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Picornaviridae represent a large family of single-stranded positive RNA viruses of which different members can infect both humans and animals. These include the enteroviruses (e.g., poliovirus, coxsackievirus, and rhinoviruses) as well as the cardioviruses (e.g., encephalomyocarditis virus). Picornaviruses have evolved to interact with, use, and/or evade cellular host systems to create the optimal environment for replication and spreading. It is known that viruses modify kinase activity during infection, but a proteome-wide overview of the (de)regulation of cellular kinases during picornavirus infection is lacking. To study the kinase activity landscape during picornavirus infection, we here applied dedicated targeted mass spectrometry-based assays covering ∼40% of the human kinome. Our data show that upon infection, kinases of the MAPK pathways become activated (e.g., ERK1/2, RSK1/2, JNK1/2/3, and p38), while kinases involved in regulating the cell cycle (e.g., CDK1/2, GWL, and DYRK3) become inactivated. Additionally, we observed the activation of CHK2, an important kinase involved in the DNA damage response. Using pharmacological kinase inhibitors, we demonstrate that several of these activated kinases are essential for the replication of encephalomyocarditis virus. Altogether, the data provide a quantitative understanding of the regulation of kinome activity induced by picornavirus infection, providing a resource important for developing novel antiviral therapeutic interventions., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of terminal mucin glycans sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry., Competing Interests: The authors declare no competing interests., (Copyright: © 2023 Chatterjee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Preclinical and clinical studies suggest that consumption of long chain omega-3 polyunsaturated fatty acids (PUFAs) reduces severity of chronic inflammatory and autoimmune diseases. While these ameliorative effects are conventionally associated with downregulated expression of proinflammatory cytokine and chemokine genes, our laboratory has recently identified Type 1 interferon (IFN1)-regulated gene expression to be another key target of omega-3 PUFAs. Here we used single cell RNA sequencing (scRNAseq) to gain new mechanistic perspectives on how the omega-3 PUFA docosahexaenoic acid (DHA) influences TLR4-driven proinflammatory and IFN1-regulated gene expression in a novel self-renewing murine fetal liver-derived macrophage (FLM) model. FLMs were cultured with 25 µM DHA or vehicle for 24 h, treated with modest concentration of LPS (20 ng/ml) for 1 and 4 h, and then subjected to scRNAseq using the 10X Chromium System. At 0 h (i.e., in the absence of LPS), DHA increased expression of genes associated with the NRF2 antioxidant response (e.g. Sqstm1 , Hmox1, Chchd10 ) and metal homeostasis (e.g. Mt1 , Mt2, Ftl1, Fth1 ), both of which are consistent with DHA-induced polarization of FLMs to a more anti-inflammatory phenotype. At 1 h post-LPS treatment, DHA inhibited LPS-induced cholesterol synthesis genes (e.g. Scd1, Scd2 , Pmvk, Cyp51, Hmgcs1 , and Fdps) which potentially could contribute to interference with TLR4-mediated inflammatory signaling. At 4 h post-LPS treatment, LPS-treated FLMs reflected a more robust inflammatory response including upregulation of proinflammatory cytokine (e.g. Il1a, Il1b, Tnf ) and chemokine (e.g. Ccl2, Ccl3, Ccl4, Ccl7 ) genes as well as IFN1-regulated genes (e.g. Irf7, Mx1, Oasl1, Ifit1 ), many of which were suppressed by DHA. Using single-cell regulatory network inference and clustering (SCENIC) to identify gene expression networks, we found DHA modestly downregulated LPS-induced expression of NF-κB-target genes. Importantly, LPS induced a subset of FLMs simultaneously expressing NF-κB- and IRF7/STAT1/STAT2-target genes that were conspicuously absent in DHA-pretreated FLMs. Thus, DHA potently targeted both the NF-κB and the IFN1 responses. Altogether, scRNAseq generated a valuable dataset that provides new insights into multiple overlapping mechanisms by which DHA may transcriptionally or post-transcriptionally regulate LPS-induced proinflammatory and IFN1-driven responses in macrophages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wierenga, Riemers, Westendorp, Harkema and Pestka.)

