Shady Younis

All Publications

• Epstein-Barr virus as a potentiator of autoimmune diseases. Nature reviews. Rheumatology Robinson, W. H., Younis, S., Love, Z. Z., Steinman, L., Lanz, T. V. 2024

  Abstract

  The Epstein-Barr virus (EBV) is epidemiologically associated with development of autoimmune diseases, including systemic lupus erythematosus, Sjögren syndrome, rheumatoid arthritis and multiple sclerosis. Although there is well-established evidence for this association, the underlying mechanistic basis remains incompletely defined. In this Review, we discuss the role of EBV infection as a potentiator of autoimmune rheumatic diseases. We review the EBV life cycle, viral transcription programmes, serological profiles and lytic reactivation. We discuss the epidemiological and mechanistic associations of EBV with systemic lupus erythematosus, Sjögren syndrome, rheumatoid arthritis and multiple sclerosis. We describe the potential mechanisms by which EBV might promote autoimmunity, including EBV nuclear antigen 1-mediated molecular mimicry of human autoantigens; EBV-mediated B cell reprogramming, including EBV nuclear antigen 2-mediated dysregulation of autoimmune susceptibility genes; EBV and host genetic factors, including the potential for autoimmunity-promoting strains of EBV; EBV immune evasion and insufficient host responses to control infection; lytic reactivation; and other mechanisms. Finally, we discuss the therapeutic implications and potential therapeutic approaches to targeting EBV for the treatment of autoimmune disease.

  View details for DOI 10.1038/s41584-024-01167-9

  View details for PubMedID 39390260

  View details for PubMedCentralID 3528287

• Oxidative Stress is a shared characteristic of ME/CFS and Long COVID. bioRxiv : the preprint server for biology Shankar, V., Wilhelmy, J., Curtis, E. J., Michael, B., Cervantes, L., Mallajosyula, V. A., Davis, R. W., Snyder, M., Younis, S., Robinson, W. H., Shankar, S., Mischel, P. S., Bonilla, H., Davis, M. M. 2024

  Abstract

  More than 65 million individuals worldwide are estimated to have Long COVID (LC), a complex multisystemic condition, wherein patients of all ages report fatigue, post-exertional malaise, and other symptoms resembling myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS). With no current treatments or reliable diagnostic markers, there is an urgent need to define the molecular underpinnings of these conditions. By studying bioenergetic characteristics of peripheral blood lymphocytes in over 16 healthy controls, 15 ME/CFS, and 15 LC, we find both ME/CFS and LC donors exhibit signs of elevated oxidative stress, relative to healthy controls, especially in the memory subset. Using a combination of flow cytometry, bulk RNA-seq analysis, mass spectrometry, and systems chemistry analysis, we also observed aberrations in ROS clearance pathways including elevated glutathione levels, decreases in mitochondrial superoxide dismutase levels, and glutathione peroxidase 4 mediated lipid oxidative damage. Critically, these changes in redox pathways show striking sex-specific trends. While females diagnosed with ME/CFS exhibit higher total ROS and mitochondrial calcium levels, males with an ME/CFS diagnosis have normal ROS levels, but larger changes in lipid oxidative damage. Further analyses show that higher ROS levels correlates with hyperproliferation of T cells in females, consistent with the known role of elevated ROS levels in the initiation of proliferation. This hyperproliferation of T cells can be attenuated by metformin, suggesting this FDA-approved drug as a possible treatment, as also suggested by a recent clinical study of LC patients. Thus, we report that both ME/CFS and LC are mechanistically related and could be diagnosed with quantitative blood cell measurements. We also suggest that effective, patient tailored drugs might be discovered using standard lymphocyte stimulation assays.

