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Tribbles homolog 3 (TRIB3) is pseudokinase involved in intracellular regulatory processes and has been implicated in several diseases. In this article, we report that human TRIB3 promoter contains a 33-bp variable number tandem repeat (VNTR) and characterize the heterogeneity and function of this genetic element. Analysis of human populations around the world uncovered the existence of alleles ranging from 1 to 5 copies of the repeat, with 2-, 3- and 5-copy alleles being the most common but displaying considerable geographical differences in frequency. The repeated sequence overlaps a C/EBP-ATF transcriptional regulatory element and is highly conserved, but not repeated, in various mammalian species, including great apes. The repeat is however evident in Neanderthal and Denisovan genomes. Reporter plasmid experiments in human cell culture reveal that an increased copy number of the TRIB3 promoter 33-bp repeat results in increased transcriptional activity. In line with this, analysis of whole genome sequencing and RNA-Seq data from human cohorts demonstrates that the copy number of TRIB3 promoter 33-bp repeats is positively correlated with TRIB3 mRNA expression level in many tissues throughout the body. Moreover, the copy number of the TRIB3 33-bp repeat appears to be linked to known TRIB3 eQTL SNPs as well as TRIB3 SNPs reported in genetic association studies. Taken together, the results indicate that the promoter 33-bp VNTR constitutes a causal variant for TRIB3 expression variation between individuals and could underlie the results of SNP-based genetic studies.

The ability of intracellular pathogens to manipulate host-cell viability is critical to successful infection. Some pathogens promote host-cell survival to protect their replicative niche, whereas others trigger host-cell death to facilitate release and dissemination of the pathogen after intracellular replication has occurred. We previously showed that the intracellular fungal pathogen Histoplasma capsulatum (Hc) uses the secreted protein Cbp1 to actively induce apoptosis in macrophages; interestingly, cbp1 mutant strains are unable to kill macrophages and display severely reduced virulence in the mouse model of Hc infection. To elucidate the mechanism of Cbp1-induced host-cell death, we performed a comprehensive alanine scanning mutagenesis and identified all amino acid residues that are required for Cbp1 to trigger macrophage lysis. Here we demonstrate that Hc strains expressing lytic CBP1 alleles activate the integrated stress response (ISR) in infected macrophages, as indicated by an increase in eIF2α phosphorylation as well as induction of the transcription factor CHOP and the pseudokinase Tribbles 3 (TRIB3). In contrast, strains bearing a non-lytic allele of CBP1 fail to activate the ISR, whereas a partially lytic CBP1 allele triggers intermediate levels of activation. We further show that macrophages deficient for CHOP or TRIB3 are partially resistant to lysis during Hc infection, indicating that the ISR is critical for susceptibility to Hc-mediated cell death. Moreover, we show that CHOP-dependent macrophage lysis is critical for efficient spread of Hc infection to other macrophages. Notably, CHOP knockout mice display reduced macrophage apoptosis and diminished fungal burden and are markedly resistant to Hc infection. Together, these data indicate that Cbp1 is required for Hc to induce the ISR and mediate a CHOP-dependent virulence pathway in the host.

Activating Transcription Factor 4 (ATF4) is an important regulator of gene expression in stress responses and developmental processes in many cell types. Here, we catalogued ATF4 binding sites in the human genome and identified overlaps with trait-associated genetic variants. We probed these genetic variants for allelic regulatory activity using a massively parallel reporter assay (MPRA) in HepG2 hepatoma cells exposed to tunicamycin to induce endoplasmic reticulum stress and ATF4 upregulation. The results revealed that in the majority of cases, the MPRA allelic activity of these SNPs was in agreement with the nucleotide preference seen in the ATF4 binding motif from ChIP-Seq. Luciferase and electrophoretic mobility shift assays in additional cellular models further confirmed ATF4-dependent regulatory effects for the SNPs rs532446 ( GADD45A intronic; linked to hematological parameters), rs7011846 ( LPL upstream; myocardial infarction), rs2718215 (diastolic blood pressure), rs281758 (psychiatric disorders) and rs6491544 (educational attainment). CRISPR-Cas9 disruption and/or deletion of the regulatory elements harboring rs532446 and rs7011846 led to the downregulation of GADD45A and LPL , respectively. Thus, these SNPs could represent examples of GWAS genetic variants that affect gene expression by altering ATF4-mediated transcriptional activation.

