Assay For Transposase-accessible Chromatin Research Articles

ATAC-seq reveals the roles of chromatin accessibility in the chondrocytes of Kashin-Beck disease compared with primary osteoarthritis.

Objective: This study aimed to investigate the roles of accessible chromatin in understanding the different pathogeneses between Kashin-Beck disease (KBD) and primary osteoarthritis (OA). Methods: Articular cartilages of KBD and OA patients were collected, and after tissue digestion, primary chondrocytes were cultured in vitro. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed to compare the accessible chromatin differences of chondrocytes between KBD and OA groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were executed for the promoter genes. Then, the IntAct online database was used to generate networks of significant genes. Finally, we overlapped the analysis of differentially accessible region (DAR)-associated genes and differentially expressed genes (DEGs) obtained from whole-genomic microarray. Results: We obtained 2,751 total DARs, which contained 1,985 loss and 856 gain DARs and belonged to 11 location distributions. We obtained 218 motifs associated with loss DARs, 71 motifs associated with gain DARs, 30 motif enrichments of loss DARs, and 30 motif enrichments of gain DARs. In total, 1,749 genes are associated with loss DARs, and 826 genes are associated with gain DARs. Among them, 210 promoter genes are associated with loss DARs, and 112 promoter genes are associated with gain DARs. We obtained 15 terms of GO enrichment and 5 terms of KEGG pathway enrichment from loss DAR promoter genes, and 15 terms of GO enrichment and 3 terms of KEGG pathway enrichment from gain DAR promoter genes. We obtained CAPN6 and other 2 overlap genes from loss DARs-vs-down DEGs, AMOTL1 from gain DARs-vs-down DEGs, EBF3 and other 12 overlap genes from loss DARs-vs-up DEGs, and ADARB1 and other 10 overlap genes from 101 gain DARs-vs-up DEGs. These overlap genes were built into 4 gene interaction networks. Conclusion: FGF7, GPD1L, NFIB, RUNX2, and VCAM1 were the overlapped genes from the DAR-associated genes and DEGs. These genes were associated with the abnormal chondrocyte function, which may play crucial roles in different processes between KBD and OA in the way of accessible chromatin.

Optimization of ATAC-seq in wheat seedling roots using INTACT-isolated nuclei

BackgroundThe genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however, cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification.ResultsIn this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks and cis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels.ConclusionsWe have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome.

SeATAC: a tool for exploring the chromatin landscape and the role of pioneer factors

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) reveals chromatin accessibility across the genome. Currently, no method specifically detects differential chromatin accessibility. Here, SeATAC uses a conditional variational autoencoder model to learn the latent representation of ATAC-seq V-plots and outperforms MACS2 and NucleoATAC on six separate tasks. Applying SeATAC to several pioneer factor-induced differentiation or reprogramming ATAC-seq datasets suggests that induction of these factors not only relaxes the closed chromatin but also decreases chromatin accessibility of 20% to 30% of their target sites. SeATAC is a novel tool to accurately reveal genomic regions with differential chromatin accessibility from ATAC-seq data.

Unsupervised contrastive peak caller for ATAC-seq.

The assay for transposase-accessible chromatin with sequencing (ATAC-seq) is a common assay to identify chromatin accessible regions by using a Tn5 transposase that can access, cut, and ligate adapters to DNA fragments for subsequent amplification and sequencing. These sequenced regions are quantified and tested for enrichment in a process referred to as "peak calling". Most unsupervised peak calling methods are based on simple statistical models and suffer from elevated false positive rates. Newly developed supervised deep learning methods can be successful, but they rely on high quality labeled data for training, which can be difficult to obtain. Moreover, though biological replicates are recognized to be important, there are no established approaches for using replicates in the deep learning tools, and the approaches available for traditional methods either cannot be applied to ATAC-seq, where control samples may be unavailable, or are post-hoc and do not capitalize on potentially complex, but reproducible signal in the read enrichment data. Here, we propose a novel peak caller that uses unsupervised contrastive learning to extract shared signals from multiple replicates. Raw coverage data are encoded to obtain low-dimensional embeddings and optimized to minimize a contrastive loss over biological replicates. These embeddings are passed to another contrastive loss for learning and predicting peaks and decoded to denoised data under an autoencoder loss. We compared our Replicative Contrastive Learner (RCL) method with other existing methods on ATAC-seq data, using annotations from ChromHMM genome and transcription factor ChIP-seq as noisy truth. RCL consistently achieved the best performance.

