ATAC-seq normalization method can significantly affect differential accessibility analysis and interpretation

ABSTRACTSaccharomyces cerevisiae undergoes robust oscillations to regulate its physiology for adaptation and survival under nutrient-limited conditions. Environmental cues can induce rhythmic metabolic alterations in order to facilitate the coordination of dynamic metabolic behaviors. Of such metabolic processes, the yeast metabolic cycle enables adaptation of the cells to varying nutritional status through oscillations in gene expression and metabolite production levels. In this process, yeast metabolism is altered between diverse cellular states based on changing oxygen consumption levels: quiescent (reductive charging [RC]), growth (oxidative [OX]), and proliferation (reductive building [RB]) phases. We characterized metabolic alterations during the yeast metabolic cycle using a variety of approaches. Gene expression levels are widely used for condition-specific metabolic simulations, whereas the use of epigenetic information in metabolic modeling is still limited despite the clear relationship between epigenetics and metabolism. This prompted us to investigate the contribution of epigenomic information to metabolic predictions for progression of the yeast metabolic cycle. In this regard, we determined altered pathways through the prediction of regulated reactions and corresponding model genes relying on differential chromatin accessibility levels. The predicted metabolic alterations were confirmed via data analysis and literature. We subsequently utilized RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) data sets in the contextualization of the yeast model. The use of ATAC-seq data considerably enhanced the predictive capability of the model. To the best of our knowledge, this is the first attempt to use genome-wide chromatin accessibility data in metabolic modeling. The preliminary results showed that epigenomic data sets can pave the way for more accurate metabolic simulations.IMPORTANCE Dynamic chromatin organization mediates the emergence of condition-specific phenotypes in eukaryotic organisms. Saccharomyces cerevisiae can alter its metabolic profile via regulation of genome accessibility and robust transcriptional oscillations under nutrient-limited conditions. Thus, both epigenetic information and transcriptomic information are crucial in the understanding of condition-specific metabolic behavior in this organism. Based on genome-wide alterations in chromatin accessibility and transcription, we investigated the yeast metabolic cycle, which is a remarkable example of coordinated and dynamic yeast behavior. In this regard, we assessed the use of ATAC-seq and RNA-seq data sets in condition-specific metabolic modeling. To our knowledge, this is the first attempt to use chromatin accessibility data in the reconstruction of context-specific metabolic models, despite the extensive use of transcriptomic data. As a result of comparative analyses, we propose that the incorporation of epigenetic information is a promising approach in the accurate prediction of metabolic dynamics.

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.

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

Introduction: Subpopulations of highly tumorigenic, drug resistant cancer stem-like cells (CSCs) play a key role in cancer recurrence and progression. Surprisingly, these aggressive cells can arise repeatedly de novo from bulk tumor cells independently of mutational events. We investigated whether transition to and from a cancer stem-like state is associated with epigenetic alterations, such as DNA methylation and chromatin accessibility. Materials and methods: Using FACS and Hoechst dye staining, side population (SP) cells marked by high tumorigenicity and drug resistance vs. non-side population (NSP) cells lacking these properties were isolated from bladder cancer cell lines. Global DNA methylation and chromatin accessibility were mapped for SP vs. NSP cells using AcceSssIble assay on the Infinium EPIC platform. Differential gene expression for SP vs. NSP cells was assayed by HuEx array. AcceSssIble and HuEx results were overlapped to generate a subset of genes with differential accessible promoter regions and expression. One such gene, E2F3, was validated by qRT-PCR and analyzed clinically using the Oncomine and Basespace online data mining engines, as well as functionally by lentiviral shRNA depletion in bladder cancer cells for cell migration, invasion and drug resistance. 5-AZA was used to modulate global DNA methylation in bladder cancer cells. Results: SP and NSP subpopulations exhibited differential DNA methylation and chromatin accessibility at thousands of gene loci by AcceSssIble assay, and a majority of differential chromatin accessibility was independent of DNA methylation. When overlapped with HuEx data, 38 genes exhibited differential expression associated with differential promoter chromatin accessibility. One such gene validated by qRT-PCR was E2F3, a cell cycle regulator that was found to be associated with bladder cancer in the Oncomine and Basespace databases. In vitro E2F3 depletion by lentiviral shRNA resulted in smaller SP subpopulations and reduced cell migration, invasiveness, and drug resistance. Epigenetic interference with 5-AZA blocked the phenotypic transition, reduced SP subpopulations, and lowered E2F3 expression. Conclusion: Rapid cyclical transition between the SP and NSP subpopulations was associated with dramatic shifts in chromatin accessibility with concomitant changes in gene expression of clinically relevant genes that drove the SP phenotype, cell migration, invasiveness, and drug resistance. These observations implicate epigenetic plasticity as a driver of the switch between cancer stem-like and non-cancer stem-like states, suggesting new potential therapeutic avenues to control cancer resistance and progression. Citation Format: Tong Xu, Hongtao Li, Rong Xu, Jenny Wei, Meng Li, Gangning Liang, Amir Goldkorn. Epigenetic plasticity potentiates a rapid cyclical shift to and from an aggressive cancer phenotype [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 4335.

