Project description:We studied miRNAs and their gene targets affecting SARS-CoV-2 pathogenesis in CF airway epithelial cell models in response to TGF-β1. Small RNAseq in CF human bronchial epithelial cell line treated with TGF-β1 and miRNA profiling characterized TGF-β1 effects on the SARS-CoV-2 pathogenesis pathways. Among the effectors, we identified and validated two miRNAs targeting ACE2 mRNA using different CF and non-CF human bronchial epithelial cell models. We have shown that TGF-β1 inhibits ACE2 expression by miR-136-3p and miR-369-5p. ACE2 levels were higher in cells expressing F508del-CFTR, compared to wild-type(WT)-CFTR and TGF-β1 inhibited ACE2 in both cell types. The ACE2 protein levels were still higher in CF, compared to non-CF cells after TGF-β1 treatment. TGF-β1 prevented the functional rescue of F508del-CFTR by ETI in primary human bronchial epithelial cells while ETI did not prevent the TGF-β1 inhibition of ACE2 protein. Finally, TGF-β1 reduced binding of ACE2 to the recombinant monomeric spike RBD. Our results may help to explain, at least in part, the role of TGF-β1 on the SARS-CoV-2 entry via ACE2 in the CF and non-CF airway.
Project description:We demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and MERS-CoV entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of monkey and human origin and an in vivo mouse model.
Project description:Genetically Engineered Mouse Models (GEMMs) aid in understanding human pathologies and developing new therapeutics, yet recapitulating human diseases authentically in mice is challenging to design and execute. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences controlling spatiotemporal gene expression patterns and splicing to human diseases. It is thus apparent that including regulatory genomic regions during the engineering of GEMMs is highly preferable for disease modeling, with the prerequisite of large-scale genome engineering ability. Existing genome engineering methods have limits on the size and efficiency of DNA delivery, hampering routine creation of highly informative GEMMs. Here, we describe mSwAP-In (mammalian Switching Antibiotic resistance markers Progressively for Integration), a method for efficient genome rewriting in mouse embryonic stem cells. We first demonstrated the use of mSwAP-In for iterative genome rewriting of up to 115 kb of the Trp53 locus, as well as for genomic humanization of up to 180 kb ACE2 locus in response to the COVID-19 pandemic. Second, we showed the hACE2 GEMM authentically recapitulated human ACE2 expression patterns and splicing, and importantly, presented milder symptoms without mortality when challenged with SARS-CoV-2 compared to the K18-ACE2 model, thus representing a more authentic model of infection. Lastly, we demonstrated serial genome writing by humanizing mouse Tmprss2 in a biallelic fashion, highlighting the versatility of mSwAP-In in mouse genome writing.
Project description:Genetically Engineered Mouse Models (GEMMs) aid in understanding human pathologies and developing new therapeutics, yet recapitulating human diseases authentically in mice is challenging to design and execute. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences controlling spatiotemporal gene expression patterns and splicing to human diseases. It is thus apparent that including regulatory genomic regions during the engineering of GEMMs is highly preferable for disease modeling, with the prerequisite of large-scale genome engineering ability. Existing genome engineering methods have limits on the size and efficiency of DNA delivery, hampering routine creation of highly informative GEMMs. Here, we describe mSwAP-In (mammalian Switching Antibiotic resistance markers Progressively for Integration), a method for efficient genome rewriting in mouse embryonic stem cells. We first demonstrated the use of mSwAP-In for iterative genome rewriting of up to 115 kb of the Trp53 locus, as well as for genomic humanization of up to 180 kb ACE2 locus in response to the COVID-19 pandemic. Second, we showed the hACE2 GEMM authentically recapitulated human ACE2 expression patterns and splicing, and importantly, presented milder symptoms without mortality when challenged with SARS-CoV-2 compared to the K18-ACE2 model, thus representing a more authentic model of infection. Lastly, we demonstrated serial genome writing by humanizing mouse Tmprss2 in a biallelic fashion, highlighting the versatility of mSwAP-In in mouse genome writing.
