Chromatin Accessibility Analysis Research Articles

Integrative Analysis of Chromatin Accessibility and Transcriptional Landscape Identifies Key Genes During Muscle Development in Pigs

Many efforts have been made to reveal the mechanisms underlying skeletal muscle development because of its importance in animals. However, knowledge on chromatin accessibility, a prerequisite for gene expression, remains limited. Here, dynamic changes in chromatin accessibility were analyzed in the skeletal muscles of Min pigs at the ages of 30, 90, and 210 d using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). A total of 16,301 differentially accessible regions (DARs) associated with 7455 genes were identified among three developmental stages. Seven out of eight DARs selected for a functional analysis were found to regulate reporter gene expression significantly (p < 0.05), indicating that DARs are active in gene expression. A total of 2219 differentially expressed genes (DEGs) were identified with RNA sequencing (RNA-seq). Through integrated analyses of ATAC-seq and RNA-seq data, 54 DEG_DAR_genes and 61 transcription factors (TFs) were characterized as critical for muscle development. Among them, Kruppel-like factor 5 (KLF5), targeted to 36 DEG_DAR_genes, was the most important TF. The effects of KLF5 on DEG_DAR_gene expression were then analyzed with molecular biology techniques. KLF5 was found to regulate SLPI (secretory leukocyte proteinase inhibitor) expression by directly binding to the promoter; KLF5 was also involved in APOA1 (apolipoprotein A-I) expression through affecting the regulatory role of DAR located in the intron. These results indicate that the TFs identified were functional. Altogether, the chromatin accessibility region, TFs, and genes important for muscle development in Min pigs were identified. The results provide novel data for further revealing the mechanisms underlying the epigenetic regulation of muscle development.

Impact of c-JUN deficiency on thalamus development in mice and human neural models

Backgroundc-Jun is a key regulator of gene expression. Through the formation of homo- or heterodimers, c-JUN binds to DNA and regulates gene transcription. While c-Jun plays a crucial role in embryonic development, its impact on nervous system development in higher mammals, especially for some deep structures, for example, thalamus in diencephalon, remains unclear.MethodsTo investigate the influence of c-JUN on early nervous system development, c-Jun knockout (KO) mice and c-JUN KO H1 embryonic stem cells (ESCs)-derived neural progenitor cells (NPCs), cerebral organoids (COs), and thalamus organoids (ThOs) models were used. We detected the dysplasia via histological examination and immunofluorescence staining, omics analysis, and loss/gain of function analysis.ResultsAt embryonic day 14.5, c-Jun knockout (KO) mice exhibited sparseness of fibers in the brain ventricular parenchyma and malformation of the thalamus in the diencephalon. The absence of c-JUN accelerated the induction of NPCs but impaired the extension of fibers in human neuronal cultures. COs lacking c-JUN displayed a robust PAX6+/NESTIN+ exterior layer but lacked a fibers-connected core. Moreover, the subcortex-like areas exhibited defective thalamus characteristics with transcription factor 7 like 2-positive cells. Notably, in guided ThOs, c-JUN KO led to inadequate thalamus patterning with sparse internal nerve fibers. Chromatin accessibility analysis confirmed a less accessible chromatin state in genes related to the thalamus. Overexpression of c-JUN rescued these defects. RNA-seq identified 18 significantly down-regulated genes including RSPO2, WNT8B, MXRA5, HSPG2 and PLAGL1 while 24 genes including MSX1, CYP1B1, LMX1B, NQO1 and COL2A1 were significantly up-regulated.ConclusionOur findings from in vivo and in vitro experiments indicate that c-JUN depletion impedes the extension of nerve fibers and renders the thalamus susceptible to dysplasia during early mouse embryonic development and human ThO patterning. Our work provides evidence for the first time that c-JUN is a key transcription regulator that play important roles in the thalamus/diencephalon development.

Ancestral Genomic Functional Differences in Oligodendroglia: Implications for Alzheimer's Disease.

