Characterization of the chromatin accessibility in an Alzheimer\u2019s disease (AD) mouse model

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.

BackgroundChromatin dysregulation is associated with developmental disorders and cancer. Numerous methods for measuring genome-wide chromatin accessibility have been developed in the genomic era to interrogate the function of chromatin regulators. A recent technique which has gained widespread use due to speed and low input requirements with native chromatin is the Assay for Transposase-Accessible Chromatin, or ATAC-seq. Biologists have since used this method to compare chromatin accessibility between two cellular conditions. However, approaches for calculating differential accessibility can yield conflicting results, and little emphasis is placed on choice of normalization method during differential ATAC-seq analysis, especially when global chromatin alterations might be expected.ResultsUsing an in vivo ATAC-seq data set generated in our recent report, we observed differences in chromatin accessibility patterns depending on the data normalization method used to calculate differential accessibility. This observation was further verified on published ATAC-seq data from yeast. We propose a generalized workflow for differential accessibility analysis using ATAC-seq data. We further show this workflow identifies sites of differential chromatin accessibility that correlate with gene expression and is sensitive to differential analysis using negative controls.ConclusionsWe argue that researchers should systematically compare multiple normalization methods before continuing with differential accessibility analysis. ATAC-seq users should be aware of the interpretations of potential bias within experimental data and the assumptions of the normalization method implemented.

BackgroundCancer stem cells (CSCs) are related to the patient’s prognosis, recurrence and therapy resistance in oesophageal squamous cell carcinoma (ESCC). Although increasing evidence suggests that aspirin (acetylsalicylic acid, ASA) could lower the incidence and improve the prognosis of ESCC, the mechanism(s) remains to be fully understood.MethodsWe investigated the role of ASA in chemotherapy/chemoprevention in human ESCC cell lines and an N-nitrosomethylbenzylamine-induced rat ESCC carcinogenesis model. The effects of combined treatment with ASA/cisplatin on ESCC cell lines were examined in vitro and in vivo. Sphere-forming cells enriched with putative CSCs (pCSCs) were used to investigate the effect of ASA in CSCs. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) was performed to determine the alterations in chromatin accessibility caused by ASA in ESCC cells.ResultsASA inhibits the CSC properties and enhances cisplatin treatment in human ESCC cells. ATAC-seq indicates that ASA treatment results in remarkable epigenetic alterations on chromatin in ESCC cells, especially their pCSCs, through the modification of histone acetylation levels. The epigenetic changes activate Bim expression and promote cell death in CSCs of ESCC. Furthermore, ASA prevents the carcinogenesis of NMBzA-induced ESCC in the rat model.ConclusionsASA could be a potential chemotherapeutic adjuvant and chemopreventive drug for ESCC treatment.

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.

BackgroundFilamentous actin (F‐actin) is the major cytoskeleton protein present in dendritic spines and it is an essential element for defining dendritic spine structure. We have shown that synaptosomal F‐actin levels were significantly decreased in APP/PS1 mice as early as 1 month of age. Further, reduced form of F‐actin is diminished and glutathionylated F‐actin is significantly increased in synaptosomes of 1 month old APP/PS1 mice. Synaptic neurotransmission and synaptic plasticity are dependent on the dynamic regulation of the actin through actin‐interacting and actin‐modulating proteins. Recent studies highlight that F‐actin nanoarchitecture is susceptible to several key regulators such as reactive oxygen species, cofilin and drebrin that are present in the dendritic spines. Yet, how synaptosomal F‐actin organization is regulated remains poorly understood. To address this, we characterized actin interactome in synaptosomes prepared from wild type and APPSwe/PS1ΔE9 (APP/PS1) mice.MethodsWe utilized age matched wild type (WT) and APP/PS1 mice for our experiments. Synaptosomes and highly enriched F‐actin and G‐actin fractions from synaptosomes were isolated from WT and APP/PS1 mice. We performed immunoprecipitation using all these fractions with an anti‐actin polyclonal antibody and eluted in Laemmli sample buffer. The IP elutes were resolved using SDS‐PAGE gels, all proteins purified from gels, and analysed by LC‐MS/MS spectrometry.ResultsWe detected approximately 620 synaptic proteins in synaptosomes from both wild type and APP/PS1 mice at the age of six months. About 110 proteins were upregulated and 198 proteins down regulated in synaptosomes of APP/PS1 mice. We identified 69 proteins that were associated with F‐actin in WT mice while only 26 proteins interacted with F‐actin in APP/PS1 mice in synaptosomal F‐actin fraction. Further, we detected G‐actin‐interaction only with 10 proteins in WT and 15 proteins in APP/PS1 mice in synaptosomal G‐actin fraction. However, we were able to identify 401 proteins in WT and 444 proteins with F/G‐actin‐association in synaptosomes of APP/PS1 mice compared with WT mice.ConclusionsOur result defines a provisional synaptosomal actin interactome in mouse brain cortex of WT and APP/PS1 mice and identifies several critical regulator proteins that may be involved in the progression of AD pathogenesis.

