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A major goal in the field of transcriptional regulation is the mapping of changes in the binding of transcription factors to the resultant changes in gene expression. Recently, methods for measuring chromatin accessibility have enabled us to measure changes in accessibility across the genome, which are thought to correspond to transcription factor‐binding events. In concert with RNA‐sequencing, these data in principle enable such mappings; however, few studies have looked at their concordance over short‐duration treatments with specific perturbations. Here, we used tandem, bulk ATAC‐seq, and RNA‐seq measurements from MCF‐7 breast carcinoma cells to systematically evaluate the concordance between changes in accessibility and changes in expression in response to retinoic acid and TGF‐β. We found two classes of genes whose expression showed a significant change: those that showed some changes in the accessibility of nearby chromatin, and those that showed virtually no change despite strong changes in expression. The peaks associated with genes in the former group had lower baseline accessibility prior to exposure to signal. Focusing the analysis specifically on peaks with motifs for transcription factors associated with retinoic acid and TGF‐β signaling did not reduce the lack of correspondence. Analysis of paired chromatin accessibility and gene expression data from distinct paths along the hematopoietic differentiation trajectory showed a much stronger correspondence, suggesting that the multifactorial biological processes associated with differentiation may lead to changes in chromatin accessibility that reflect rather than driving altered transcriptional status. Together, these results show many gene expression changes can happen independently of changes in the accessibility of local chromatin in the context of a single‐factor perturbation. Synopsis Systematic analysis of tandem, bulk ATAC‐seq and RNA‐seq measurements from cells exposed to single‐factor perturbations shows two groups of genes: those with concordance between accessibility and expression changes and those without. MCF‐7 cells exposed to retinoic acid or TGF‐β have genes that show high expression changes without changes in local chromatin. By comparison, data from hematopoietic differentiation show much more concordance between chromatin accessibility changes and gene expression changes. Genes in the single‐factor perturbation data set that demonstrated more concordance had peaks that were less accessible at baseline prior to retinoic acid or TGF‐β exposure. Graphical Abstract Systematic analysis of tandem, bulk ATAC‐seq, and RNA‐seq measurements from cells exposed to single‐factor perturbations shows two groups of genes: those with concordance between accessibility and expression changes and those without.

T-cell exhaustion is a progressive loss of effector function and memory potential due to persistent antigen exposure, which occurs in chronic viral infections and cancer. Here we investigate the relation between gene expression and chromatin accessibility in CD8⁺ tumor-infiltrating lymphocytes (TILs) that recognize a model tumor antigen and have features of both activation and functional exhaustion. By filtering out accessible regions observed in bystander, nonexhausted TILs and in acutely restimulated CD8⁺ T cells, we define a pattern of chromatin accessibility specific for T-cell exhaustion, characterized by enrichment for consensus binding motifs for Nr4a and NFAT transcription factors. Anti–PD-L1 treatment of tumor-bearing mice results in cessation of tumor growth and partial rescue of cytokine production by the dysfunctional TILs, with only limited changes in gene expression and chromatin accessibility. Our studies provide a valuable resource for the molecular understanding of T-cell exhaustion in cancer and other inflammatory settings.

Across mammals, the epigenome is highly predictive of chronological age. These “epigenetic clocks,” most of which have been built using DNA methylation (DNAm) profiles, have gained traction as biomarkers of aging and organismal health. While the ability of DNAm to predict chronological age has been repeatedly demonstrated, the ability of other epigenetic features to predict age remains unclear. Here, we use two types of epigenetic information—DNAm, and chromatin accessibility as measured by ATAC‐seq—to develop age predictors in peripheral blood mononuclear cells sampled from a population of domesticated dogs. We measured DNAm and ATAC‐seq profiles for 71 dogs, building separate predictive clocks from each, as well as the combined dataset. We also use fluorescence‐assisted cell sorting to quantify major lymphoid populations for each sample. We found that chromatin accessibility can accurately predict chronological age (R2ATAC = 26%), though less accurately than the DNAm clock (R2DNAm = 33%), and the clock built from the combined datasets was comparable to both (R2combined = 29%). We also observed various populations of CD62L+ T cells significantly correlated with dog age. Finally, we found that all three clocks selected features that were in or near at least two protein‐coding genes: BAIAP2 and SCARF2, both previously implicated in processes related to cognitive or neurological impairment. Taken together, these results highlight the potential of chromatin accessibility as a complementary epigenetic resource for modeling and investigating biologic age. We used two types of epigenetic information—DNA methylation (DNAm), and chromatin accessibility as measured by ATAC‐seq—to develop age predictors in peripheral blood mononuclear cells sampled from a population of 71 domesticated dogs. Chromatin accessibility can predict chronological age, though less accurately than the DNAm clock or the clock built from the combined datasets. The types of meta data features selected per clock also varied by data type. These results suggest that chromatin accessibility may act as a complementary epigenetic resource for investigating biological age.