  View details for DOI 10.1101/2024.05.04.592477

  View details for PubMedID 38746454

• Low mutation load in a supergene underpinning alternative male mating strategies in ruff (Calidris pugnax). Molecular biology and evolution Hill, J., Enbody, E. D., Bi, H., Lamichhaney, S., Lei, W., Chen, J., Wei, C., Liu, Y., Schwochow, D., Younis, S., Widemo, F., Andersson, L. 2023

  Abstract

  A paradox in evolutionary biology is how supergenes can maintain high fitness despite reduced effective population size, the suppression of recombination, and the expected accumulation of mutational load. The ruff supergene involves two rare inversion haplotypes (Satellite and Faeder). These are recessive lethals but with dominant effects on male mating strategies, plumage, and body size. Sequence divergence to the wild-type (Independent) haplotype indicates that the inversion could be as old as 4 million years. Here we have constructed a highly contiguous genome assembly of the inversion region for both the Independent and Satellite haplotypes. Based on the new data we estimate that the recombination event(s) creating the Satellite haplotype occurred only about 70,000 years ago. Contrary to expectations for supergenes, we find no substantial expansion of repeats and only a modest mutation load on the Satellite and Faeder haplotypes despite high sequence divergence to the non-inverted haplotype (1.46%). The essential centromere protein N gene CENPN is disrupted by the inversion, and is as well conserved on the inversion haplotypes as on the noninversion haplotype. These results suggest that the inversion may be much younger than previously thought. The low mutation load, despite recessive lethality, may be explained by the introgression of the inversion from a now extinct lineage.

  View details for DOI 10.1093/molbev/msad224

  View details for PubMedID 37804117

• Identification of a Mast Cell-high Synovial Pathotype of Osteoarthritis Younis, S., Bai, A., Wong, H., Love, Z., Wang, Q., Robinson, W. WILEY. 2023: 1766-1767

  View details for Web of Science ID 001190014301377

• The RNA-binding protein ZC3H11A interacts with the nuclear poly(A)-binding protein PABPN1 and alters polyadenylation of viral transcripts. The Journal of biological chemistry Kases, K., Schubert, E., Hajikhezri, Z., Larsson, M., Devi, P., Darweesh, M., Andersson, L., Akusjärvi, G., Punga, T., Younis, S. 2023: 104959

  Abstract

  Nuclear mRNA metabolism is regulated by multiple proteins, which either directly bind to RNA or form multi-protein complexes. The RNA-binding protein ZC3H11A is involved in nuclear mRNA export, NF-κB signaling and is essential during mouse embryo development. Furthermore, previous studies have shown that ZC3H11A is important for nuclear-replicating viruses. However, detailed biochemical characterization of the ZC3H11A protein has been lacking. In this study, we established the ZC3H11A protein interactome in human and mouse cells. We demonstrate that the nuclear poly(A)-binding protein PABPN1 interacts specifically with the ZC3H11A protein and controls ZC3H11A localization into nuclear speckles. We report that ZC3H11A specifically interacts with the human adenovirus type 5 (HAdV-5) capsid mRNA in a PABPN1-dependent manner. Notably, ZC3H11A uses the same zinc finger motifs to interact with PABPN1 and viral mRNA. Further, we demonstrate that the lack of ZC3H11A alters the polyadenylation of HAdV-5 capsid mRNA. Taken together, our results suggest that the ZC3H11A protein may act as a novel regulator of polyadenylation of nuclear mRNA.

  View details for DOI 10.1016/j.jbc.2023.104959

  View details for PubMedID 37356722

• Ablation of ZC3H11A causes early embryonic lethality and dysregulation of metabolic processes. Proceedings of the National Academy of Sciences of the United States of America Younis, S., Jouneau, A., Larsson, M., Oudin, J., Adenot, P., Omar, J., Brochard, V., Andersson, L. 2023; 120 (23): e2216799120

  Abstract

  ZC3H11A (zinc finger CCCH domain-containing protein 11A)is a stress-induced mRNA-binding protein required for efficient growth of nuclear-replicating viruses. The cellular functions of ZC3H11A during embryonic development are unknown. Here, we report the generation and phenotypic characterization of Zc3h11a knockout (KO) mice. Heterozygous null Zc3h11a mice were born at the expected frequency without distinguishable phenotypic differences compared with wild-type mice. In contrast, homozygous null Zc3h11a mice were missing, indicating that Zc3h11a is crucial for embryonic viability and survival. Zc3h11a -/- embryos were detected at the expected Mendelian ratios up to late preimplantation stage (E4.5). However, phenotypic characterization at E6.5 revealed degeneration of Zc3h11a -/- embryos, indicating developmental defects around the time of implantation. Transcriptomic analyses documented a dysregulation of glycolysis and fatty acid metabolic pathways in Zc3h11a-/- embryos at E4.5. Proteomic analysis indicated a tight interaction between ZC3H11A and mRNA-export proteins in embryonic stem cells. CLIP-seq analysis demonstrated that ZC3H11A binds a subset of mRNA transcripts that are critical for metabolic regulation of embryonic cells. Furthermore, embryonic stem cells with an induced deletion of Zc3h11a display an impaired differentiation toward epiblast-like cells and impaired mitochondrial membrane potential. Altogether, the results show that ZC3H11A is participating in export and posttranscriptional regulation of selected mRNA transcripts required to maintain metabolic processes in embryonic cells. While ZC3H11A is essential for the viability of the early mouse embryo, inactivation of Zc3h11a expression in adult tissues using a conditional KO did not lead to obvious phenotypic defects.