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

V(D)J recombination is believed to be regulated by alterations in chromatin accessibility to the recombinase machinery, but the mechanisms responsible remain unclear. We previously proposed that antisense intergenic transcription, activated throughout the mouse Igh VH region in pro-B cells, remodels chromatin for VH-to-DJH recombination. Using RNA fluorescence in situ hybridization, we now show that antisense intergenic transcription occurs throughout the Igh DHJH region before D-to-J recombination, indicating that this is a widespread process in V(D)J recombination. Transcription initiates near the Igh intronic enhancer Emu and is abrogated in mice lacking this enhancer, indicating that Emu regulates DH antisense transcription. Emu was recently demonstrated to regulate DH-to-JH recombination of the Igh locus. Together, these data suggest that Emu controls DH-to-JH recombination by activating this form of germ line Igh transcription, thus providing a long-range, processive mechanism by which Emu can regulate chromatin accessibility throughout the DH region. In contrast, Emu deletion has no effect on VH antisense intergenic transcription, which is rarely associated with DH antisense transcription, suggesting differential regulation and separate roles for these processes at sequential stages of V(D)J recombination. These results support a directive role for antisense intergenic transcription in enabling access to the recombination machinery.

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.

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.

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

BackgroundAlzheimer’s disease (AD) is a neurodegenerative disorder that can result in neurotoxicity and an imbalance in gut microbiota. Probiotics have been shown to play an important role in regulating the gut microbiota, but their viability and bioactivity are often compromised as they traverse the gastrointestinal tract, thereby reducing their efficacy and limiting their clinical utility.ResultsIn this work, layer-by-layer (LbL) encapsulation technology was used to encapsulate Lactiplantibacillus plantarum (LP) to improve the above shortcomings. Studies in APPswe/PS1dE9 (APP/PS1) transgenic mice show that LbL-encapsulated LP ((CS/SP)2-LP) protects LP from gastrointestinal damage while (CS/SP)2-LP treatment It improves brain neuroinflammation and neuronal damage in AD mice, reduces Aβ deposition, improves tau protein phosphorylation levels, and restores intestinal barrier damage in AD mice. In addition, post-synaptic density protein 95 (PSD-95) expression increased in AD mice after treatment, indicating enhanced synaptic plasticity. Fecal metabolomic and microbiological analyzes showed that the disordered intestinal microbiota composition of AD mice was restored and short-chain fatty acids (SCFAs) levels were significantly increased after (CS/SP)2-LP treatment.ConclusionOverall, the above evidence suggests that (CS/SP)2-LP can improve AD symptoms by restoring the balance of intestinal microbiota, and (CS/SP)2-LP treatment will provide a new method to improve the symptoms of AD patients.

Integrated analysis of ATAC-seq and RNA-seq reveals the chromatin accessibility and transcriptional landscape of immunoglobulin a nephropathy.

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

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.

BackgroundDecoding the genetic mechanisms of late‐onset Alzheimer’s disease (LOAD) is a major challenge in the post‐GWAS era, since the majority of the LOAD associated SNPs are in noncoding regions. Noncoding disease‐associated loci have been shown to be enriched for regulatory elements in tissues and cells relevant to the disease. Thus, post‐GWAS research requires an in‐depth characterization of cell‐type‐specific DNA regulatory elements and gene expression patterns.MethodsWe conducted ATAC‐seq profiling using NeuN sorted‐nuclei from 40 frozen brain tissues to determine LOAD‐specific changes in chromatin accessibility landscape in a cell‐type specific manner. We complemented these experiments by generating single‐nuclei(sn)RNA‐seq data from a subset group of the same brain samples.ResultsThe study has five major findings for the field of LOAD epigenetics. First, we generated a map of LOAD‐associated cell‐type specific chromatin accessibility sites. Second, we identified 211 LOAD‐specific differential chromatin accessibility sites in neuronal‐nuclei. Third, we provided a catalogue of 842 female‐specific disease‐associated chromatin accessibility sites in non‐neuronal cells. Forth, we showed that the LOAD‐associated cell‐type‐specific changes in chromatin accessibility overlapped with ∼25% of the LOAD‐GWAS regions. Thus, suggesting a noncoding regulatory mechanism, namely chromatin accessibility, by which several LOAD‐GWAS loci may exert their pathogenic effect. Fifth, we functionally validated the ATAC‐seq findings using snRNA‐seq datasets. Data integration identified overlaps between the LOAD‐specific ATAC‐seq signals with the transcriptome profiles of known and new LOAD loci and demonstrated that LOAD‐associated changes in chromatin accessibility can result in gene dysregulation.ConclusionTo our knowledge, this study represents the first and most comprehensive systematic interrogation of the chromatin accessibility landscape in LOAD and the functional effect on gene dysregulation. Moreover, the outcomes unveiled how cell‐type‐specific and sex‐dependent alterations in chromatin structure and transcription programs are associated with LOAD. These findings enhance the interpretation of LOAD‐GWAS discoveries towards translating association to causation, provide potential pathomechanisms, and suggest novel LOAD‐loci. Furthermore, our results convey mechanistic insights into sex differences in LOAD risk and clinicopathology.