Megakaryocytopoiesis is a complex differentiation process driven by the hormone thrombopoietin by which haematopoietic progenitor cells give rise to megakaryocytes, the giant bone marrow cells that in turn break down to form blood platelets. The Tribbles Pseudokinase 3 gene ( TRIB3 ) encodes a pleiotropic protein increasingly implicated in the regulation of cellular differentiation programmes. Previous studies have hinted that TRIB3 could be also involved in megakaryocytopoiesis but its role in this process has so far not been investigated. Using cellular model systems of haematopoietic lineage differentiation here we demonstrate that TRIB3 is a negative modulator of megakaryocytopoiesis. We found that in primary cultures derived from human haematopoietic progenitor cells, thrombopoietin-induced megakaryocytic differentiation led to a time and dose-dependent decrease in TRIB3 mRNA levels. In the haematopoietic cell line UT7/mpl, silencing of TRIB3 increased basal and thrombopoietin-stimulated megakaryocyte antigen expression, as well as basal levels of ERK1/2 phosphorylation. In primary haematopoietic cell cultures, silencing of TRIB3 facilitated megakaryocyte differentiation. In contrast, over-expression of TRIB3 in these cells inhibited the differentiation process. The in-vitro identification of TRIB3 as a negative regulator of megakaryocytopoiesis suggests that in-vivo this gene could be important for the regulation of platelet production.

An excessive accumulation of hepatic lipids is a characteristic feature of metabolic dysfunction-associated steatotic liver disease (MASLD) and its severe form, metabolic dysfunction-associated steatohepatitis (MASH). Acyl-CoA:glycerol-sn-3-phosphate acyltransferase 3 (GPAT3) and other members of the GPAT family are enzymes which play an important role in glycerolipid synthesis. Previous articles have reported that GPAT3 mRNA and ER stress marker genes are upregulated in patients with MASH. Here, we study the regulatory mechanism of GPAT3 gene expression in human hepatoma cells suffering from ER stress. Transcriptome profiling showed that among the genes implicated in the formation of glycerolipids, GPAT3 is one of the most strongly activated genes in response to tunicamycin, an inducer of ER stress. CRISPR/Cas9-mediated disruption of activating transcription factor 4 ( ATF4 ) resulted in reduced GPAT3 expression under ER stress. Luciferase reporter assays of GPAT3 gene fragments encompassing ATF4 ChIP-seq peaks and mutational analysis revealed that activator protein-1 (AP-1) sites located in GPAT3 promoter and intron 2 mediate the activation of transcription in response to ER stress and ATF4. CRISPR/Cas9-mediated deletion of the region containing AP-1 sites from GPAT3 intron 2 caused a reduction of GPAT3 expression and triglyceride content in both unstressed cells and under ER stress. Thus, the results indicate that the induction of GPAT3 expression in response to ER stress is mediated by ATF4 via AP-1 elements in the promoter and second intron.