This study aims to elucidate ancestry-specific changes to the genomic regulatory architecture in induced pluripotent stem cell (iPSC)-derived oligodendroglia, focusing on their implications for Alzheimer's disease (AD). This work addresses the lack of diversity in previous iPSC studies by including ancestries that contribute to African American (European/African) and Hispanic/Latino populations (Amerindian/African/European). We generated 12 iPSC lines-four African, four Amerindian, and four European- from both AD patients and non-cognitively impaired individuals, with varying APOE genotypes (APOE3/3 and APOE4/4). These lines were differentiated into neural spheroids containing oligodendrocyte lineage cells. Single-nuclei RNA sequencing and ATAC sequencing were employed to analyze transcriptional and chromatin accessibility profiles, respectively. Differential gene expression, chromatin accessibility, and Hi-C analyses were conducted, followed by pathway analysis to interpret the results. We identified ancestry-specific differences in gene expression and chromatin accessibility. Notably, numerous AD GWAS-associated genes were differentially expressed across ancestries. The largest number of differentially expressed genes (DEGs) were found in European vs. Amerindian and African vs. Amerindian iPSC-derived oligodendrocyte progenitor cells (OPCs). Pathway analysis of APOE4/4 carriers vs APOE3/3 carriers exhibited upregulation of a large number of disease and metabolic pathways in APOE4/4 individuals of all ancestries. Of particular interest was that APOE4/4 carriers had significantly upregulated cholesterol biosynthesis genes relative to APOE3/3 individuals across all ancestries, strongest in iOPCs. Comparison of iOPC and iOL transcriptome data with corresponding human frontal cortex data demonstrated a high correlation (R2 > 0.85). This research emphasizes the importance of including diverse ancestries in AD research to uncover critical gene expression differences between populations and ancestries that may influence disease susceptibility and therapeutic interventions. The upregulation of cholesterol biosynthesis genes in APOE4/4 carriers of all three ancestries supports the concept that APOE4 may produce disease effects early in life, which could have therapeutic implications as we move forward towards specific therapy for APOE4 carriers. These findings and the high correlation between brain and iPSC-derived OPC and OL transcriptomes support the relevance of this approach as a model for disease study.

Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties.

Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here, we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2 knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single-cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested hepatocyte nuclear factor 4 alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9, a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.

Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties

Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here, we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2 knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single-cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested hepatocyte nuclear factor 4 alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9, a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.

Differential chromatin accessibility and transcriptional dynamics define breast cancer subtypes and their lineages

Breast cancer (BC) is defined by distinct molecular subtypes with different cells of origin. The transcriptional networks that characterize the subtype-specific tumor-normal lineages are not established. In this work, we applied bulk, single-cell and single-nucleus multi-omic techniques as well as spatial transcriptomics and multiplex imaging on 61 samples from 37 patients with BC to show characteristic links in gene expression and chromatin accessibility between BC subtypes and their putative cells of origin. Regulatory network analysis of transcription factors underscored the importance of BHLHE40 in luminal BC and luminal mature cells and KLF5 in basal-like tumors and luminal progenitor cells. Furthermore, we identify key genes defining the basal-like (SOX6 and KCNQ3) and luminal A/B (FAM155A and LRP1B) lineages. Exhausted CTLA4-expressing CD8+ T cells were enriched in basal-like BC, suggesting an altered means of immune dysfunction. These findings demonstrate analysis of paired transcription and chromatin accessibility at the single-cell level is a powerful tool for investigating cancer lineage and highlight transcriptional networks that define basal and luminal BC lineages.

Single-nucleus transcriptional and chromatin accessibility analyses of maturing mouse Achilles tendon uncover the molecular landscape of tendon stem/progenitor cells.

Tendons and ligaments are crucial connective tissues linking bones and muscles, yet achieving full functional recovery after injury remains challenging. We investigated the characteristics of tendon stem/progenitor cells (TSPCs) by focusing on the declining tendon repair capacity with growth. Using single-cell RNA sequencing on Achilles tendon cells from 2- and 6-week-old mice, we identified Cd55 and Cd248 as novel surface antigen markers for TSPCs. Combining single-nucleus ATAC and RNA sequencing analyses revealed that Cd55 and Cd248 positive fractions in tendon tissue are TSPCs, with this population decreasing at 1 weeks. We also identified candidate upstream transcription factors regulating these fractions. Functional analyses of isolated CD55/CD248 positive cells demonstrated high clonogenic potential and tendon differentiation capacity, forming functional tendon-like tissue in vitro . This study establishes CD55 and CD248 as novel TSPC surface antigens, potentially advancing tendon regenerative medicine and contributing to the development of new treatment strategies for tendon and ligament injuries.

Chromatin remodelling drives immune cell-fibroblast communication in heart failure.

