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Population-scale gene expression analysis reveals the contribution of expression diversity to the modern wheat improvement.
Gene expression diversity is crucial for crop breeding, yet population genomics has focused primarily on sequence polymorphisms. A single reference genome for RNA-seq cannot handle introgression bias. Here, we conduct RNA-seq for 328 wheat lines, including landraces and elite cultivars from China and the United States, to investigate the expression variation underlying agronomic traits. Leveraging pan-genome resources, we identify 23,296 more transcripts than using the Chinese Spring reference. We construct a pan-gene regulatory atlas through eQTL analysis, revealing the tight regulation of introgressed genes. We identify 299 high-confidence candidate genes for 34 agronomic traits and resistance to 8 Blumeria graminis f. sp. tritici isolates, more than one-fifth of which were absent from the Chinese Spring. Utilizing the Kenong 9204 mutant library, 73.7% of the candidates show significant phenotypic effects. Our work mitigates the reference bias and highlights the impact of breeding-driven directional expression changes on wheat adaptation and improvement.
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An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.
Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.
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Single-cell transcriptomic atlas of the human testis across the reproductive lifespan.
Testicular aging is associated with declining reproductive health, but the molecular mechanisms are unclear. Here we generate a dataset of 214,369 single-cell transcriptomes from testicular cells of 35 individuals aged 21-69, offering a resource for studying testicular aging and physiology. Machine learning analysis reveals a stronger aging response in somatic cells compared to germ cells. Two waves of aging-related changes are identified: the first in peritubular cells of donors in their 30s, marked by increased basement membrane thickness, indicating a priming state for aging. In their 50s, testicular cells exhibit functional changes, including altered steroid metabolism in Leydig cells and immune responses in macrophages. Further analyses reveal the impact of body mass index on spermatogenic capacity as age progresses, particularly after age 45. Altogether, our findings illuminate molecular alterations during testis aging and their relationship with body mass index, providing a foundation for future research and offering potential diagnostic markers and therapeutic targets.
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Single-cell RNA sequencing of OSCC primary tumors and lymph nodes reveals distinct origin and phenotype of fibroblasts.
Desmoplasia in fibroblasts within metastatic lymph nodes (MLNs) serves as an indicator of extranodal extension (ENE), which led mortality in oral squamous cell carcinoma (OSCC). However, systematic studies on fibroblasts in MLNs are lacking. Therefore, this study characterized the differences in phenotype, function, and origin of fibroblasts between primary tumors (PTs) and lymph nodes (LNs) in OSCC. We generated single-cell maps of PTs and paired MLNs and draining LNs from three OSCC patients. The transcriptomic atlas, pseudotime analysis, intercellular communication networks and enrichment analysis of the single cells were characterized. Phenotype and function heterogeneity of fibroblast cells between PTs and MLNs were further verified in vitro. Among 44,052 fibroblasts, we identified two distinct subpopulations of cancer-associated myofibroblastic cells (mCAFs): RGS4+ mCAF1 and COMP + mCAF2. Notably, they exhibited distinct distributions, with mCAF1 predominantly localized in the PTs and mCAF2 in the MLNs. Moreover, pseudotime analysis revealed their distinct origins: mCAF1 originated from inherent normal myofibroblastic cells in the PT, whereas mCAF2 originated from fibroblastic reticular cells in the LNs. Further functional experiments using primary fibroblasts revealed that, compared to mCAF1, mCAF2 in MLNs exhibited weaker crosstalk with immune cells but enhanced extracellular matrix activity, which is closely linked to ENE formation in OSCC. Additionally, we identified two fibroblast subgroups in a transforming state, indicating a potential epithelial-mesenchymal transition. Our research offers profound insights into the heterogeneity of fibroblasts between the PT and MLN in OSCC, serving as an essential resource for future drug discovery endeavors.
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A single-cell atlas of the normal and malformed human brain vasculature.
Cerebrovascular diseases are a leading cause of death and neurologic disability. Further understanding of disease mechanisms and therapeutic strategies requires a deeper knowledge of cerebrovascular cells in humans. We profiled transcriptomes of 181,388 cells to define a cell atlas of the adult human cerebrovasculature, including endothelial cell molecular signatures with arteriovenous segmentation and expanded perivascular cell diversity. By leveraging this reference, we investigated cellular and molecular perturbations in brain arteriovenous malformations, which are a leading cause of stroke in young people, and identified pathologic endothelial transformations with abnormal vascular patterning and the ontology of vascularly derived inflammation. We illustrate the interplay between vascular and immune cells that contributes to brain hemorrhage and catalog opportunities for targeting angiogenic and inflammatory programs in vascular malformations.