Integration of ATAC-Seq and RNA-Seq reveals FOSL2 drives human liver progenitor-like cell aging by regulating inflammatory factors

BackgroundHuman primary hepatocytes (PHCs) are considered to be the best cell source for cell-based therapies for the treatment of end-stage liver disease and acute liver failure. To obtain sufficient and high-quality functional human hepatocytes, we have established a strategy to dedifferentiate human PHCs into expandable hepatocyte-derived liver progenitor-like cells (HepLPCs) through in vitro chemical reprogramming. However, the reduced proliferative capacity of HepLPCs after long-term culture still limits their utility. Therefore, in this study, we attempted to explore the potential mechanism related to the proliferative ability of HepLPCs in vitro culture.ResultsIn this study, analysis of assay for transposase accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) were performed for PHCs, proliferative HepLPCs (pro-HepLPCs) and late-passage HepLPCs (lp-HepLPCs). Genome-wide transcriptional and chromatin accessibility changes during the conversion and long-term culture of HepLPCs were studied. We found that lp-HepLPCs exhibited an aged phenotype characterized by the activation of inflammatory factors. Epigenetic changes were found to be consistent with our gene expression findings, with promoter and distal regions of many inflammatory-related genes showing increased accessibility in the lp-HepLPCs. FOSL2, a member of the AP-1 family, was found to be highly enriched in the distal regions with increased accessibility in lp-HepLPCs. Its depletion attenuated the expression of aging- and senescence-associated secretory phenotype (SASP)-related genes and resulted in a partial improvement of the aging phenotype in lp-HepLPCs.ConclusionsFOSL2 may drive the aging of HepLPCs by regulating inflammatory factors and its depletion may attenuate this phenotypic shift. This study provides a novel and promising approach for the long-term in vitro culture of HepLPCs.

Single-Cell Analysis in Immuno-Oncology.

The complexity of the cellular and non-cellular milieu surrounding human tumors plays a decisive role in the course and outcome of disease. The high variability in the distribution of the immune and non-immune compartments within the tumor microenvironments (TME) among different patients governs the mode of their response or resistance to current immunotherapeutic approaches. Through deciphering this diversity, one can tailor patients' management to meet an individual's needs. Single-cell (sc) omics technologies have given a great boost towards this direction. This review gathers recent data about how multi-omics profiling, including the utilization of single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin with sequencing (scATAC-seq), T-cell receptor sequencing (scTCR-seq), mass, tissue-based, or microfluidics cytometry, and related bioinformatics tools, contributes to the high-throughput assessment of a large number of analytes at single-cell resolution. Unravelling the exact TCR clonotype of the infiltrating T cells or pinpointing the classical or novel immune checkpoints across various cell subsets of the TME provide a boost to our comprehension of adaptive immune responses, their antigen specificity and dynamics, and grant suggestions for possible therapeutic targets. Future steps are expected to merge high-dimensional data with tissue localization data, which can serve the investigation of novel multi-modal biomarkers for the selection and/or monitoring of the optimal treatment from the current anti-cancer immunotherapeutic armamentarium.

Nono deficiency impedes the proliferation and adhesion of H9c2 cardiomyocytes through Pi3k/Akt signaling pathway

Congenital heart disease (CHD) is the most common type of birth defect and the main noninfectious cause of death during the neonatal stage. The non-POU domain containing, octamer-binding gene, NONO, performs a variety of roles involved in DNA repair, RNA synthesis, transcriptional and post-transcriptional regulation. Currently, hemizygous loss-of-function mutation of NONO have been described as the genetic origin of CHD. However, essential effects of NONO during cardiac development have not been fully elucidated. In this study, we aim to understand role of Nono in cardiomyocytes during development by utilizing the CRISPR/Cas9 gene editing system to deplete Nono in the rat cardiomyocytes H9c2. Functional comparison of H9c2 control and knockout cells showed that Nono deficiency suppressed cell proliferation and adhesion. Furthermore, Nono depletion significantly affected the mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, resulting in H9c2 overall metabolic deficits. Mechanistically we demonstrated that the Nono knockout impeded the cardiomyocyte function by attenuating phosphatidyl inositol 3 kinase-serine/threonine kinase (Pi3k/Akt) signaling via the assay for transposase-accessible chromatin using sequencing in combination with RNA sequencing. From these results we propose a novel molecular mechanism of Nono to influence cardiomyocytes differentiation and proliferation during the development of embryonic heart. We conclude that NONO may represent an emerging possible biomarkers and targets for the diagnosis and treatment of human cardiac development defects.