Chronic inflammation and tissue fibrosis are common responses that worsen organ function, yet the molecular mechanisms governing their cross-talk are poorly understood. In diseased organs, stress-induced gene expression changes fuel maladaptive cell state transitions1 and pathological interaction between cellular compartments. Although chronic fibroblast activation worsens dysfunction in the lungs, liver, kidneys and heart, and exacerbates many cancers2, the stress-sensing mechanisms initiating transcriptional activation of fibroblasts are poorly understood. Here we show that conditional deletion of the transcriptional co-activator Brd4 in infiltrating Cx3cr1+ macrophages ameliorates heart failure in mice and significantly reduces fibroblast activation. Analysis of single-cell chromatin accessibility and BRD4 occupancy in vivo in Cx3cr1+ cells identified a large enhancer proximal to interleukin-1β (IL-1β, encoded by Il1b), and a series of CRISPR-based deletions revealed the precise stress-dependent regulatory element that controls Il1b expression. Secreted IL-1β activated a fibroblast RELA-dependent (also known as p65) enhancer near the transcription factor MEOX1, resulting in a profibrotic response in human cardiac fibroblasts. In vivo, antibody-mediated IL-1β neutralization improved cardiac function and tissue fibrosis in heart failure. Systemic IL-1β inhibition or targeted Il1b deletion in Cx3cr1+ cells prevented stress-induced Meox1 expression and fibroblast activation. The elucidation of BRD4-dependent cross-talk between a specific immune cell subset and fibroblasts through IL-1β reveals how inflammation drives profibrotic cell states and supports strategies that modulate this process in heart disease and other chronic inflammatory disorders featuring tissue remodelling.

Heat stress causes chromatin accessibility and related gene expression changes in crown tissues of barley (Hordeum vulgare)

Plant responses to stress caused by high temperatures involve changes occurring at the molecular, metabolic, and physiological levels. Understanding the mechanisms by which plants recognize signals to activate this response is a prerequisite for identifying key genes and signaling pathways and for obtaining heat-tolerant plants. We demonstrated the first implementation of an assay for transposase-accessible chromatin to identify open chromatin regions (OCRs) in crown tissues of barley using three genotypes carrying different allelic forms of the sdw1 gene encoding gibberellin 20-oxidase subjected to elevated temperatures. In parallel, we performed gene expression analysis, which allowed us to relate changes in chromatin state to changes in transcriptional activity. The obtained data revealed that the hypersensitive chromatin regions within the genes were more repeatable than those outside the gene intervals. We observed that prolonged exposure to high temperatures increased chromatin accessibility. Genes with OCRs in their regulatory regions were involved in stress signaling and tolerance, including calcium-dependent protein kinase, mitogen-activated protein kinase (MAPK3), receptor-like cytoplasmic kinase (RLK), TIFY domain-containing transcriptional regulator, bZIP transcription factor, and regulatory protein NPR1. The effect of genotype on gene expression was not as pronounced as that of temperature. By combining results from the differential analysis of chromatin accessibility and expression profiles, we identified genes with high temperature-induced changes in chromatin accessibility associated with expression alterations. Importantly, our data revealed a relationship between the loss of chromatin accessibility in response to heat and the downregulation of genes related to gibberellin signaling.

The MaNAP1-MaMADS1 transcription factor module mediates ethylene-regulated peel softening and ripening in banana.

The banana (Musa spp.) peel undergoes rapid softening during ripening, leading to finger drop and a shortened shelf life. The regulatory mechanism behind this process remains to be elucidated. In this study, we confirmed the role of peel softening in banana finger drop and uncovered the underlying transcriptional regulatory network. Cell wall-related (CWR) genes were substantially upregulated in both the peel and finger drop zone during ethylene-induced ripening. Transcriptome analysis and genome-wide profiling of chromatin accessibility and transcription factor (TF) binding revealed that two key regulators of fruit ripening, Musa acuminata NAC-like, Activated by apetala3/Pistillata1 (MaNAP1) and MaMADS1, regulate CWR genes by directly binding to their promoters or by targeting other ripening-related TFs to form a hierarchical regulatory network. Notably, MaNAP1 and MaMADS1 were directly targeted by ETHYLENE INSENSITIVE3 (MaEIN3), and MaNAP1 and MaMADS1 associated with tissue-specific histone modifications, enabling them to integrate MaEIN3-mediated ethylene signaling and undergo epigenetic regulation. Overexpression of MaNAP1, MaMADS1, or other identified regulatory TFs upregulated CWR genes and promoted peel softening. Our findings unveil a MaNAP1-MaMADS1-centered regulatory cascade governing banana peel softening and finger drop, offering potential targets for enhancing banana texture and shelf life.