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Novel macrophage-related gene prognostic index for glioblastoma associated with M2 macrophages and T cell dysfunction.
This study aims to construct a Macrophage-Related Gene Prognostic Index (MRGPI) for glioblastoma (GBM) and explore the underlying molecular, metabolic, and immunological features. Based on the GBM dataset from The Cancer Genome Atlas (n = 156), 13 macrophage-related hub genes were identified by weighted gene co-expression network (WGCNA) analysis. 5 prognostic genes screened by Kaplan-Meire (K-M) analysis and Cox regression model were used to construct the MRGPI, including GPR84, NCF2, HK3, LILRB2, and CCL18. Multivariate Cox regression analysis found that the MRGPI was an independent risk factor (HR = 2.81, CI95: 1.13-6.98, p = 0.026), leading to an unfavorable outcome for the MRGPI-high group, which was further validated by 4 validation GBM cohorts (n = 728). Thereafter, the molecular, metabolic, and immune features and the clinical implications of the MRGPI-based groups were comprehensively characterized. Gene set enrichment analysis (GSEA) found that immune-related pathways, including inflammatory and adaptive immune response, and activated eicosanoid metabolic pathways were enriched in the MRGPI-high group. Besides, genes constituting the MRGPI was primarily expressed by monocytes and macrophages at single-cell scope and was associated with the alternative activation of macrophages. Moreover, correlation analysis and receiver operating characteristic (ROC) curves revealed the relevance between the MRGPI with the expression of immune checkpoints and T cell dysfunction. Thus, the responsiveness of samples in the MRGPI-high group to immune checkpoint inhibitors (ICI) was detected by algorithms, including Tumor Immune Dysfunction and Exclusion (TIDE) and Submap. In contrast, the MRGPI-low group had favorable outcome, was less immune active and insensitive to ICI. Together, we have developed a promising biomarker to classify the prognosis, metabolic and immune features for GBM, and provide references for facilitating the personalized application of ICI in GBM.
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Identification of immune-related targets of N6-methyladenosine regulators in hepatocellular carcinoma via RNA-seq analysis.
BACKGROUND: N6-methyladenosine (m6A) regulators are involved in hepatocellular carcinoma (HCC) development. However, the functions of m6A regulators in HCC have not yet been elucidated. This study aimed to evaluate the potential of m6A methylation regulators as biomarkers and immunotherapeutic targets in HCC. METHODS: RNA sequencing data were obtained from publicly available databases including The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Bioinformatics analysis was conducted, including Kaplan-Meier (KM) survival analysis, univariate and multivariate Cox regression, receiver operating characteristic (ROC) curve analysis and immune-related signatures were assessed using single-sample gene set enrichment analysis (ssGSEA) and drug sensitivity prediction was performed to identify potential small-molecule compounds. In vitro, small interfering RNA (siRNA)-mediated knockdown of selected m6A regulators was performed in three HCC cell lines (HepG2, HuH7 and MHCC-97H). Quantitative polymerase chain reaction (qPCR) and Western blot (WB) were subsequently conducted to validate gene expression changes. Additionally, WB was performed after treatment with candidate drugs to evaluate their functional impact. RESULTS: The five upregulated m6A regulators in HCC, KIAA1429, METTL3, PRRC2A, RBMX, and ZC3H3, were related to worse outcomes in HCC. ROC curves showed that m6A regulators could accurately distinguish between normal and HCC tumor tissues. Furthermore, differential m6A regulator expression was affected by immune infiltration. In advanced HCC patients, m6A regulators were important in determining clinical outcomes. Drug response analysis demonstrated m6A regulators as potential therapeutic agents that could be used as guidance for patients with HCC. PCR and WB analysis showed that m6A regulators could act on immune-related sites, interact with immune tolerance, and inhibit it. CONCLUSIONS: This study indicates that KIAA1429, METTL3, PRRC2A, RBMX, and ZC3H3 may be potential immunotherapeutic targets and biomarkers in HCC.
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Atlas of Fshr expression from novel reporter mice.