Screening for functional regulatory variants in open chromatin using GenIE-ATAC.

Understanding the effects of genetic variation in gene regulatory elements is crucial to interpreting genome function. This is particularly pertinent for the hundreds of thousands of disease-associated variants identified by GWAS, which frequently sit within gene regulatory elements but whose functional effects are often unknown. Current methods are limited in their scalability and ability to assay regulatory variants in their endogenous context, independently of other tightly linked variants. Here, we present a new medium-throughput screening system: genome engineering based interrogation of enhancers assay for transposase accessible chromatin (GenIE-ATAC), that measures the effect of individual variants on chromatin accessibility in their endogenous genomic and chromatin context. We employ this assay to screen for the effects of regulatory variants in human induced pluripotent stem cells, validating a subset of causal variants, and extend our software package (rgenie) to analyse these new data. We demonstrate that this methodology can be used to understand the impact of defined deletions and point mutations within transcription factor binding sites. We thus establish GenIE-ATAC as a method to screen for the effect of gene regulatory element variation, allowing identification and prioritisation of causal variants from GWAS for functional follow-up and understanding the mechanisms of regulatory element function.

Abstract 163: Thrombocytopenia Independently Leads To Monocyte Immune Dysfunction

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and leukocytes, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. We have now found that thrombocytopenia (a low platelet count) leads to monocyte dysfunction, independent of the cause of thrombocytopenia, in a manner that is dependent on direct platelet-monocyte CD47 interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) responses, including increased IL-6 production. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming and durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed persistently open chromatin at LPS response genes. Using mouse models of thrombocytopenia and sepsis we found that normal platelet numbers are needed to limit immune dysregulation in sepsis. IL6 and TNF expression in monocytes from human patients with sepsis also inversely correlated with patient platelet counts. Our studies demonstrate that in healthy conditions resting platelets maintain monocyte immune tolerance by regulating monocyte immunometabolic processes that lead to epigenetic changes in TLR-related genes. Platelets therefore function as immune rheostats in both health and disease.

GTAC enables parallel genotyping of multiple genomic loci with chromatin accessibility profiling in single cells

Understanding clonal evolution and cancer development requires experimental approaches for characterizing the consequences of somatic mutations on gene regulation. However, no methods currently exist that efficiently link high-content chromatin accessibility with high-confidence genotyping in single cells. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), enabling accurate mutation detection at multiple amplified loci, coupled with robust chromatin accessibility readout. We applied GTAC to primary acute myeloid leukemia, obtaining high-quality chromatin accessibility profiles and clonal identities for multiple mutations in 88% of cells. We traced chromatin variation throughout clonal evolution, showing the restriction of different clones to distinct differentiation stages. Furthermore, we identified switches in transcription factor motif accessibility associated with a specific combination of driver mutations, which biased transformed progenitors toward a leukemia stem cell-like chromatin state. GTAC is a powerful tool to study clonal heterogeneity across a wide spectrum of pre-malignant and neoplastic conditions.

B lymphocytes in treatment-naive paediatric patients with lupus are epigenetically distinct from healthy children

BackgroundSLE is likely triggered by gene–environment interactions. We have shown that most SLE-associated haplotypes encompass genomic regions enriched for epigenetic marks associated with enhancer function in lymphocytes, suggesting genetic risk...

Efficient Generation of Paired Single-Cell Multiomics Profiles by Deep Learning.

Recent advances in single-cell sequencing technology have made it possible to measure multiple paired omics simultaneously in a single cell such as cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-nucleus chromatin accessibility and mRNA expression sequencing (SNARE-seq). However, the widespread application of these single-cell multiomics profiling technologies has been limited by their experimental complexity, noise in nature, and high cost. In addition, single-omics sequencing technologies have generated tremendous and high-quality single-cell datasets but have yet to be fully utilized. Here, single-cell multiomics generation (scMOG), a deep learning-based framework to generate single-cell assay for transposase-accessible chromatin (ATAC)data in silico is developed from experimentally available single-cell RNA-seq measurements and vice versa. The results demonstrate that scMOG can accurately perform cross-omics generation between RNA and ATAC, and generate paired multiomics data with biological meanings when one omics is experimentally unavailable and out of training datasets. The generated ATAC, either alone or in combination with measured RNA, exhibits equivalent or superior performance to that of the experimentally measured counterparts throughout multiple downstream analyses. scMOG is also applied to human lymphoma data, which proves to be more effective in identifying tumor samples than the experimentally measured ATAC data. Finally, the performance of scMOG is investigated in other omics such as proteomics and it still shows robust performance on surface protein generation.