FACS sorting followed by single-cell RNA-sequencing (SORT-Seq) is a popular procedure to select cells of interest for single-cell transcriptomics. However, FACS is not suitable for measurement of subcellular distribution of fluorescence or for small samples (<1,000 cells). The VYCAP puncher system overcomes these limitations. Here, we describe a workflow to capture, image, and collect fluorescent human retina pigment epithelium cells for SORT-Seq using this system. The workflow can be used for any cell type with a diameter of ∼5-50 μm. For complete details on the use and execution of this protocol, please refer to Segeren et al. (2020)., Competing Interests: H.A.S. and B.W. declare no competing interests. L.O. and K.C.A. are employed by VYCAP B.V., (© 2021 The Author(s).)

E2F-transcription factors activate many genes involved in cell cycle progression, DNA repair, and apoptosis. Hence, E2F-dependent transcription must be tightly regulated to prevent tumorigenesis, and therefore metazoan cells possess multiple E2F regulation mechanisms. The best-known is the Retinoblastoma protein (RB), which is mutated in many cancers. Atypical E2Fs (E2F7 and -8) can repress E2F-target gene expression independently of RB and are rarely mutated in cancer. Therefore, they may act as emergency brakes in RB-mutated cells to suppress tumor growth. Currently, it is unknown if and how RB and atypical E2Fs functionally interact in vivo . Here, we demonstrate that mice with liver-specific combinatorial deletion of Rb and E2f7/8 have reduced life-spans compared to E2f7/8 or Rb deletion alone. This was associated with increased proliferation and enhanced malignant progression of liver tumors. Hence, atypical repressor E2Fs and RB cooperatively act as tumor suppressors in hepatocytes. In contrast, loss of either E2f7 or E2f8 largely prevented the formation of pituitary tumors in Rb +/- mice. To test whether atypical E2Fs can also function as oncogenes independent of RB loss, we induced long-term overexpression of E2f7 or E2f8 in mice. E2F7 and -8 overexpression increased the incidence of tumors in the lungs, but not in other tissues. Collectively, these data show that atypical E2Fs can promote but also inhibit tumorigenesis depending on tissue type and RB status. We propose that the complex interactions between atypical E2Fs and RB on maintenance of genetic stability underlie this context-dependency.

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. This multi-protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome-induced p53-activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro-tumorigenic effect of PIDDosome-mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts poor survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and that tumor cell density may serve as a novel prognostic marker for recurrence-free survival in HCC patients., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/C Cdh1 via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/C Cdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

E2F transcription factors control the cytokinesis machinery and thereby ploidy in hepatocytes. If or how these proteins limit proliferation of polyploid cells with extra centrosomes remains unknown. Here, we show that the PIDDosome, a signaling platform essential for caspase-2-activation, limits hepatocyte ploidy and is instructed by the E2F network to control p53 in the developing as well as regenerating liver. Casp2 and Pidd1 act as direct transcriptional targets of E2F1 and its antagonists, E2F7 and E2F8, that together co-regulate PIDDosome expression during juvenile liver growth and regeneration. Of note, whereas hepatocyte aneuploidy correlates with the basal ploidy state, the degree of aneuploidy itself is not limited by PIDDosome-dependent p53 activation. Finally, we provide evidence that the same signaling network is engaged to control ploidy in the human liver after resection. Our study defines the PIDDosome as a primary target to manipulate hepatocyte ploidy and proliferation rates in the regenerating liver., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