The proteasome is an appealing target for anticancer therapy and the proteasome inhibitor bortezomib has been approved for the treatment of several types of malignancies. However, the molecular mechanisms underlying cancer cell resistance to bortezomib remain poorly understood. In the current article, we investigate how modulation of the eIF2α–ATF4 stress pathway affects hepatoma cell response to bortezomib. Transcriptome profiling revealed that many ATF4 transcriptional target genes are among the most upregulated genes in bortezomib-treated HepG2 human hepatoma cells. While pharmacological enhancement of the eIF2α–ATF4 pathway activity results in the elevation of the activities of all branches of the unfolded protein response (UPR) and sensitizes cells to bortezomib toxicity, the suppression of ATF4 induction delays bortezomib-induced cell death. The pseudokinase TRIB3, an inhibitor of ATF4, is expressed at a high basal level in hepatoma cells and is strongly upregulated in response to bortezomib. To map genome-wide chromatin binding loci of TRIB3 protein, we fused a Flag tag to endogenous TRIB3 in HepG2 cells and performed ChIP-Seq. The results demonstrate that TRIB3 predominantly colocalizes with ATF4 on chromatin and binds to genomic regions containing the C/EBP–ATF motif. Bortezomib treatment leads to a robust enrichment of TRIB3 binding near genes induced by bortezomib and involved in the ER stress response and cell death. Disruption of TRIB3 increases C/EBP–ATF-driven transcription, augments ER stress and cell death upon exposure to bortezomib, while TRIB3 overexpression enhances cell survival. Thus, TRIB3, colocalizing with ATF4 and limiting its transcriptional activity, functions as a factor increasing resistance to bortezomib, while pharmacological over-activation of eIF2α–ATF4 can overcome the endogenous restraint mechanisms and sensitize cells to bortezomib.

Genome-wide association studies have successfully mapped thousands of loci associated with complex traits. During the last decade, functional genomics approaches combining genotype information with bulk RNA-sequencing data have identified genes regulated by GWAS loci through expression quantitative trait locus (eQTL) analysis. Single-cell RNA-Sequencing (scRNA-Seq) technologies have created new exciting opportunities for spatiotemporal assessment of changes in gene expression at the single-cell level in complex and inherited conditions. A growing number of studies have demonstrated the power of scRNA-Seq in eQTL mapping across different cell types, developmental stages and stimuli that could be obscured when using bulk RNA-Seq methods. In this review, we outline the methodological principles, advantages, limitations and the future experimental and analytical considerations of single-cell eQTL studies. We look forward to the explosion of single-cell eQTL studies applied to large-scale population genetics to take us one step closer to understanding the molecular mechanisms of disease.

Background Regulatory networks controlling aging and disease trajectories remain incompletely understood. MicroRNAs (miRNAs) are a class of regulatory non-coding RNAs that contribute to the regulation of tissue homeostasis by modulating the stability and abundance of their target mRNAs. MiRNA genes are transcribed similarly to protein-coding genes which has facilitated their annotation and quantification from bulk transcriptomes. Here, we show that droplet, spatial, and plate-based single-cell RNA-sequencing platforms can be used to decipher miRNA gene signatures at cellular resolution to reveal their expression dynamics in vivo. Methods We first benchmarked the approach examining concordance between platforms, species, and cell type-specific bulk expression data. To discover changes in miRNA gene expression that could contribute to the progressive loss of cellular homeostasis during aging and disease development, we annotated the comprehensive aging mouse dataset, Tabula Muris Senis, with cell type-specific miRNA expression and acquired transcriptome and translatome profiles from an atherosclerosis disease model. Results We generated an openly available workflow and aging-profile resource to characterize miRNA expression from single-cell genomics studies. Comparing immune cells in spleen tissue between young and old mice revealed concordance with previous functional studies, highlighting the upregulation of mmu-mir-146a, mmu-mir-101a, and mmu-mir-30 family genes involved in senescence and inflammatory pathways. Atherosclerosis progression is reflected within adipose tissue as expansion of the myeloid compartment, with elevated pro-inflammatory mmu-mir-511 expression in several macrophage subtypes. Upregulation of the immunosuppressive mmu-mir-23b ~ mir-24–2 ~ mir-27b locus was specific to Trem2 + lipid-associated macrophages, prevalent at late disease. Accordingly, ribosome-associated RNA profiling from myeloid cells in vivo validated significant mmu-mir-23b target gene enrichment in disease-regulated translatomes. Prominent tissue infiltration of monocytes led to upregulated mmu-mir-1938 and mmu-mir-22 expression and in classical monocytes activated mmu-mir-221 ~ 222, mmu-mir-511, and mmu-mir-155 gene loci, confirmed by bulk nascent transcriptomics data from ex vivo macrophage cultures. Overall, the monocyte-associated changes in miRNA expression represented the most significant target gene associations in the disease-trajectory translatome profiles. Conclusions We demonstrate that miRNA gene transcriptional activity is widely impacted in immune cells by aging and during disease development and further identify the corresponding translatome signature of inflamed adipose tissue.