A systems biology approach uncovers novel disease mechanisms in age-related macular degeneration

Age-related macular degeneration (AMD) is a leading cause of blindness, affecting 200 million people worldwide. To identify genes that could be targeted for treatment, we created a molecular atlas at different stages of AMD. Our resource is comprised of RNA sequencing (RNA-seq) and DNA methylation microarrays from bulk macular retinal pigment epithelium (RPE)/choroid of clinically phenotyped normal and AMD donor eyes (n= 85), single-nucleus RNA-seq (164,399 cells), and single-nucleus assay for transposase-accessible chromatin (ATAC)-seq (125,822 cells) from the retina, RPE, and choroid of 6 AMD and 7 control donors. We identified 23 genome-wide significant loci differentially methylated in AMD, over 1,000 differentially expressed genes across different disease stages, and an AMD Müller state distinct from normal or gliosis. Chromatin accessibility peaks in genome-wide association study (GWAS) loci revealed putative causal genes for AMD, including HTRA1 and C6orf223. Our systems biology approach uncovered molecular mechanisms underlying AMD, including regulators of WNT signaling, FRZB and TLE2, as mechanistic players in disease.

TEAD1 trapping by the Q353R-Lamin A/C causes dilated cardiomyopathy.

Mutations in the LMNA gene encoding Lamin A and C (Lamin A/C), major components of the nuclear lamina, cause laminopathies including dilated cardiomyopathy (DCM), but the underlying molecular mechanisms have not been fully elucidated. Here, by leveraging single-cell RNA sequencing (RNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), protein array, and electron microscopy analysis, we show that insufficient structural maturation of cardiomyocytes owing to trapping of transcription factor TEA domain transcription factor 1 (TEAD1) by mutant Lamin A/C at the nuclear membrane underlies the pathogenesis of Q353R-LMNA-related DCM. Inhibition of the Hippo pathway rescued the dysregulation of cardiac developmental genes by TEAD1 in LMNA mutant cardiomyocytes. Single-cell RNA-seq of cardiac tissues from patients with DCM with the LMNA mutation confirmed the dysregulated expression of TEAD1 target genes. Our results propose an intervention for transcriptional dysregulation as a potential treatment of LMNA-related DCM.

Evaluation of MYBL1 as the master regulator for pachytene spermatocyte genes dysregulated in interspecific hybrid dzo

Misregulation of spermatogenesis transcription factors (TF) in hybrids can lead to misexpression, which is a mechanism for hybrid male sterility (HMS). We used dzo (male offspring of Bos taurus ♂ × Bos grunniens ♀) in bovines to investigate the relationship of the key TF with HMS via RNA sequencing and assay for transposase-accessible chromatin with high-throughput sequencing analyses. RNA sequencing showed that the widespread misexpression in dzo was associated with spermatogenesis-related genes and somatic or progenitor genes. The transition from leptotene or zygotene spermatocytes to pachytene spermatocytes may be the key stage for meiosis arrest in dzo. The analysis of TF-binding motif enrichment revealed that the male meiosis-specific master TF MYB proto-oncogene like 1 (MYBL1, known as A-MYB) motif was enriched on the promoters of downregulated pachytene spermatocyte genes in dzo. Assay for transposase-accessible chromatin with high-throughput sequencing revealed that TF-binding sites for MYBL1, nuclear transcription factor Y, and regulatory factor X were enriched in the low-chromatin accessibility region of dzo. The target genes of the MYBL1-binding motif were associated with meiosis-specific genes and significantly downregulated in dzo testis. The transcription factor MYBL1 may be the candidate master regulator for pachytene spermatocyte genes dysregulated in interspecific HMS dzo. This study reported that a few upstream TF regulation changes might exert a cascading effect downstream in a regulatory network as a mechanism for HMS.

FOXK1 regulates Wnt signalling to promote cardiogenesis.