An increase in reactive oxygen species underlies neonatal cerebellum repair.

The neonatal mouse cerebellum shows remarkable regenerative potential upon injury at birth, wherein a subset of Nestin-expressing progenitors (NEPs) undergoes adaptive reprogramming to replenish granule cell progenitors that die. Here, we investigate how the microenvironment of the injured cerebellum changes upon injury and contributes to the regenerative potential of normally gliogenic-NEPs and their adaptive reprogramming. Single cell transcriptomic and bulk chromatin accessibility analyses of the NEPs from injured neonatal cerebella compared to controls show a temporary increase in cellular processes involved in responding to reactive oxygen species (ROS), a known damage-associated molecular pattern. Analysis of ROS levels in cerebellar tissue confirm a transient increased one day after injury at postanal day 1, overlapping with the peak cell death in the cerebellum. In a transgenic mouse line that ubiquitously overexpresses human mitochondrial catalase (mCAT), ROS is reduced 1 day after injury to the granule cell progenitors, and we demonstrate that several steps in the regenerative process of NEPs are curtailed leading to reduced cerebellar growth. We also provide evidence that microglia are involved in one step of adaptive reprogramming by regulating NEP replenishment of the granule cell precursors. Collectively, our results highlight that changes in the tissue microenvironment regulate multiple steps in adaptative reprogramming of NEPs upon death of cerebellar granule cell progenitors at birth, highlighting the instructive roles of microenvironmental signals during regeneration of the neonatal brain.

Dynamic chromatin accessibility and transcriptome changes following PDGF-BB treatment of bone-marrow derived mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) are multipotent stem cells that are under investigation for use in clinical trials because they are capable of self-renewal and differentiating into different cell types under defined conditions. Nonetheless, the therapeutic effects of MSCs have been constrained by low engraftment rates, cell fusion, and cell survival. Various strategies have been explored to improve the therapeutic efficacy of MSCs, with platelet-derived growth factor (PDGF)-BB emerging as a promising candidate. To enhance our comprehension of the impact of PDGF-BB on the gene expression profile and chromosomal accessibility of MSCs, RNA-sequencing and analysis of chromatin accessibility profiles were conducted on three human primary MSCs in culture, both with and without stimulation by PDGF-BB.ResultsIntegrative analysis of gene expression and chromatin accessibility demonstrated that PDGF-BB treatment modified the chromatin accessibility landscape, marking regions for activation or repression through the AP-1 family transcription factors TEAD, CEBP, and RUNX2. These changes in AP-1 transcription factor expression, in turn, led to cell proliferation and differentiation potential towards osteoblasts, adipocytes, or chondrocytes. The degree of MSC differentiation varies among cells isolated from different donors. The presence of an enrichment of exosome-related genes is also noted among all the differentially expressed genes.ConclusionsIn conclusion, the observed changes in AP-1 transcription factor expression not only induced cellular proliferation and differentiation, but also revealed variations in the degree of MSC differentiation based on donor-specific differences. Moreover, the enrichment of exosome-related genes among differentially expressed genes suggests a potential significant role for PDGF-BB in facilitating intercellular communication.

Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties.

Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2-knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested Hepatocyte nuclear factor 4-alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9, a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.

Analysis of chromatin accessibility in peripheral blood mononuclear cells from patients with early-stage breast cancer.

Objective: This study was aimed at exploring a specific open region of chromatin in the peripheral blood mononuclear cells (PBMCs) of patients with breast cancer and evaluating its feasibility as a biomarker for diagnosing and predicting breast cancer prognosis. Methods: We obtained PBMCs from breast cancer patients and healthy people for the assay for transposase-accessible chromatin (ATAC) sequencing (n = 3) and obtained the GSE27562 chip sequencing data for secondary analyses. Through bioinformatics analysis, we mined the pattern changes for chromatin accessibility in the PBMCs of breast cancer patients. Results: A total of 1,906 differentially accessible regions (DARs) and 1,632 differentially expressed genes (DEGs) were identified via ATAC sequencing. The upregulated DEGs in the disease group were mainly distributed in the cells, organelles, and cell-intima-related structures and were mainly responsible for biological functions such as cell nitrogen complex metabolism, macromolecular metabolism, and cell communication, in addition to functions such as nucleic acid binding, enzyme binding, hydrolase reaction, and transferase activity. Combined with microarray data analysis, the following set of nine DEGs showed intersection between the ATAC and microarray data: JUN, MSL2, CDC42, TRIB1, SERTAD3, RAB14, RHOB, RAB40B, and PRKDC. HOMER predicted and identified five transcription factors that could potentially bind to these peak sites, namely NFY, Sp 2, GFY, NRF, and ELK 1. Conclusion: Chromatin accessibility analysis of the PBMCs from patients with early-stage breast cancer underscores its potential as a significant avenue for biomarker discovery in breast cancer diagnostics and treatment. By screening the transcription factors and DEGs related to breast cancer, this study provides a comprehensive theoretical foundation that is expected to guide future clinical applications and therapeutic developments.