Neuroendocrine carcinoma (NEC) is a highly aggressive subtype of the neuroendocrine tumor with an extremely poor prognosis. We have previously conducted a comprehensive genomic analysis of over 100 cases of NEC of the gastrointestinal system (GIS‐NEC) and unraveled its unique and organ‐specific genomic drivers. However, the epigenomic features of GIS‐NEC remain unexplored. In this study, we have described the epigenomic landscape of GIS‐NEC and small cell lung carcinoma (SCLC) by integrating motif enrichment analysis from the assay of transposase‐accessible chromatin sequencing (ATAC‐seq) and enhancer profiling from a novel cleavage under targets and tagmentation (CUT&Tag) assay for H3K27ac and identified ELF3 as one of the super‐enhancer–related transcriptional factors in NEC. By combining CUT&Tag and knockdown RNA sequencing for ELF3, we uncovered the transcriptional network regulated by ELF3 and defined its distinctive gene signature, including AURKA, CDC25B, CLDN4, ITGB6, and YWAHB. Furthermore, a loss‐of‐function assay revealed that ELF3 depletion led to poor cell viability. Finally, using gene expression of clinical samples, we successfully divided GIS‐NEC patients into two subgroups according to the ELF3 signature and demonstrated that tumor‐promoting pathways were activated in the ELF3 signature–high group. Our findings highlight the transcriptional regulation of ELF3 as an oncogenic transcription factor and its tumor‐promoting properties in NEC. Multiorgan comprehensive epigenomic analysis identified ELF3 as a super‐enhancer–associated transcription factor and revealed its oncogenic properties in neuroendocrine carcinoma.

Animal studies have shown that virus infection causes changes in host chromatin accessibility, but little is known about changes in chromatin accessibility of plants infected by viruses and its potential impact. Here, rice infected by rice stripe virus (RSV) was used to investigate virus‐induced changes in chromatin accessibility. Our analysis identified a total of 6462 open‐ and 3587 closed‐differentially accessible chromatin regions (DACRs) in rice under RSV infection by ATAC‐seq. Additionally, by integrating ATAC‐seq and RNA‐seq, 349 up‐regulated genes in open‐DACRs and 126 down‐regulated genes in closed‐DACRs were identified, of which 34 transcription factors (TFs) were further identified by search of upstream motifs. Transcription levels of eight of these TFs were validated by reverse transcription‐PCR. Importantly, four of these TFs (OsWRKY77, OsWRKY28, OsZFP12 and OsERF91) interacted with RSV proteins and are therefore predicted to play important roles in RSV infection. This is the first application of ATAC‐seq and RNA‐seq techniques to analyse changes in rice chromatin accessibility caused by RSV infection. Integrating ATAC‐seq and RNA‐seq provides a new approach to select candidate TFs in response to virus infection. Changes in chromatin accessibility of rice infected by RSV were analysed by ATAC‐seq; four transcription factors (OsWRKY77, OsWRKY28, OsZFP12 and OsERF91) related to RSV infection were identified by integrating ATAC‐seq and RNA‐seq.

Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) is a simple protocol for detection of open chromatin. Computational footprinting, the search for regions with depletion of cleavage events due to transcription factor binding, is poorly understood for ATAC-seq. We propose the first footprinting method considering ATAC-seq protocol artifacts. HINT-ATAC uses a position dependency model to learn the cleavage preferences of the transposase. We observe strand-specific cleavage patterns around transcription factor binding sites, which are determined by local nucleosome architecture. By incorporating all these biases, HINT-ATAC is able to significantly outperform competing methods in the prediction of transcription factor binding sites with footprints.

BackgroundNeutrophils play an important role in the immune system by sensing environmental perturbations, including pathogens. Upon activation, neutrophils extrude their chromatin to form neutrophil extracellular traps (NETs), which trap and remove pathogens. Chromatin decondensation during NET formation is a regulated process that reflects both the inducing pathways and the cellular environment. However, most studies rely on non-physiological stimuli like phorbol 12-myristate 13-acetate (PMA), which bypass key regulatory mechanisms. As a result, how physiologically relevant inflammatory signals change neutrophil chromatin accessibility and relate to disease associated transcriptional states remains poorly understood.MethodsWe used the Assay for Transposase-Accessible Chromatin with sequencing (ATAC-Seq) to profile chromatin accessibility in neutrophils stimulated in whole blood with PMA and physiologically relevant inflammatory natural factors (NFs), including TNF-α, GM-CSF, fMLP, C5a, and IL-1β, alone and in combination. Chromatin responses were compared across conditions and integrated with publicly available transcriptomic sepsis cohorts.ResultsNF stimulation induced stimulus specific chromatin accessibility programs distinct from PMA. Individual NFs enriched specific transcription factor (TF) motif enrichments in a stimulus dependent manner, with GM-CSF associated with STAT motifs, TNF-α with NF-κB, and C5a/fMLP with AP-1, while the combined condition showed a cooperative response including CEBP. Integration with sepsis transcriptomic datasets revealed that promoter accessibility changes under NF stimulation corresponded to transcriptional states associated with disease severity, highlighting the upstream regulatory programs linked to clinical outcomes.ConclusionsThese findings demonstrate that NF stimulation in whole blood reveals chromatin accessibility programs in neutrophils that correlate with disease severity in sepsis. This approach provides a framework for linking cytokine driven neutrophil regulation to heterogenous inflammatory states in sepsis and other NET-associated diseases.