We construct a simple workflow for fluent genomics data analysis using the R/Bioconductor ecosystem. This involves three core steps: importthe data into an appropriate abstraction, model the data with respect to the biological questions of interest, and integrate the results with respect to their underlying genomic coordinates. Here we show how to implement these steps to integrate published RNA-seq and ATAC-seq experiments on macrophage cell lines. Using tximeta, we importRNA-seq transcript quantifications into an analysis-ready data structure, called the SummarizedExperiment, that contains the ranges of the reference transcripts and metadata on their provenance. Using SummarizedExperiments to represent the ATAC-seq and RNA-seq data, we model differentially accessible (DA) chromatin peaks and differentially expressed (DE) genes with existing Bioconductor packages. Using plyrangeswe then integratethe results to see if there is an enrichment of DA peaks near DE genes by finding overlaps and aggregating over log-fold change thresholds. The combination of these packages and their integration with the Bioconductor ecosystem provide a coherent framework for analysts to iteratively and reproducibly explore their biological data.

We construct a simple workflow for fluent genomics data analysis using the R/Bioconductor ecosystem. This involves three core steps:import the data into an appropriate abstraction,model the data with respect to the biological questions of interest, andintegrate the results with respect to their underlying genomic coordinates. Here we show how to implement these steps to integrate published RNA-seq and ATAC-seq experiments on macrophage cell lines. Usingtximeta, weimport RNA-seq transcript quantifications into an analysis-ready data structure, called theSummarizedExperiment, that contains the ranges of the reference transcripts and metadata on their provenance. UsingSummarizedExperiments to represent the ATAC-seq and RNA-seq data, wemodel differentially accessible (DA) chromatin peaks and differentially expressed (DE) genes with existing Bioconductor packages. Usingplyranges we thenintegrate the results to see if there is an enrichment of DA peaks near DE genes by finding overlaps and aggregating over log-fold change thresholds. The combination of these packages and their integration with the Bioconductor ecosystem provide a coherent framework for analysts to iteratively and reproducibly explore their biological data.

