC-seq). Our sample cohort consist 11 benign prostatic hyperplasia (BPH), 16 primary prostate cancer (PC), and 11 castration resistant prostate cancer (CRPC) samples. We identified 23,307 to 136,104 regions of accessible chromatin per sample using MACS2 peak calling. Utilizing a peak unification method resulted in a unified set of 178,333 high confidence peaks across sample set. To find out which loci are differentially accessible during disease progression, we further compared normalized ATAC-seq signal between sample groups across the genome. We identified 4747 and 9445 differentially accessible regions (DARs) for BPH to PC and PC to CRPC comparison, respectively. Out of these, in 2961 and 6652 chromatin was opening and in 1786 and 2793 chromatin was closing in respective comparison. Using DARs, we observe clear separation of the sample groups. Earlier, we have characterized this cohort using DNA, RNA and DNA methylation sequencing as well as SWATH proteomics. Using these data and the same analysis approach as with DARs, we identified 2061 and 2723 differentially methylated regions (DMRs) in BPH to PC and PC to CRPC comparisons, respectively. We compared locations of DARs and DMRs and found out that these occur in different loci overlapping only in 27 and 35 loci in respective comparisons. When integrated with gene expression data, the chromatin accessibility correlated (|coefficient| &gt0.5) with the expression of at least one gene located in the same topologically associating domain (TAD) in altogether 2713 DARs. Next, we examined which transcription factors (TFs) are binding to DARs and thus putatively regulating gene-expression. Using HOMER database, we found several TFs with binding motif enrichment in our DARs. In BPH to PC comparison, opening DARs contain binding sites e.g. for AR and FOXA1 and, in PC to CRPC comparison, opening DARs contain binding sites e.g. for HOXB13, as expected. Interestingly, in PC to CRPC comparison closing DARs contain binding sites for AR and FOXA1 indicating that these TFs have smaller role or alternative regulatory programs when disease progresses. We utilized publicly available CHIP-seq datasets to study this more closely in DARs where ATAC-seq signal correlates with gene-expression within a TAD. Here, while the total number of AR binding sites doubles, the number of AR binding sites in closing DARs is ten times higher in CRPC to PC comparison than in PC to BPH comparison. These results suggest that chromatin accessibility is an important regulator of prostate cancer progression and changes occur in specific loci to where several relevant prostate cancer TFs can bind. Citation Format: Joonas I. Tuominen, Ebrahim Afyounian, Francesco Tabaro, Tomi Häkkinen, Anastasia Shcherban, Matti Annala, Kati Kivinummi, Teuvo Tammela, Juha Kesseli, Leena Latonen, Kirsi Granberg, Tapio Visakorpi, Matti Nykter. Chromatin alterations in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-096.

Epigenetic modifications are critical in precisely regulating gene expression. The common carp (Cyprinus carpio) is an economically important fish species, and females exhibit faster growth rates than males. However, the studies related to epigenetic modifications in the common carp gonads are limited. In this study, we conducted the Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) and Bisulfite sequencing (BS-seq) to explore the roles of epigenetic modifications in the common carp gonads. We identified 84,207 more accessible regions and 77,922 less accessible regions in ovaries compared to testes, and some sex-biased genes showed differential chromatin accessibility in their promoter regions, such as sox9a and zp3. Motif enrichment analysis showed that transcription factors (TFs) associated with embryonic development and cell proliferation were heavily enriched in ovaries, and the TFs Foxl2 and SF1 were only identified in ovaries. We also analyzed the possible regulations between chromatin accessibility and gene expression. By BS-seq, we identified 2087 promoter differentially methylated genes (promoter-DMGs) and 5264 gene body differentially methylated genes (genebody-DMGs) in CG contexts. These genebody-DMGs were significantly enriched in the Wnt signaling pathway, TGF-beta signaling pathway, and GnRH signaling pathway, indicating that methylation in gene body regions could play an essential role in sex maintenance, just like methylation in promoter regions. Combined with transcriptomes, we revealed that the expression of dmrtb1-like, spag6, and fels was negatively correlated with their methylation levels in promoter regions. Our study on the epigenetic modifications of gonads contributes to elucidating the molecular mechanism of sex differentiation and sex maintenance in the common carp.