E2F7 and E2F8 act as tumor suppressors via transcriptional repression of genes involved in S-phase entry and progression. Previously, we demonstrated that these atypical E2Fs are degraded by APC/C C dh1 during G1 phase of the cell cycle. However, the mechanism driving the downregulation of atypical E2Fs during G2 phase is unknown. Here, we show that E2F7 is targeted for degradation by the E3 ubiquitin ligase SCF cyclin F during G2. Cyclin F binds via its cyclin domain to a conserved C-terminal CY motif on E2F7. An E2F7 mutant unable to interact with SCF cyclin F remains stable during G2. Furthermore, SCF cyclin F can also interact and induce degradation of E2F8. However, this does not require the cyclin domain of SCF cyclin F nor the CY motifs in the C-terminus of E2F8, implying a different regulatory mechanism than for E2F7. Importantly, depletion of cyclin F causes an atypical-E2F-dependent delay of the G2/M transition, accompanied by reduced expression of E2F target genes involved in DNA repair. Live cell imaging of DNA damage revealed that cyclin F-dependent regulation of atypical E2Fs is critical for efficient DNA repair and cell cycle progression., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Atypical E2F transcription factors (E2F7 and E2F8) function as key regulators of cell cycle progression and their inactivation leads to spontaneous cancer formation in mice. However, the mechanism of the tumor suppressor functions of E2F7/8 remain obscure. In this study we discovered that atypical E2Fs control tumor angiogenesis, one of the hallmarks of cancer. We genetically inactivated atypical E2Fs in epithelial and mesenchymal neoplasm and analyzed blood vessel formation in three different animal models of cancer. Tumor formation was either induced by application of 7,12-Dimethylbenz(a)anthracene/12-O-Tetradecanoylphorbol-13-acetate or by Myc/Ras overexpression. To our surprise, atypical E2Fs suppressed tumor angiogenesis in all three cancer models, which is in a sharp contrast to previous findings showing that atypical E2Fs promote angiogenesis during fetal development in mice and zebrafish. Real-time imaging in zebrafish displayed that fluorescent-labeled blood vessels showed enhanced intratumoral branching in xenografted E2f7/8-deficient neoplasms compared with E2f7/8-proficient neoplasms. DLL4 expression, a key negative inhibitor of vascular branching, was decreased in E2f7/8-deficient neoplastic cells, indicating that E2F7/8 might inhibit intratumoral vessel branching via induction of DLL4.

Background & Aims: Upon intestinal epithelial damage a complex wound healing response is initiated to restore epithelial integrity and defend against pathogenic invasion. Epithelium-derived Indian Hedgehog (Ihh) functions as a critical sensor in this process. Signaling occurs in a paracrine manner because the receptor for Ihh is expressed only in the mesenchyme, but the exact Hedgehog target cell has remained elusive. The aim of this study was to elucidate further the nature of this target cell in the context of intestinal inflammation., Methods: Hedgehog activity was modulated genetically in both cell type-specific and body-wide models and the resulting animals were analyzed for gene expression profiles and sensitivity for dextran sodium sulfate (DSS) colitis. To characterize the Hedgehog target cell, Gli1-CreERT2-Rosa26-ZsGreen animals were generated, which express ZsGreen in all Hedgehog-responsive cells. These cells were characterized using flow cytometry and immunofluorescence., Results: Loss of Indian Hedgehog from the intestinal epithelium resulted in a rapid increase in expression of inflammation-related genes, accompanied by increased influx of immune cells. Animals with epithelium-specific deletion of Ihh or lacking the Hedgehog receptor Smoothened from Hedgehog target cells were more sensitive to DSS colitis. In contrast, specific deletion of Smoothened in the myeloid compartment did not alter the response to DSS. This suggests that Hedgehog signaling does not repress intestinal immunity through an effect on myeloid cells. Indeed, we found that Hedgehog-responsive cells expressed gp38, smooth muscle actin, and desmin, indicating a fibroblastic nature. Ihh signaling inhibited expression of C-X-C motif chemokine ligand 12 (CXCL12) in fibroblasts in vitro and in vivo, thereby impairing the recruitment of immune cells., Conclusions: We show that epithelium-derived Indian Hedgehog signals exclusively to fibroblasts in the intestine. Loss of Ihh leads to a rapid immune response with up-regulation of fibroblast-derived CXCL12, and migration of immune cells into the lamina propria.