Congenital heart disease (CHD) is the most common genetic birth defect, which has considerable morbidity and mortality. We focused on deciphering key regulators that govern cardiac progenitors and cardiogenesis. FOXK1 is a forkhead/winged helix transcription factor known to regulate cell cycle kinetics and is restricted to mesodermal progenitors, somites, and heart. In the present study, we define an essential role for FOXK1 during cardiovascular development. We used the mouse embryoid body system to differentiate control and Foxk1 KO embryonic stem cells into mesodermal, cardiac progenitor cells and mature cardiac cells. Using flow cytometry, immunohistochemistry, cardiac beating, transcriptional and chromatin immunoprecipitation quantitative polymerase chain reaction assays, bulk RNA sequencing (RNAseq) and assay for transposase-accessible chromatin using sequencing (ATACseq) analyses, FOXK1 was observed to be an important regulator of cardiogenesis. Flow cytometry analyses revealed perturbed cardiogenesis in Foxk1 KO embryoid bodies (EBs). Bulk RNAseq analysis at two developmental stages showed a significant reduction of the cardiac molecular program in Foxk1 KO EBs compared to the control EBs. ATACseq analysis during EB differentiation demonstrated that the chromatin landscape nearby known important regulators of cardiogenesis was significantly relaxed in control EBs compared to Foxk1 KO EBs. Furthermore, we demonstrated that in the absence of FOXK1, cardiac differentiation was markedly impaired by assaying for cardiac Troponin T expression and cardiac contractility. We demonstrate that FOXK1 is an important regulator of cardiogenesis by repressing the Wnt/β-catenin signalling pathway and thereby promoting differentiation. These results identify FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development. Mechanistically, FOXK1 represses Wnt signalling to promote the development of cardiac progenitor cells.

Abstract 1506: Decoding multi-omics genetic profiling reveals heterogeneity in adult pan-gliomas

Abstract Adult-type diffuse gliomas (Glioblastoma (GBM), Astrocytoma, Oligodendroglioma) are the most common malignant brain tumor in adults with dismal prognoses. Recent large-scale genomic studies have established molecular classification of glioma based on coding mutations and copy number variations. However, whole-genome landscape and multi-omics profiles of gliomas are not well analyzed. To understand the multi-omics genetic landscape of gliomas, we performed deep whole-genome sequencing (≥ ×120 coverage) of 357 cases with adult-type diffuse gliomas (162 GBMs, 96 astrocytomas, 99 oligodendrogliomas) along with EPIC DNA methylation profiling. RNA-seq, whole-genome bisulfate sequencing, and assay for transposase-accessible chromatin with sequencing (ATAC-seq) were performed on 349, 40, and 40 of these tumors, respectively. Deep WGS delineated a fine view of clonal architecture demonstrating mutational order during tumor initiation of each glioma. Mutational signature varies between clonal and subclonal mutations, supporting a model that tumors acquire genetic alterations by distinct mechanisms based on their developmental stage. Structural variants (SVs) are more frequently detected in higher malignant gliomas. While a complex SV is rare in Astrocytoma and Oligodendroglioma (35% of samples), a large proportion of GBM cases (85%) have complex SV with ≥ 10 breakpoints, which involves multiple different chromosomes including driver genes. CDKN2A homozygous deletions and focal CDK4 amplifications are often induced by complex SVs in GBM. These results suggest that complex SVs could play a pivotal role in the initiation and progression of GBM. Integrative analysis of transcriptomic and epigenomic profiles by similarity network fusion (SNF) identifies homogenous clusters in each glioma where those clusters are associated with mutational and SV signatures, suggesting that specific cell states may have distinct sensitivities to mutational processes. In oligodendrogliomas, the SNF classification detected a cluster with poor prognosis which has an enrichment of stem cell-like signature by CIBERSORTx deconvolution analysis. ATAC-seq demonstrated distinct features of genome-wide chromatin accessibility in each glioma which may reflect the difference of cell of the origin. Most of the focally amplified genes have open chromatin status suggesting that tumors take advantage of not only gene duplication but also a transcriptional activity to activate driver genes. Our integrated analysis uncovers molecular mechanisms of gliomas which will help to understand glioma-genesis. Citation Format: Takuma Nakashima, Yusuke Funakoshi, Atsuhito Uneda, Yuriko Sugihara, Shohei Nambu, Manabu Kinoshita, Yoshiki Arakawa, Shota Tanaka, Joji Ishida, Ryuta Saito, Ryosuke Hanaya, Koji Yoshimoto, Yoshitaka Narita, Hiromichi Suzuki. Decoding multi-omics genetic profiling reveals heterogeneity in adult pan-gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1506.