An Atlas of Accessible Chromatin in Advanced Prostate Cancer Reveals the Epigenetic Evolution during Tumor Progression.

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal disease that resists therapy targeting androgen signaling, the primary driver of prostate cancer. mCRPC resists androgen receptor (AR) inhibitors by amplifying AR signaling or by evolving into therapy-resistant subtypes that do not depend on AR. Elucidation of the epigenetic underpinnings of these subtypes could provide important insights into the drivers of therapy resistance. In this study, we produced chromatin accessibility maps linked to the binding of lineage-specific transcription factors (TF) by performing assay for transposase-accessible chromatin sequencing on 70 mCRPC tissue biopsies integrated with transcriptome and whole-genome sequencing. mCRPC had a distinct global chromatin accessibility profile linked to AR function. Analysis of TF occupancy across accessible chromatin revealed 203 TFs associated with mCRPC subtypes. Notably, ZNF263 was identified as a putative prostate cancer TF with a significant impact on gene activity in the double-negative subtype (AR- neuroendocrine-), potentially activating MYC targets. Overall, this analysis of chromatin accessibility in mCRPC provides valuable insights into epigenetic changes that occur during progression to mCRPC. Significance: Integration of a large cohort of transcriptome, whole-genome, and ATAC sequencing characterizes the chromatin accessibility changes in advanced prostate cancer and identifies therapy-resistant prostate cancer subtype-specific transcription factors that modulate oncogenic programs.

Single-nucleus multiomics unravels the genetic mechanisms underlying musk secretion in Chinese forest musk deer (Moschus berezovskii)

Musk secreted by the musk glands in male forest musk deer (FMD; Moschus berezovskii) is highly valued for its pharmaceutical and perfumery applications. However, the regulatory mechanisms underlying musk secretion are not well understood. This study aimed to investigate the genes and transcription factors involved in musk secretion across different periods and ages. We analyzed the musk glands of adult male FMD during the non-secretory and secretory periods, as well as juvenile and adult male FMD during the secretory period, using single-cell multiome ATAC+gene expression technique. Our analysis identified 13 cell types, including acinar cells of Types 1 and 2. Chromatin accessibility analysis and gene expression data confirmed that the genes Map3k2, Hsd17b12, and Jun are critical for musk secretion. Additionally, EHF, NR4A2, and FOXO1 proteins play crucial regulatory roles. Weighted gene co-expression network analysis (WGCNA) highlighted the importance of GnRH signaling pathway in musk secretion. Gene set enrichment analysis (GSEA) showed that the steroid hormone biosynthesis pathway is notably enriched in acinar cells. Furthermore, intercellular communication appears to influence both the initiation and maintenance of musk secretion. These findings provide valuable insights into the molecular pathways of musk secretion in FMD, offering potential avenues for increasing musk production and developing treatment for inflammation and tumors.

ScaDA: A novel statistical method for differential analysis of single-cell chromatin accessibility sequencing data.

Single-cell ATAC-seq sequencing data (scATAC-seq) has been widely used to investigate chromatin accessibility on the single-cell level. One important application of scATAC-seq data analysis is differential chromatin accessibility (DA) analysis. However, the data characteristics of scATAC-seq such as excessive zeros and large variability of chromatin accessibility across cells impose a unique challenge for DA analysis. Existing statistical methods focus on detecting the mean difference of the chromatin accessible regions while overlooking the distribution difference. Motivated by real data exploration that distribution difference exists among cell types, we introduce a novel composite statistical test named "scaDA", which is based on zero-inflated negative binomial model (ZINB), for performing differential distribution analysis of chromatin accessibility by jointly testing the abundance, prevalence and dispersion simultaneously. Benefiting from both dispersion shrinkage and iterative refinement of mean and prevalence parameter estimates, scaDA demonstrates its superiority to both ZINB-based likelihood ratio tests and published methods by achieving the highest power and best FDR control in a comprehensive simulation study. In addition to demonstrating the highest power in three real sc-multiome data analyses, scaDA successfully identifies differentially accessible regions in microglia from sc-multiome data for an Alzheimer's disease (AD) study that are most enriched in GO terms related to neurogenesis and the clinical phenotype of AD, and AD-associated GWAS SNPs.

