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BACKGROUND: Gene expression profiling of uterus tissue has been performed in various contexts, but a significant amount of the data remains underutilized as it is not covered by the existing general resources. METHODOLOGY/PRINCIPAL FINDINGS: We curated 2254 datasets from 325 uterus related mass scale gene expression studies on human, mouse, rat, cow and pig species. We then computationally derived a 'reliability score' for each gene's expression status (transcribed/dormant), for each possible combination of conditions and locations, based on the extent of agreement or disagreement across datasets. The data and derived information has been compiled into the Mammalian Gene Expression Uterus database (MGEx-Udb, http://resource.ibab.ac.in/MGEx-Udb/). The database can be queried with gene names/IDs, sub-tissue locations, as well as various conditions such as the cervical cancer, endometrial cycles and disorders, and experimental treatments. Accordingly, the output would be a) transcribed and dormant genes listed for the queried condition/location, or b) expression profile of the gene of interest in various uterine conditions. The results also include the reliability score for the expression status of each gene. MGEx-Udb also provides information related to Gene Ontology annotations, protein-protein interactions, transcripts, promoters, and expression status by other sequencing techniques, and facilitates various other types of analysis of the individual genes or co-expressed gene clusters. CONCLUSIONS/SIGNIFICANCE: In brief, MGEx-Udb enables easy cataloguing of co-expressed genes and also facilitates bio-marker discovery for various uterine conditions.

Aims: Vascular smooth muscle cells (SMCs) and their derivatives are key contributors to the development of atherosclerosis. However, studying changes in SMC gene expression in heterogeneous vascular tissues is challenging due to the technical limitations and high cost associated with current approaches. In this paper, we apply translating ribosome affinity purification sequencing to profile SMC-specific gene expression directly from tissue., Methods and Results: To facilitate SMC-specific translatome analysis, we generated SMCTRAP mice, a transgenic mouse line expressing enhanced green fluorescent protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a) under the control of the SMC-specific αSMA promoter. These mice were further crossed with the atherosclerosis model Ldlr-/-, ApoB100/100 to generate SMCTRAP-AS mice and used to profile atherosclerosis-associated SMCs in thoracic aorta samples of 15-month-old SMCTRAP and SMCTRAP-AS mice. Our analysis of SMCTRAP-AS mice showed that EGFP-L10a expression was localized to SMCs in various tissues, including the aortic wall and plaque. The TRAP fraction demonstrated high enrichment of known SMC-specific genes, confirming the specificity of our approach. We identified several genes, including Cemip, Lum, Mfge8, Spp1, and Serpina3, which are known to be involved in atherosclerosis-induced gene expression. Moreover, we identified several novel genes not previously linked to SMCs in atherosclerosis, such as Anxa4, Cd276, inter-alpha-trypsin inhibitor-4 (Itih4), Myof, Pcdh11x, Rab31, Serpinb6b, Slc35e4, Slc8a3, and Spink5. Among them, we confirmed the SMC-specific expression of Itih4 in atherosclerotic lesions using immunofluorescence staining of mouse aortic roots and spatial transcriptomics of human carotid arteries. Furthermore, our more detailed analysis of Itih4 showed its link to coronary artery disease through the colocalization of genome-wide association studies, splice quantitative trait loci (QTL), and protein QTL signals., Conclusion: We generated a SMC-specific TRAP mouse line to study atherosclerosis and identified Itih4 as a novel SMC-expressed gene in atherosclerotic plaques, warranting further investigation of its putative function in extracellular matrix stability and genetic evidence of causality., Competing Interests: Conflict of interest: All authors confirm that there are no conflicts of interest related to this manuscript., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells., Methods: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL , we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells., Results: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production., Conclusions: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses., Competing Interests: Disclosures None.