Maternal diet alters long-term innate immune cell memory in fetal and juvenile hematopoietic stem and progenitor cells in nonhuman primate offspring

Maternal overnutrition increases inflammatory and metabolic disease risk in postnatal offspring. This constitutes a major public health concern due to increasing prevalence of these diseases, yet mechanisms remain unclear. Here, using nonhuman primate models, we show that maternal Western-style diet (mWSD) exposure is associated with persistent pro-inflammatory phenotypes at the transcriptional, metabolic, and functional levels in bone marrow-derived macrophages (BMDMs) from 3-year-old juvenile offspring and in hematopoietic stem and progenitor cells (HSPCs) from fetal and juvenile bone marrow and fetal liver. mWSD exposure is also associated with increased oleic acid in fetal and juvenile bone marrow and fetal liver. Assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling of HSPCs and BMDMs from mWSD-exposed juveniles supports a model in which HSPCs transmit pro-inflammatory memory to myeloid cells beginning in utero. These findings show that maternal dietalters long-term immune cell developmental programming in HSPCs with proposed consequences for chronic diseases featuring altered immune/inflammatory activation across the lifespan.

Oncogenic epidermal growth factor receptor signal-induced histone deacetylation suppresses chemokine gene expression in human lung adenocarcinoma

Epidermal growth factor receptor (EGFR)-mutated (mt) lung adenocarcinoma (LA) is refractory to immune checkpoint inhibitors (ICIs). However, the mechanisms have not been fully elucidated. CD8+ T cell infiltration was significantly lower in EGFR-mt than in EGFR-wild-type LA, which was associated with suppression of chemokine expression. Since this T cell-deserted tumor microenvironment may lead to the refractoriness of ICIs against EGFR-mt LA, we investigated the mechanism by focusing on the regulation of chemokine expression. The expression of C-X-C motif ligand (CXCL) 9, 10 and 11, which constitute a gene cluster on chromosome 4, was suppressed under EGFR signaling. The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) revealed open chromatin peaks near this gene cluster following EGFR-tyrosine kinase inhibitor (TKI) treatment. The histone deacetylase (HDAC) inhibitor recovered the expression of CXCL9, 10 and 11 in EGFR-mt LA. Nuclear HDAC activity, as well as histone H3 deacetylation, were dependent on oncogenic EGFR signaling. Furthermore, the Cleavage Under Targets and Tagmentation (CUT & Tag) assay revealed a histone H3K27 acetylation peak at 15 kb upstream of CXCL11 after treatment with EGFR-TKI, which corresponded to one of the open chromatin peaks detected by ATAC-seq. The data suggest that EGFR-HDAC axis mediates silencing of the chemokine gene cluster through chromatin conformational change, which might be relevant to the ICI resistance by creating T cell-deserted tumor microenvironment. Targeting this axis may develop a new therapeutic strategy to overcome the ICI resistance of EGFR-mt LA.

CD226 identifies functional CD8+T cells in the tumor microenvironment and predicts a better outcome for human gastric cancer.

It is well-known that CD226 serves as a critical activating receptor on various immune cells, such as lymphocytes and monocytes, and it is suggested to promote anti-tumor immunity in the tumor microenvironment (TME). Herein, we showed a crucial regulatory role of CD226 in CD8+T cell-mediated anti-tumor response in TME of human gastric cancer (GC). Specifically, the increased CD226 expression in cancer tissues was significantly associated with better clinical outcomes in GC patients. Moreover, the increased infiltrating CD226+CD8+T cells and the increased ratio of infiltrating CD226+CD8+T cells in CD8+T subpopulation within cancer tissues could also be valuable prognostic predictors for GC patients. Mechanically, the assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis revealed that the chromatin accessibility of CD226 in CD4+ and CD8+TILs was significantly higher than that in CD8+T cells in normal tissues. Further analysis showed that CD8+TILs highly expressed immune checkpoint molecules, such as TIGIT, LAG3, and HAVCR2, which means CD8+TILs are more exhausted. In addition, our multi-color immunohistochemical staining (mIHC) revealed that GC patients with higher frequency of IFN-γ+CD226+CD8+TILs showed poorer prognosis. Combined with the single-cell transcriptome sequencing (scRNA-seq) data analysis, we found that the expressions of IFN-γ and TIGIT in CD8+TILs were significantly and positively correlated. The expression of TIGIT in IFN-γ+CD226+CD8+TILs was higher, while that in IFN-γ-CD226+CD8+TILs was significantly lower. The correlation analysis showed that the expression of CD226 was positively correlated with the score of effector T cells but negatively correlated with that of immunosuppressive factors, such as Tregs and tumor-associated macrophages (TAMs). Collectively, we showed that the frequency of CD226+CD8+TILs was an excellent prognostic predictor for GC patients. Our findings provided insights into the interaction pattern between co-stimulatory receptor CD226 and tumor cells as well as the infiltrating immune cells in the TME in GC.