Background: Salt-sensitivity of blood pressure (SSBP) puts both normotensive and hypertensive individuals at a higher risk for cardiovascular morbidity and mortality. Environmental and dietary factors, such as oxidative stress and salt, induce epigenetic regulation changes in the development of SSBP. Isolevuglandins (IsoLGs) are increased in multiple oxidative-stress-associated diseases, including SSBP. IsoLGs adduct histones, specifically H3 and H4 histone proteins. However, the impact of IsoLG-modified histones on gene transcription remains unclear. We hypothesized that elevated sodium in myeloid cells leads to IsoLG-adducted histones, thereby epigenetically contributing to salt-sensitive hypertension. Methods: RNA sequencing analysis was performed on I solated monocytes from 11 hypertensive individuals cultured in either normal sodium (150mM) or high sodium (190mM). In vivo studies were performed on 19 hypertensive subject enrolled in an acute inpatient protocol to assess response to salt loading (460 mmoL/24 hours) and salt depletion (10 mmoL/24 hours, and furosemide 40 mg x 3). IsoLG-containing dendritic cells (DCs) were identified using flow cytometry as CD45+MerTK-HLADR+CD1c+. Isolated PBMCs from a salt-sensitive subject (ΔSBPSD-SL = -12.9) and a salt-resistant subject (ΔSBPSD-SL = 0.35) were analyzed using an assay for transposase-accessible chromatin with high-throughput sequencing (ATACseq). Histones were isolated from splenic MHC-II+ cells treated in vitro with either normal or high sodium media and Western blotted for IsoLGs using a selective ScFv (D11). Results: Utilizing the bulk RNA sequencing of isolated monocytes, combined with the EpiFactors database, we identified differentially expressed genes related to histones and their modifications. High sodium exposure resulted in an overall upregulation of histone modifications, notably HIST4H4 (25.4 ± 4.6 vs. 65.5 ± 6.5; n=11, q=1.5 x 10-3, FDR < 0.05). IsoLG-containing DCs during the salt sensitivity protocol correlated with ∆SBP (R2=0.25, p=0.03). ATAC-seq data revealed differential chromatin accessibility and distinct motif enrichment in cDC2 and CD14+ monocyte clusters, highlighting significant epigenetic changes between salt-sensitive and salt-resistant individuals in response to salt status. Additionally, IsoLG-adducted histones and histone H4 expression levels increased in response to high sodium environments compared to normal sodium. Conclusions: Our results suggest the high sodium environment increase in IsoLG-adducts and chromatin accessibility may epigenetically regulate innate immune cells in SSBP. Funding: NIH-R01HL147818, This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Light is an important environmental factor affecting the ripening and quality of strawberry fruit. Previous studies have shown that red light treatment can promote strawberry ripening. Gene expression is closely associated with chromatin openness, and changes in chromatin accessibility are crucial for the binding of transcription factors to downstream regulatory sequences. However, the changes in chromatin accessibility in response to different light treatments in octoploid strawberry plants are still unclear. In this study, the landscape of chromatin accessibility of octoploid strawberry under red (R) and yellow-green (YG) light conditions was analyzed by the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Through bioinformatics and Venn diagram analyses, a total of 1456 and 1854 group-specific genes (GSGs) were screened in the R and YG groups, respectively. By using RNA sequencing (RNA-seq), 440 differentially expressed genes (DEGs) were identified. Among these genes, 194 were upregulated under red light treatment. Through joint analysis of ATAC-seq and RNA-seq data, three red group-specific genes with increased expression were identified, namely, FaTIP1, FaQKY and FaLBD1. Through gene expression and transient transformation analyses of strawberry fruit, we further demonstrated that FaTIP1 can respond to red light induction and promote the ripening process of strawberry fruit. Our results provide a reference for the study of chromatin accessibility in octoploid strawberry and reveal new factors involved in the fruit's response to red light and the regulation of the ripening process of strawberry fruit.

Comparative analysis of chromatin accessibility in porcine oocytes from follicles of different sizes.

1508 Single-cell epigenetic profiling highlights genetic impact on chromatin accessibility in SLE

The Osteosarcoma (OS) genome is characterized by aneuploidy and dramatic structural rearrangements often leading to copy number gain and loss. There is significant heterogeneity between patients. Additionally, there is less known regarding the epigenetic heterogeneity within osteosarcoma and how this may impact critical phenotypes such as therapy response and metastasis. To characterize the osteosarcoma epigenome, we integrated ATAC-seq and RNA-seq across a unique set of patient samples, PDXs and PDX-derived cell lines obtained across the disease continuum and including biopsies, resections, and metastases. Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) analysis revealed the presence of two distinct epigenetic cell states. Differential chromatin accessibility between the clusters showed ~3000 unique open regions per cluster. Using GREAT to associate the differential genomic regions to genes, the enriched pathways of Cluster 1 (C1) are related to mesenchymal cell proliferation, skeletal system morphogenesis, osteoblast differentiation, and Wnt signaling pathway, whereas Cluster 2 (C2) enriched pathways are related to cell motility and migration, extracellular matrix organization and regulation of protein serine/threonine kinase activity. Accessible regions in the chromatin were enriched for a unique set of transcription factor binding sites. C1 accessible regions are enriched with transcription factor binding motifs (TFBM) related to development including RUNX2/3, MEOX2, HOXA2/5, VAX1/2, MEF, VSX and DLX; whereas members of AP1 complex including FOSL1/2, c-FOS, JUN are TFBM enriched in open regions in C2. This is correlated with high protein expression of these transcription factors in a cluster-specific manner and differential binding to chromatin. As expected, gene expression showed a positive correlation with accessible regions in promoters. RUNX2 plays a major role in chromatin remodeling in C1, modifying the chromatin accessibility of 5300 regions, most located in <1kb from the promoters. In conclusion, we uncovered two epigenetically cell states regulated by a cluster-specific set of transcription factors which are able to remodel the chromatin. Citation Format: Eunice Lopez Fuentes, Andrew Clugston, Leanne C. Sayles, E. Alejandro Sweet-Cordero. Two epigenetically distinct cellular states in osteosarcoma are regulated by a cluster-specific set of pioneer transcription factors [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B011.