Hypertension or elevated blood pressure (BP) is a worldwide clinical challenge and the leading primary risk factor for kidney dysfunctions, heart failure, and cerebrovascular disease. The kidney is a central regulator of BP by maintaining sodium-water balance. Multiple genome-wide association studies revealed that BP is a heritable quantitative trait, modulated by several genetic, epigenetic, and environmental factors. The SNPs identified in genome-wide association studies predominantly (>95%) reside within noncoding genomic regions, making it difficult to understand how they regulate BP. Given the central role of the kidney in regulating BP, we hypothesized that chromatin-accessible regions in renal tissue would be enriched for BP-associated single nucleotide polymorphisms. We manually dissected 2 important kidney segments that maintain the sodium-water balance: proximal tubules and medullary thick ascending limbs from the human and rat kidneys. To delineate their chromatin and transcriptomic profiles, we performed the assay for transposase-accessible chromatin and RNA sequencing, respectively. The chromatin accessibility maps revealed the shared and unique cis-regulatory elements that modulate the chromatin accessibility in proximal tubule and medullary thick ascending limbs of humans and rats. We developed a visualization tool to compare the cross-species epigenomic maps to identify potential regulatory targets for hypertension pathogenesis. We also identified a significant enrichment of BP-associated single nucleotide polymorphisms (1064 for human proximal tubule and 1172 for human medullary thick ascending limbs) within accessible chromatin regions of both segments, including rs1173771 and rs1421811 at the NPR3 locus and rs1800470 at the TGFb1 locus. Collectively, this study lays a foundation for interrogating how intergenic single nucleotide polymorphisms may regulate polygenic traits such as BP.

The primary effector cells involved in the formation of hypertrophic scars are fibroblasts. A potential peptide, ADSCP2 (adipose-derived stem cell peptide 2, the peptide fragment of ALCAM protein), derived from adipose-derived stem cell-conditioned medium, has been identified as having the potential to mitigate hypertrophic scar formation by targeting pyruvate carboxylase. However, the underlying mechanisms remain incompletely understood. Whether ADSCP2 attenuates hypertrophic scar fibrosis at the transcription level remains unclear. Consequently, this study sought to elucidate the potential mechanism associated with ADSCP2 by examining genome-wide transcriptional alterations and changes in chromatin accessibility in fibroblasts. This was achieved through the integrated analysis of assay for transposase accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq). In the ADSCP2 treatment group, ATAC-seq identified a total of 7,805 differential peaks associated with 3,176 genes. RNA-seq analysis revealed 345 upregulated and 399 downregulated transcripts in the same group. A combined Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of both downregulated genes and close-ACRs (accessible chromatin regions) genes within the ADSCP2 treatment group indicated regulation of the oxidative phosphorylation pathway (OXPHOS) by ADSCP2. The amalgamation of ATAC-seq and RNA-seq data elucidates that two OXPHOS associated genes, namely COX6B1 (cytochrome c oxidase subunit 6B1) and NDUFA1 (NADH dehydrogenase (ubiquinone) alpha subcomplex-1), demonstrate significant downregulation in the presence of ADSCP2. Further analysis using the integrative genomics viewer indicates that the promoter regions of both COX6B1 and NDUFA1 exhibit a higher degree of closure in the ADSCP2 treatment group. Quantitative PCR analysis demonstrated that ADSCP2 treatment resulted in a reduction of COX6B1 and NDUFA1 mRNA expression levels. Furthermore, cellular ATP and lactic acid concentrations were diminished in the ADSCP2-treated group. Collectively, these findings suggest potential avenues for future research into the therapeutic application of the peptide ADSCP2 in the treatment of hypertrophic scars.

Understanding gene regulation is fundamental to plant improvement. However, the lack of plant-specific single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) frameworks and cross-species databases has limited insights into cell-type-specific cellular regulation. Here we present 'scPlantReg', an integrated framework and database for plant scATAC-seq data. scPlantReg supports end-to-end analyses from raw data processing to biological interpretation and features 'scATACtor', a supervised machine-learning approach that outperforms existing tools for cell-type annotation. We applied scPlantReg to pearl millet to characterize cell-type-specific chromatin accessibility and identify validated activating and repressing accessible chromatin regions (ACRs), revealing WRKY transcription factors as potential regulators of xylem development. Furthermore, we reanalysed scATAC-seq datasets from 8 plant species, spanning 11 tissues and multiple developmental stages, enabling cross-species comparisons. These analyses uncovered conserved regulatory programmes, including AP2/EREBP-associated ACRs linked to cell wall development and cell-type-conserved TFs across grasses. Collectively, scPlantReg provides a general framework and resource for comparative regulatory analysis in plants.