  View details for DOI 10.1073/pnas.2216799120

  View details for PubMedID 37252988

• Clonally Expanded Cytotoxic CD8<SUP>+</SUP> T cells Target Citrullinated Antigens In ACPA<SUP>+</SUP> Rheumatoid Arthritis Moon, J., Younis, S., Sharpe, O., Rao, N. L., Carman, J. A., James, E. A., Buckner, J. H., Deane, K. D., Holers, V., Donlin, L. T., Davis, M. M., Robinson, W. H. AMER ASSOC IMMUNOLOGISTS. 2023

  View details for Web of Science ID 001106506500443

• Cytotoxic CD8+ T cells target citrullinated antigens in rheumatoid arthritis. Nature communications Moon, J. S., Younis, S., Ramadoss, N. S., Iyer, R., Sheth, K., Sharpe, O., Rao, N. L., Becart, S., Carman, J. A., James, E. A., Buckner, J. H., Deane, K. D., Holers, V. M., Goodman, S. M., Donlin, L. T., Davis, M. M., Robinson, W. H. 2023; 14 (1): 319

  Abstract

  The immune mechanisms that mediate synovitis and joint destruction in rheumatoid arthritis (RA) remain poorly defined. Although increased levels of CD8+ T cells have been described in RA, their function in pathogenesis remains unclear. Here we perform single cell transcriptome and T cell receptor (TCR) sequencing of CD8+ T cells derived from anti-citrullinated protein antibodies (ACPA)+ RA blood. We identify GZMB+CD8+ subpopulations containing large clonal lineage expansions that express cytotoxic and tissue homing transcriptional programs, while a GZMK+CD8+ memory subpopulation comprises smaller clonal expansions that express effector T cell transcriptional programs. We demonstrate RA citrullinated autoantigens presented by MHC class I activate RA blood-derived GZMB+CD8+ T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrate that these clonally expanded GZMB+CD8+ cells are present in RA synovium. These findings suggest that cytotoxic CD8+ T cells targeting citrullinated antigens contribute to synovitis and joint tissue destruction in ACPA+ RA.

  View details for DOI 10.1038/s41467-022-35264-8

  View details for PubMedID 36658110

  View details for PubMedCentralID PMC9852471

• ZC3H11A loss of function enhances NF-κB signaling through defective IκBα protein expression. Frontiers in immunology Darweesh, M., Younis, S., Hajikhezri, Z., Ali, A., Jin, C., Punga, T., Gupta, S., Essand, M., Andersson, L., Akusjärvi, G. 2022; 13: 1002823

  Abstract

  ZC3H11A is a cellular protein associated with the transcription export (TREX) complex that is induced during heat-shock. Several nuclear-replicating viruses exploit the mRNA export mechanism of ZC3H11A protein for their efficient replication. Here we show that ZC3H11A protein plays a role in regulation of NF-κB signal transduction. Depletion of ZC3H11A resulted in enhanced NF-κB mediated signaling, with upregulation of numerous innate immune related mRNAs, including IL-6 and a large group of interferon-stimulated genes. IL-6 upregulation in the absence of the ZC3H11A protein correlated with an increased NF-κB transcription factor binding to the IL-6 promoter and decreased IL-6 mRNA decay. The enhanced NF-κB signaling pathway in ZC3H11A deficient cells correlated with a defect in IκBα inhibitory mRNA and protein accumulation. Upon ZC3H11A depletion The IκBα mRNA was retained in the cell nucleus resulting in failure to maintain normal levels of the cytoplasmic IκBα mRNA and protein that is essential for its inhibitory feedback loop on NF-κB activity. These findings indicate towards a previously unknown mechanism of ZC3H11A in regulating the NF-κB pathway at the level of IkBα mRNA export.