The FSH-FSHR pathway has been considered an essential regulator in reproductive development and fertility. But there has been emerging evidence of FSHR expression in extragonadal organs. This poses new questions and long-term debates regarding the physiological role of the FSH-FSHR, and underscores the need for reliable, in vivo analysis of FSHR expression in animal models. However, conventional methods have proven insufficient for examining FSHR expression due to several limitations. To address this challenge, we developed Fshr-ZsGreen reporter mice under the control of Fshr endogenous promoter using CRISPR-Cas9. With this novel genetic tool, we provide a reliable readout of Fshr expression at single-cell resolution level in vivo and in real time. Reporter animals were also subjected to additional analyses,to define the accurate expression profile of FSHR in gonadal and extragonadal organs/tissues. Our compelling results not only demonstrated Fshr expression in intragonadal tissues but also, strikingly, unveiled notably increased expression in Leydig cells, osteoblast lineage cells, endothelial cells in vascular structures, and epithelial cells in bronchi of the lung and renal tubes. The genetic decoding of the widespread pattern of Fshr expression highlights its physiological relevance beyond reproduction and fertility, and opens new avenues for therapeutic options for age-related disorders of the bones, lungs, kidneys, and hearts, among other tissues. Exploiting the power of the Fshr knockin reporter animals, this report provides the first comprehensive genetic record of the spatial distribution of FSHR expression, correcting a long-term misconception about Fshr expression and offering prospects for extensive exploration of FSH-FSHR biology.
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Integrated bioinformatics and experimental analysis of CHAF1B as a novel biomarker and immunotherapy target in LUAD.
The prognosis and treatment efficacy of lung adenocarcinoma (LUAD), a disease with a high incidence, remains unsatisfactory. Identifying new biomarkers and therapeutic targets for LUAD is essential. Chromosomal assembly factor 1B (CHAF1B), a p60 component of the CAF-1 complex, is closely linked to tumor incidence and cell proliferation. However, CHAF1B's biological role and molecular mechanism in LUAD remain unclear. Here, CHAF1B expression in LUAD was examined using the GEPIA2 and UALCAN databases. Using The Cancer Genome Atlas (TCGA) LUAD database, we analyzed the diagnostic and prognostic significance of CHAF1B and its association with immune infiltration and immunological checkpoints. Gene ontology (GO) enrichment and single-cell function analyses were employed to investigate CHAF1B's possible biological roles. Drug sensitivity analysis predicted CHAF1B's effect on chemotherapeutic drug sensitivity. We also predicted lncRNAs-miRNA-CHAF1B axis to explore the molecular mechanism of CHAF1B in LUAD. Preliminary in vitro studies using qRT-PCR, CCK8, Transwell, glucose, and lactate metabolism confirmed CHAF1B's expression and role in LUAD. Its expression is associated with drug sensitivity, immunological checkpoints, and immune cell infiltration. We predicted that three miRNAs (miR-29c-3p, miR-145-5p, miR-1247-5p) and three lncRNAs (AL139287.1, NEAT1, SHG1) may be target miRNAs and target lncRNAs that regulate CHAF1B. In vitro tests showed that CHAF1B suppression decreased LUAD's migration, invasion, proliferation, and glycolysis. Overall, CHAF1B may be an innovative biomarker and therapeutic target for LUAD.
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Single-cell transcriptomic analysis reveals disparate effector differentiation pathways in human Treg compartment.
Human FOXP3+ regulatory T (Treg) cells are central to immune tolerance. However, their heterogeneity and differentiation remain incompletely understood. Here we use single-cell RNA and T cell receptor sequencing to resolve Treg cells from healthy individuals and patients with or without acute graft-versus-host disease (aGVHD) who undergo stem cell transplantation. These analyses, combined with functional assays, separate Treg cells into naïve, activated, and effector stages, and resolve the HLA-DRhi, LIMS1hi, highly suppressive FOXP3hi, and highly proliferative MKI67hi effector subsets. Trajectory analysis assembles Treg subsets into two differentiation paths (I/II) with distinctive phenotypic and functional programs, ending with the FOXP3hi and MKI67hi subsets, respectively. Transcription factors FOXP3 and SUB1 contribute to some Path I and Path II phenotypes, respectively. These FOXP3hi and MKI67hi subsets and two differentiation pathways are conserved in transplanted patients, despite having functional and migratory impairments under aGVHD. These findings expand the understanding of Treg cell heterogeneity and differentiation and provide a single-cell atlas for the dissection of Treg complexity in health and disease.