Project description:Angiotensin-Converting Enzyme 2 (ACE2) binds SARS-CoV-2 spike protein and facilitates viral entry into the cell. ACE2 is expressed in the kidney and renal injury strongly impacts COVID-19 prognosis. In addition, a short, interferon-inducible isoform of ACE2, dACE2 was recently identified. Change in ACE2 expression has already been linked to several human nephropathies. However, these changes were not analyzed in context of dACE2 and regulation of ACE2 expression in the kidney remains unclear. Human primary Renal Proximal Tubule cells were used for all experiments. ChIP-seq analysis of histone modifications (H3K27ac, H3K4me3, K3K4me1), RNA polymerase loading and DNAse hypersensitive sites (DHS) were used to identify gene regulatory elements in the ACE2 locus. qRT-PCR was used to assess mRNA expression of ACE2, dACE2, TMPRSS2 and STAT1. RNA-seq was used to identify changes in global gene expression after cytokine treatment. Putative regulatory elements controlling dACE2 expression were identified using ChIP-seq profiles and RNA-seq mapping. qRT-PCR differentiating between ACE2 and dACE2 revealed that only dACE2 is interferon inducible. dACE2 mRNA was upregulated 300- and 600-fold by IFNα and IFNβ, respectively, while full length ACE2 was unchanged. Interferon stimulation also resulted in elevated STAT1 expression. JAK inhibitor ruxolitinib ablated both STAT1 and dACE2 expression after interferon treatment. RNA-seq analysis revealed approximately 1200 genes induced by IFNβ, largely innate immune response-related. We showed that human primary proximal tubules express ACE2 and its interferon-inducible short isoform, dACE2, from an intronic promoter. We also deliver new datasets identifying cytokine response genes in proximal tubule cells.
Project description:The homologous Ace2 and Swi5 transcription factors of Saccharomyces cerevisiae have identical DNA-binding domains, and both are cell cycle regulated. There are common target genes, as well as genes activated only by Ace2 and other genes activated only by Swi5. Keywords: genetic modification RNA was isolated from four strains: wild type, ace2 gene deletion, swi5 gene deletion, and the ace2 swi5 double gene deletion. RNAs from the three mutant strains were compared to wild type RNA in a microarray hybridization experiment.
Project description:Humanized mouse models and mouse-adapted SARS-CoV-2 virus are increasingly used to study COVID-19 pathogenesis, and it is therefore important to learn where the SARS-CoV-2 receptor ACE2 is expressed. Here we mapped ACE2 expression during mouse postnatal development and in adulthood. Pericytes in the central nervous system, heart and pancreas express ACE2 strongly, as do perineurial and adrenal fibroblasts, whereas endothelial cells do not at any location analyzed. In a number of other organs pericytes do not express ACE2, including in the lung where ACE2 instead is expressed in bronchial epithelium and alveolar type-II cells. The onset of ACE2 expression is organ-specific: in bronchial epithelium already at birth, in brain pericytes before and in heart pericytes after postnatal day 10.5. Establishing the vascular localization of ACE2 expression is central to correctly interpret data from modelling COVID-19 in the mouse and may shed light on the cause of vascular COVID-19 complications.
Project description:LC-MS/MS based investigation of protein abundance changes, induced by DZNep treatment in A549-ACE2 cell line (0.75 uM DZNep, vehicle PBS; 6h pre-treatment, harvested 24 h.p.i. with mock/SARS-CoV/SARS-CoV-2 at MOI 3) or primary human bronchial epithelial cells (NHBEs) (1.5 uM DZNep, vehicle PBS; 6h pre-treatment, harvested 36 h.p.i. with mock/SARS-CoV/SARS-CoV-2 at MOI 3), or by Tubercidin in A549-ACE2 cell line (1 uM Tubercidin, vehicle DMSO; 3h pre-treatment, harvested 24 h.p.i. with mock/SARS-CoV at MOI 0.01/SARS-CoV-2 at MOI 0.1).
Project description:SARS-CoV-2, the agent causing COVID-19, invades epithelial cells, including those of the respiratory and gastrointestinal mucosa, using angiotensin-converting enzyme-2 (ACE2) as a receptor. Subsequent inflammation can promote rapid virus clearance. However, severe cases of COVID-19 are characterized by an inefficient immune response that fails to clear infection. Using primary epithelial organoids from the colon, we explored how IFN-γ, a central antiviral mediator elevated in COVID-19, affects differentiation, ACE2 expression, and infectivity with SARS-CoV-2. ACE2 is mainly expressed by surface enterocytes of mouse and human colon. Inducing enterocyte differentiation in organoid culture resulted in increased ACE2 production. IFN-γ treatment promoted differentiation into mature KRT20+ enterocytes expressing high levels of ACE2. Similarly, IFN-γ promoted expression of ACE2 in human primary lung cells. IFN-y driven differentiation increased susceptibility to SARS-CoV-2 infection and electron microscopy revealed that the virus can efficiently complete its full life cycle in IFN-γ-treated enterocytes. Furthermore, infection-induced epithelial interferon signaling promoted enterocyte maturation and enhanced ACE2 expression. We reveal a mechanism by which IFN-y-driven inflammatory responses may increase susceptibility to SARS-CoV-2 and promote its replication.
Project description:We evaluate heterozygous transgenic mice expressing human ACE2 receptor driven by the epithelial cell promoter cytokeratin-18 promoter (K18-hACE2) as a model of SARS-CoV-2 infection. Intranasal inoculation of K18-hACE2 mice with SARS-CoV-2 results in high levels of infection in the lung parenchyma with spread to other organs.