Disruption of the retinoblastoma (RB) tumor suppressor pathway, either through genetic mutation of upstream regulatory components or mutation of RB1 itself, is believed to be a required event in cancer. However, genetic alterations in the RB-regulated E2F family of transcription factors are infrequent, casting doubt on a direct role for E2Fs in driving cancer. In this work, a mutation analysis of human cancer revealed subtle but impactful copy number gains in E2F1 and E2F3 in hepatocellular carcinoma (HCC). Using a series of loss- and gain-of-function alleles to dial E2F transcriptional output, we have shown that copy number gains in E2f1 or E2f3b resulted in dosage-dependent spontaneous HCC in mice without the involvement of additional organs. Conversely, germ-line loss of E2f1 or E2f3b, but not E2f3a, protected mice against HCC. Combinatorial mapping of chromatin occupancy and transcriptome profiling identified an E2F1- and E2F3B-driven transcriptional program that was associated with development and progression of HCC. These findings demonstrate a direct and cell-autonomous role for E2F activators in human cancer.

E2F-mediated transcriptional repression of cell cycle-dependent gene expression is critical for the control of cellular proliferation, survival, and development. E2F signaling also interacts with transcriptional programs that are downstream of genetic predictors for cancer development, including hepatocellular carcinoma (HCC). Here, we evaluated the function of the atypical repressor genes E2f7 and E2f8 in adult liver physiology. Using several loss-of-function alleles in mice, we determined that combined deletion of E2f7 and E2f8 in hepatocytes leads to HCC. Temporal-specific ablation strategies revealed that E2f8's tumor suppressor role is critical during the first 2 weeks of life, which correspond to a highly proliferative stage of postnatal liver development. Disruption of E2F8's DNA binding activity phenocopied the effects of an E2f8 null allele and led to HCC. Finally, a profile of chromatin occupancy and gene expression in young and tumor-bearing mice identified a set of shared targets for E2F7 and E2F8 whose increased expression during early postnatal liver development is associated with HCC progression in mice. Increased expression of E2F8-specific target genes was also observed in human liver biopsies from HCC patients compared to healthy patients. In summary, these studies suggest that E2F8-mediated transcriptional repression is a critical tumor suppressor mechanism during postnatal liver development.

E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phases. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Here, we demonstrate that E2F7 and E2F8 are substrates of the anaphase-promoting complex/cyclosome (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN mutant E2F7 during G1 impairs S phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/C(C) (dc20). Importantly, atypical E2Fs can activate APC/C(C) (dh1) by repressing its inhibitors cyclin A, cyclin E, and Emi1. In conclusion, we discovered a feedback loop between atypical E2Fs and APC/C(C) (dh1), which ensures balanced expression of cell cycle genes and normal cell cycle progression., (© 2016 The Authors.)

The E2F family of transcription factors plays an important role in controlling cell-cycle progression. While this is their best-known function, we report here novel functions for the newest members of the E2F family, E2F7 and E2F8 (E2F7/8). We show that simultaneous deletion of E2F7/8 in zebrafish and mice leads to severe vascular defects during embryonic development. Using a panel of transgenic zebrafish with fluorescent-labelled blood vessels, we demonstrate that E2F7/8 are essential for proper formation of blood vessels. Despite their classification as transcriptional repressors, we provide evidence for a molecular mechanism through which E2F7/8 activate the transcription of the vascular endothelial growth factor A (VEGFA), a key factor in guiding angiogenesis. We show that E2F7/8 directly bind and stimulate the VEGFA promoter independent of canonical E2F binding elements. Instead, E2F7/8 form a transcriptional complex with the hypoxia inducible factor 1 (HIF1) to stimulate VEGFA promoter activity. These results uncover an unexpected link between E2F7/8 and the HIF1-VEGFA pathway providing a molecular mechanism by which E2F7/8 control angiogenesis.