The advent of next-generation sequencing in crop improvement offers unprecedented insights into the chromatin landscape closely linked to gene activity governing key traits in plant development and adaptation. Particularly in maize, its dynamic chromatin structure is found to collaborate with massive transcriptional variations across tissues and developmental stages, implying intricate regulatory mechanisms, which highlights the importance of integrating chromatin information into breeding strategies for precise gene controls. The depiction of maize chromatin architecture using Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) provides great opportunities to investigate cis-regulatory elements, which is crucial for crop improvement. In this context, we developed an easy-to-implement ATAC-seq protocol for maize with fewer nuclei and simple equipment. We demonstrate a streamlined ATAC-seq protocol with four key steps for maize in which nuclei purification can be achieved without cell sorting and using only a standard bench-top centrifuge. Our protocol, coupled with the bioinformatic analysis, including validation by read length periodicity, key metrics, and correlation with transcript abundance, provides a precise and efficient assessment of the maize chromatin landscape. Beyond its application to maize, our testing design holds the potential to be applied to other crops or other tissues, especially for those with limited size and amount, establishing a robust foundation for chromatin structure studies in diverse crop species.

We present Model-based AnalysEs of Transcriptome and RegulOme (MAESTRO), a comprehensive open-source computational workflow ( http://github.com/liulab-dfci/MAESTRO ) for the integrative analyses of single-cell RNA-seq (scRNA-seq) and ATAC-seq (scATAC-seq) data from multiple platforms. MAESTRO provides functions for pre-processing, alignment, quality control, expression and chromatin accessibility quantification, clustering, differential analysis, and annotation. By modeling gene regulatory potential from chromatin accessibilities at the single-cell level, MAESTRO outperforms the existing methods for integrating the cell clusters between scRNA-seq and scATAC-seq. Furthermore, MAESTRO supports automatic cell-type annotation using predefined cell type marker genes and identifies driver regulators from differential scRNA-seq genes and scATAC-seq peaks.

We compared 24 primary pediatric T‐cell acute lymphoblastic leukemias (T‐ALL) collected at the time of initial diagnosis and relapse from 12 patients and 24 matched patient‐derived xenografts (PDXs). DNA methylation profile was preserved in PDX mice in 97.5% of the promoters (ρ = 0.99). Similarly, the genome‐wide chromatin accessibility (ATAC‐Seq) was preserved remarkably well (ρ = 0.96). Interestingly, both the ATAC regions, which showed a significant decrease in accessibility in PDXs and the regions hypermethylated in PDXs, were associated with immune response, which might reflect the immune deficiency of the mice and potentially the incomplete interaction between murine cytokines and human receptors. The longitudinal approach of this study allowed an observation that samples collected from patients who developed a type 1 relapse (clonal mutations maintained at relapse) preserved their genomic composition; whereas in patients who developed a type 2 relapse (subset of clonal mutations lost at relapse), the preservation of the leukemia's composition was more variable. In sum, this study underlines the remarkable genomic stability, and for the first time documents the preservation of the epigenomic landscape in T‐ALL‐derived PDX models. Synopsis Although patient‐derived xenografts (PDXs) of pediatric T‐cell acute lymphoblastic leukemias (T‐ALL) are generally stable at the genomic level, little is known about conservation of their epigenetic features and chromatin architecture. This study investigates epigenomic profiles of T‐ALL PDXs. Pediatric T‐ALL cells largely remain stable in their genomic and epigenomic profile when xenografted into NSG mice. PDXs from patients with type 1 relapse (all clonal mutations maintained at relapse) are more stable than PDXs from patients with type 2 relapse (subset of clonal mutations lost at relapse). PDX models of pediatric T‐ALL largely preserve the DNA methylation and the chromatin accessibility profiles (ATAC‐Seq) of the original leukemias. Hypoaccessible/hypermethylated regions in PDX compared to primary samples are associated with immune response functions, possibly reflecting the immune‐deficiency of the mice and the incomplete interaction between murine cytokines and human receptors. Graphical Abstract Although patient‐derived xenografts (PDXs) of pediatric T‐cell acute lymphoblastic leukemias (T‐ALL) are generally stable at the genomic level, little is known about conservation of their epigenetic features and chromatin architecture. This study investigates epigenomic profiles of T‐ALL PDXs.