The Kelch-like ECH-associated protein 1 (KEAP1) - Nuclear factor erythroid 2 -related factor 2 (NRF2) pathway is the major transcriptional stress response system in cells against oxidative and electrophilic stress. NRF2 is frequently constitutively active in many cancers, rendering the cells resistant to chemo- and radiotherapy. Loss-of-function (LOF) mutations in the repressor protein KEAP1 are common in non-small cell lung cancer, particularly adenocarcinoma. While the mutations can occur throughout the gene, they are enriched in certain areas, indicating that these may have unique functional importance. In this study, we show that in the GSEA analysis of TCGA lung adenocarcinoma RNA-seq data, the KEAP1 mutations in R320 and R470 were associated with enhanced Tumor Necrosis Factor alpha (TNFα) - Nuclear Factor kappa subunit B (NFκB) signaling as well as MYC and MTORC1 pathways. To address the functional role of these hotspot mutations, affinity purification and mass spectrometry (AP-MS) analysis of wild type (wt) KEAP1 and its mutation forms, R320Q and R470C were employed to interrogate differences in the protein interactome. We identified TNF receptor associated factor 2 (TRAF2) as a putative protein interaction partner. Both mutant KEAP1 forms showed increased interaction with TRAF2 and other anti-apoptotic proteins, suggesting that apoptosis signalling could be affected by the protein interactions. A549 lung adenocarcinoma cells overexpressing mutant KEAP1 showed high TRAF2-mediated NFκB activity and increased protection against apoptosis, XIAP being one of the key proteins involved in anti-apoptotic signalling. To conclude, KEAP1 R320Q and R470C and its interaction with TRAF2 leads to activation of NFκB pathway, thereby protecting against apoptosis., Competing Interests: Declaration of competing interest The authors declare that there are no potential conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Non-alcoholic fatty liver disease (NAFLD) is a fast-growing, underdiagnosed, epidemic. We hypothesise that obesity-related inflammation compromises adipose tissue functions, preventing efficient fat storage, and thus driving ectopic fat accumulation into the liver., Methods: To identify adipose-based mechanisms and potential serum biomarker candidates (SBCs) for NAFLD, we utilise dual-tissue RNA-sequencing (RNA-seq) data in adipose tissue and liver, paired with histology-based NAFLD diagnosis, from the same individuals in a cohort of obese individuals. We first scan for genes that are differentially expressed (DE) for NAFLD in obese individuals' subcutaneous adipose tissue but not in their liver; encode proteins secreted to serum; and show preferential adipose expression. Then the identified genes are filtered to key adipose-origin NAFLD genes by best subset analysis, knockdown experiments during human preadipocyte differentiation, recombinant protein treatment experiments in human liver HepG2 cells, and genetic analysis., Findings: We discover a set of genes, including 10 SBCs, that may modulate NAFLD pathogenesis by impacting adipose tissue function. Based on best subset analysis, we further follow-up on two SBCs CCDC80 and SOD3 by knockdown in human preadipocytes and subsequent differentiation experiments, which show that they modulate crucial adipogenesis genes, LPL, SREBPF1, and LEP. We also show that treatment of the liver HepG2 cells with the CCDC80 and SOD3 recombinant proteins impacts genes related to steatosis and lipid processing, including PPARA, NFE2L2, and RNF128. Finally, utilizing the adipose NAFLD DE gene cis-regulatory variants associated with serum triglycerides (TGs) in extensive genome-wide association studies (GWASs), we demonstrate a unidirectional effect of serum TGs on NAFLD with Mendelian Randomization (MR) analysis. We also demonstrate that a single SNP regulating one of the SBC genes, rs2845885, produces a significant MR result by itself. This supports the conclusion that genetically regulated adipose expression of the NAFLD DE genes may contribute to NAFLD through changes in serum TG levels., Interpretation: Our results from the dual-tissue transcriptomics screening improve the understanding of obesity-related NAFLD by providing a targeted set of 10 adipose tissue-active genes as new serum biomarker candidates for the currently grossly underdiagnosed fatty liver disease., Funding: The work was supported by NIH grants R01HG010505 and R01DK132775. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The KOBS study (J. P.) was supported by the Finnish Diabetes Research Foundation, Kuopio University Hospital Project grant (EVO/VTR grants 2005-2019), and the Academy of Finland grant (Contract no. 138006). This study was funded by the European Research Council under the European Union's Horizon 2020 research and innovation program (Grant No. 802825 to M. U. K.). K. H. P. was funded by the Academy of Finland (grant numbers 272376, 266286, 314383, and 335443), the Finnish Medical Foundation, Gyllenberg Foundation, Novo Nordisk Foundation (grant numbers NNF10OC1013354, NNF17OC0027232, and NNF20OC0060547), Finnish Diabetes Research Foundation, Finnish Foundation for Cardiovascular Research, University of Helsinki, and Helsinki University Hospital and Government Research Funds. I. S. was funded by the Instrumentarium Science Foundation. Personal grants to U. T. A. were received from the Matti and Vappu Maukonen Foundation, Ella och Georg Ehrnrooths Stiftelse and the Finnish Foundation for Cardiovascular Research., Competing Interests: Declaration of interests J. N. B. received consulting fees from GLG, and support for attending meetings and/or travel from the American Gastroenterology Association (AGA), at least once during the last 36 months. The other authors declare that they have no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The NRF2 pathway is frequently activated in various cancer types, yet a comprehensive analysis of its effects across different malignancies is currently lacking. We developed a NRF2 activity metric and utilized it to conduct a pan-cancer analysis of oncogenic NRF2 signaling. We identified an immunoevasive phenotype where high NRF2 activity is associated with low interferon-gamma (IFNγ), HLA-I expression and T cell and macrophage infiltration in squamous malignancies of the lung, head and neck area, cervix and esophagus. Squamous NRF2 overactive tumors comprise a molecular phenotype with SOX2/TP63 amplification, TP53 mutation and CDKN2A loss. These immune cold NRF2 hyperactive diseases are associated with upregulation of immunomodulatory NAMPT, WNT5A, SPP1, SLC7A11, SLC2A1 and PD-L1. Based on our functional genomics analyses, these genes represent candidate NRF2 targets, suggesting direct modulation of the tumor immune milieu. Single-cell mRNA data shows that cancer cells of this subtype exhibit decreased expression of IFNγ responsive ligands, and increased expression of immunosuppressive ligands NAMPT, SPP1 and WNT5A that mediate signaling in intercellular crosstalk. In addition, we discovered that the negative relationship of NRF2 and immune cells are explained by stromal populations of lung squamous cell carcinoma, and this effect spans multiple squamous malignancies based on our molecular subtyping and deconvolution data., Competing Interests: Declaration of competing interest The authors declare no potential conflicts interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

[Figure: see text].