Increased glutamine metabolism by macrophages is associated with development of atherosclerotic lesions. Positron emission tomography/computed tomography (PET/CT) with a glutamine analog (2S,4 R )-4- 18 F-fluoroglutamine ( 18 F-FGln) allows quantification of glutamine consumption in vivo . Here, we investigated uptake of 18 F-FGln by atherosclerotic lesions in mice and compared the results with those obtained using the glucose analog 2-deoxy-2- 18 F-fluoro- D -glucose ( 18 F-FDG). Uptake of 18 F-FGln and 18 F-FDG by healthy control mice (C57BL/6JRj) and atherosclerotic low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 (LDLR −/− ApoB 100/100 ) was investigated. The mice were injected intravenously with 18 F-FGln or 18 F-FDG for in vivo PET/CT imaging. After sacrifice at 70 minutes post-injection, tracer uptake was analyzed by gamma counting of excised tissues and by autoradiography of aorta cryosections, together with histological and immunohistochemical analyses. We found that myocardial uptake of 18 F-FGln was low. PET/CT detected lesions in the aortic arch, with a target-to-background ratio (SUV max , aortic arch/SUV mean , blood) of 1.95 ± 0.42 (mean ± standard deviation). Gamma counting revealed that aortic uptake of 18 F-FGln by LDLR −/− ApoB 100/100 mice (standardized uptake value [SUV], 0.35 ± 0.06) was significantly higher than that by healthy controls (0.20 ± 0.08, P = 0.03). More detailed analysis by autoradiography revealed that the plaque-to-healthy vessel wall ratio of 18 F-FGln (2.90 ± 0.42) was significantly higher than that of 18 F-FDG (1.93 ± 0.22, P = 0.004). Immunohistochemical staining confirmed that 18 F-FGln uptake in plaques co-localized with glutamine transporter SLC7A7-positive macrophages. Collectively these data show that the 18 F-FGln PET tracer detects inflamed atherosclerotic lesions. Thus, exploiting glutamine consumption using 18 F-FGln PET may have translational relevance for studying atherosclerotic inflammation.

Folate receptor β (FR-β), a marker expressed on macrophages, is a promising target for imaging of inflammation. Here, we report the radiosynthesis and preclinical evaluation of [ 68 Ga]Ga-NOTA-folate ( 68 Ga-FOL). After determining the affinity of 68 Ga-FOL using cells expressing FR-β, we studied atherosclerotic mice with 68 Ga-FOL and 18 F-FDG PET/CT. In addition, we studied tracer distribution and co-localization with macrophages in aorta cryosections using autoradiography, histology, and immunostaining. The specificity of 68 Ga-FOL was assessed in a blocking study with folate glucosamine. As a final step, human radiation doses were extrapolated from rat PET data. We were able to produce 68 Ga-FOL with high radiochemical purity and moderate molar activity. Cell binding studies revealed that 68 Ga-FOL had 5.1 nM affinity for FR-β. Myocardial uptake of 68 Ga-FOL was 20-fold lower than that of 18 F-FDG. Autoradiography and immunohistochemistry of the aorta revealed that 68 Ga-FOL radioactivity co-localized with Mac-3–positive macrophage-rich atherosclerotic plaques. The plaque-to-healthy vessel wall ratio of 68 Ga-FOL was significantly higher than that of 18 F-FDG. Blocking studies verified that 68 Ga-FOL was specific for FR. Based on estimations from rat data, the human effective dose was 0.0105 mSv/MBq. Together, these findings show that 68 Ga-FOL represents a promising new FR-β–targeted tracer for imaging macrophage-associated inflammation.