Reversal of high-glucose-induced transcriptional and epigenetic memories through NRF2 pathway activation.

Diabetes complications such as nephropathy, retinopathy, or cardiovascular disease arise from vascular dysfunction. In this context, it has been observed that past hyperglycemic events can induce long-lasting alterations, a phenomenon termed "metabolic memory." In this study, we evaluated the genome-wide gene expression and chromatin accessibility alterations caused by transient high-glucose exposure in human endothelial cells (ECs) in vitro. We found that cells exposed to high glucose exhibited substantial gene expression changes in pathways known to be impaired in diabetes, many of which persist after glucose normalization. Chromatin accessibility analysis also revealed that transient hyperglycemia induces persistent alterations, mainly in non-promoter regions identified as enhancers with neighboring genes showing lasting alterations. Notably, activation of the NRF2 pathway through NRF2 overexpression or supplementation with the plant-derived compound sulforaphane, effectively reverses the glucose-induced transcriptional and chromatin accessibility memories in ECs. These findings underscore the enduring impact of transient hyperglycemia on ECs' transcriptomic and chromatin accessibility profiles, emphasizing the potential utility of pharmacological NRF2 pathway activation in mitigating and reversing the high-glucose-induced transcriptional and epigenetic alterations.

The evolution of ovarian somatic cells characterized by transcriptome and chromatin accessibility across rodents, monkeys, and humans

Abstract The ovary plays a crucial role in the reproductive system of female mammals by producing mature oocytes through folliculogenesis. Non-human model organisms are extensively utilized in research on human ovarian biology, thus necessitating the investigation of conservation and divergence in molecular mechanisms across species. In this study, we employed integrative single-cell analysis of transcriptome and chromatin accessibility to identify the evolutionary conservation and divergence patterns of ovaries among humans, monkeys, mice, rats, and rabbits. Our analyses revealed that theca cells exhibited the most significant changes during evolution based on scRNA-seq and scATAC-seq datasets. Furthermore, we discovered common cis-regulatory architectures in theca cells across species by conducting joint analyses of scRNA-seq and scATAC-seq datasets. These findings have potential applications in non-human biomedical and genetic research to validate molecular mechanisms found in human organisms. Additionally, our investigation into non-coding genomic regions identified intergenic highly transcribed regions (igHTRs) that may contribute to the evolution of species-specific phenotypic traits. Overall, our study provides valuable insights into understanding the molecular characteristics of adult ovaries while offering new perspectives for studying human ovarian physiology and diseases.

Limitations in mitochondrial programming restrain the differentiation and maturation of human stem cell-derived β cells.

Pluripotent stem cell (SC)-derived islets offer hope as a renewable source for β cell replacement for type 1 diabetes (T1D), yet functional and metabolic immaturity may limit their long-term therapeutic potential. Here, we show that limitations in mitochondrial transcriptional programming impede the formation and maturation of SC-derived β (SC-β) cells. Utilizing transcriptomic profiling, assessments of chromatin accessibility, mitochondrial phenotyping, and lipidomics analyses, we observed that SC-β cells exhibit reduced oxidative and mitochondrial fatty acid metabolism compared to primary human islets that are related to limitations in key mitochondrial transcriptional networks. Surprisingly, we found that reductions in glucose- stimulated mitochondrial respiration in SC-islets were not associated with alterations in mitochondrial mass, structure, or genome integrity. In contrast, SC-islets show limited expression of targets of PPARIZ and PPARγ, which regulate mitochondrial programming, yet whose functions in β cell differentiation are unknown. Importantly, treatment with WY14643, a potent PPARIZ agonist, induced expression of mitochondrial targets, improved insulin secretion, and increased the formation and maturation of SC-β cells both in vitro and following transplantation. Thus, mitochondrial programming promotes the differentiation and maturation of SC-β cells and may be a promising target to improve β cell replacement efforts for T1D.