Genetic factors underlying coronary artery disease (CAD) have been widely studied using genome-wide association studies (GWASs). However, the functional understanding of the CAD loci has been limited by the fact that a majority of GWAS variants are located within non-coding regions with no functional role. High cholesterol and dysregulation of the liver metabolism such as non-alcoholic fatty liver disease confer an increased risk of CAD. Here, we studied the function of non-coding single-nucleotide polymorphisms in CAD GWAS loci located within liver-specific enhancer elements by identifying their potential target genes using liver cis-eQTL analysis and promoter Capture Hi-C in HepG2 cells. Altogether, 734 target genes were identified of which 121 exhibited correlations to liver-related traits. To identify potentially causal regulatory SNPs, the allele-specific enhancer activity was analyzed by (1) sequence-based computational predictions, (2) quantification of allele-specific transcription factor binding, and (3) STARR-seq massively parallel reporter assay. Altogether, our analysis identified 1,277 unique SNPs that display allele-specific regulatory activity. Among these, susceptibility enhancers near important cholesterol homeostasis genes (APOB, APOC1, APOE, and LIPA) were identified, suggesting that altered gene regulatory activity could represent another way by which genetic variation regulates serum lipoprotein levels. Using CRISPR-based perturbation, we demonstrate how the deletion/activation of a single enhancer leads to changes in the expression of many target genes located in a shared chromatin interaction domain. Our integrative genomics approach represents a comprehensive effort in identifying putative causal regulatory regions and target genes that could predispose to clinical manifestation of CAD by affecting liver function., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The identification of causal variants and mechanisms underlying complex disease traits in humans is important for the progress of human disease genetics; this requires finding strategies to detect functional regulatory variants in disease-relevant cell types. To achieve this, we collected genetic and transcriptomic data from the aortic endothelial cells of up to 157 donors and four epigenomic phenotypes in up to 44 human donors representing individuals of both sexes and three major ancestries. We found thousands of expression quantitative trait loci (eQTLs) at all ranges of effect sizes not detected by the Gene-Tissue Expression Project (GTEx) in human tissues, showing that novel biological relationships unique to endothelial cells (ECs) are enriched in this dataset. Epigenetic profiling enabled discovery of over 3,000 regulatory elements whose activity is modulated by genetic variants that most frequently mutated ETS, AP-1, and NF-kB binding motifs, implicating these motifs as governors of EC regulation. Using CRISPR interference (CRISPRi), allele-specific reporter assays, and chromatin conformation capture, we validated candidate enhancer variants located up to 750 kb from their target genes, VEGFC, FGD6, and KIF26B. Regulatory SNPs identified were enriched in coronary artery disease (CAD) loci, and this result has specific implications for PECAM-1, FES, and AXL. We also found significant roles for EC regulatory variants in modifying the traits pulse pressure, blood protein levels, and monocyte count. Lastly, we present two unlinked SNPs in the promoter of MFAP2 that exhibit pleiotropic effects on human disease traits. Together, this supports the possibility that genetic predisposition for complex disease is manifested through the endothelium., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Microarrays and RNA sequencing are widely used to profile transcriptome remodeling during myocardial ischemia. However, the steady-state RNA analysis lacks in sensitivity to detect all noncoding RNA species and does not provide separation between transcriptional and post-transcriptional regulations. Here, we provide the first comprehensive analysis of nascent RNA profiles of mRNAs, primary micro-RNAs, long noncoding RNAs, and enhancer RNAs in a large animal model of acute infarction., Methods and Results: Acute infarction was induced by cardiac catheterization of domestic swine. Nuclei isolated from healthy, border zone, and ischemic regions of the affected heart were subjected to global run-on sequencing. Global run-on sequencing analysis indicated that half of affected genes are regulated at the level of transcriptional pausing. A gradient of induction of inflammatory mediators and repression of peroxisome proliferator-activated receptor signaling and oxidative phosphorylation was detected when moving from healthy toward infarcted area. In addition, we interrogated the transcriptional regulation of primary micro-RNAs and provide evidence that several arrhythmia-related target genes exhibit repression at post-transcriptional level. We identified 450 long noncoding RNAs differently regulated by ischemia, including novel conserved long noncoding RNAs expressed in antisense orientation to myocardial transcription factors GATA-binding protein 4, GATA-binding protein 6, and Krüppel-like factor 6. Finally, characterization of enhancers exhibiting differential expression of enhancer RNAs pointed a central role for Krüppel-like factor, MEF2C, ETS, NFY, ATF, E2F2, and NRF1 transcription factors in determining transcriptional responses to ischemia., Conclusions: Global run-on sequencing allowed us to follow the gradient of gene expression occurring in the ischemic heart and identify novel noncoding RNAs regulated by oxygen deprivation. These findings highlight potential new targets for diagnosis and treatment of myocardial ischemia., (© 2017 American Heart Association, Inc.)