Isolated Changes in Chromatin Accessibility and Enhancer-Promoter Contacts at the β-Globin Locus Distinguish Fetal Hemoglobin Producing F-Cells from a-Cells

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.

Single-cell chromatin analysis provides new opportunities to identify homologous cells that regulate gene expression in a similar manner in different species. The aim of this study is to analyze chromatin accessibility of embryonic brain cells in chicken and turkey. Chromatin accessibility was profiled in single nuclei isolated from day-14 embryonic brain of chicken and turkey using the 10X Genomics Chromium ATAC (Assay for Transposase Accessible Chromatin) sequencing (data availability: GEO accession # GSE269838). Cluster analysis of the single-nuclei ATAC-seq data showed differential cluster patterns of embryonic brain cells between the two species based on the variation of chromatin accessibility. Data analysis further identified a total of 60,376 peaks associated with 22,382 genes in the chicken brain. These open chromatins were identified more frequently in the distal regulatory regions (n=43,224) compared to promoter regions (n=17,152). In the turkey brain, the analysis identified a total of 60,375 peaks associated with 14,516 genes. The majority of these open chromatins, similar to chicken brain, were also identified more frequently in the distal regulatory regions (n=56,834) compared to promoter regions (n=7,871). However, there was a 14-fold greater number of intergenic peaks in the turkey brain compared to chicken brain. Motif enrichment analysis of the peak sequences showed that the binding motif of the transcription factor ARID3A (AT-Rich Interaction Domain 3A) was commonly enriched as a distal cis-regulatory element of specific genes such as AGT, ANO4, ANO5, GPR139, GRIK1, GRM3, HAAO, LRRTM3, MGAT4C, RHCG, SLC25A21, TAL2, and TRPC5 in the brain cells of both chicken and turkey. Bulk RNA-seq analysis further showed that these predicted ARID3 target genes were expressed in the embryonic brain of chicken and turkey. The chorioallantoic membrane plays vital functions for embryonic development in chicken and turkey unlike the placental functions that modulate embryo development in mice. The expression of the predicted ARID3 target genes was quantified in the placenta and fetal brain of mouse. This comparative analysis showed that the majority of the ARID3 target genes were expressed in the mouse fetal brain but suppressed in the placenta. However, three genes AGT, HAAO and SLC25A21 were expressed in the placenta but suppressed in the fetal brain. The findings of this study suggest that ARID3A plays a common regulatory role in the brain development in avian and mammalian embryos. While role of ARID3A in placenta development is documented, further research is needed to understand the role of ARID3A in the development of chorioallantoic membrane of avian embryos.

Backround and aimsComplex host-virus interactions account for adaptive and innate immunity dysfunctions and viral cccDNA mini-chromosome persistence, key features of HBV chronicity and challenges for HBV cure. The extent of HBV direct impact on liver transcriptome remains controversial. Transcriptional activation in eukaryotic cells is tightly linked with disruption of nucleosome organization at accessible genomic sites of remodeled chromatin. We sought to investigate the impact of HBV on chromatin accessibility and transcription.MethodsWe used ATAC-seq (Assay for Transposase Accessible Chromatin followed by high throughput sequencing) to detect early changes in chromatin accessibility coupled with RNA-seq in HBV-infected Primary Human Hepatocytes (PHHs).ResultsAn increasing number of genomic sites change their nucleosome organization over time after HBV infection, with a prevalent, but not exclusive, reduction of chromatin accessibility at specific sites that is partially prevented by inhibiting HBV transcription and replication. ATAC-seq and RNA-seq integration showed that HBV infection impacts on liver fatty acids, bile acids, iron metabolism and liver cancer pathways. The upregulation of iron uptake genes leads to a significant increase of iron content in HBV-infected PHHs whereas iron chelation inhibits cccDNA transcription and viral replication. The chromatin accessibility and transcriptional changes imposed by HBV early after infection persist, as an epigenetic scar, in chronic HBV (CHB) patients and in HBV-related HCCs. These changes are to a large extent independent from viral replication levels and disease activity.ConclusionsAltogether our results show that HBV infection impacts on host cell chromatin landscape and specific transcriptional programs including liver metabolism and liver cancer pathways. Re-wiring of iron metabolism boosts viral replication early after infection. The modulation of genes involved in cancer-related pathways may favor the development or the selection of a pro-neoplastic phenotype and persists in HBV-related HCCs.