  View details for DOI 10.3389/fimmu.2022.1002823

  View details for PubMedID 36439101

  View details for PubMedCentralID PMC9681899

• RNA-seq characterization of histamine-releasing mast cells as potential therapeutic target of osteoarthritis. Clinical immunology (Orlando, Fla.) Zhao, X., Younis, S., Shi, H., Hu, S., Zia, A., Wong, H. H., Elliott, E. E., Chang, T., Bloom, M. S., Zhang, W., Liu, X., Lanz, T. V., Sharpe, O., Love, Z. Z., Wang, Q., Robinson, W. H. 2022: 109117

  Abstract

  OBJECTIVE: Mast cells in the osteoarthritis (OA) synovium correlate with disease severity. This study aimed to further elucidate the role of mast cells in OA by RNA-Seq analysis and pharmacological blockade of the activity of histamine, a key mast cell mediator, in murine OA.METHODS: We examined OA synovial tissues and fluids by flow cytometry, immunostaining, single-cell and bulk RNA-Seq, qPCR, and ELISA. Cetirizine, a histamine H1 receptor (H1R) antagonist, was used to treat the destabilization of the medial meniscus (DMM) mouse model of OA.RESULTS: Flow cytometry and immunohistology analysis of OA synovial cells revealed KIT+ FcepsilonRI+ and TPSAB1+ mast cells. Single-cell RNA-Seq of OA synovial cells identified the expression of prototypical mast cell markers KIT, TPSAB1, CPA3 and HDC, as well as distinctive markers HPGD, CAVIN2, IL1RL1, PRG2, and CKLF, confirmed by bulk RNA-Seq and qPCR. A mast cell prototypical marker expression score classified 40 OA patients into three synovial pathotypes: mast cell-high, -medium, and -low. Additionally, we detected mast cell mediators including histamine, tryptase AB1, CPA3, PRG2, CAVIN2, and CKLF in OA synovial fluids. Elevated H1R expression was detected in human OA synovium, and treatment of mice with the H1 receptor antagonist cetirizine reduced the severity and OA-related mediators in DMM.CONCLUSION: Based on differential expression of prototypical and distinct mast cell markers, human OA joints can be stratified into mast cell-high, -medium, and -low synovial tissue pathotypes. Pharmacologic blockade of histamine activity holds the potential to improve OA disease outcome.

  View details for DOI 10.1016/j.clim.2022.109117

  View details for PubMedID 36109004

• Clonally Expanded Cytotoxic CD8(+) T Cells Recognize Citrullinated Antigens in ACPA(+) Rheumatoid Arthritis Younis, S., Sharpe, O., Rao, N., Carman, J., James, E., Buckner, J., Deane, K. D., Holers, M., Donlin, L., Davis, M., Robinson, W. WILEY. 2022: 3435-3437

  View details for Web of Science ID 000877386504256

• TGFbeta selects for pro-stemness over pro-invasive phenotypes during cancer cell epithelial-mesenchymal transition. Molecular oncology Tsubakihara, Y., Ohata, Y., Okita, Y., Younis, S., Eriksson, J., Sellin, M. E., Ren, J., Ten Dijke, P., Miyazono, K., Hikita, A., Imamura, T., Kato, M., Heldin, C., Moustakas, A. 2022

  Abstract

  Transforming growth factor beta (TGFbeta) induces epithelial-mesenchymal transition (EMT), which correlates with stemness and invasiveness. Mesenchymal-epithelial transition (MET) is induced by TGFbeta withdrawal and correlates with metastatic colonization. Whether TGFbeta promotes stemness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E-cadherin promoter. In 2D cultures, TGFbeta induced EMT, generating RFPlow cells with a mesenchymal transcriptome, and regained RFP, with an epithelial transcriptome, after MET induced by TGFbeta withdrawal. RFPlow cells generated robust mammospheres, with epithelio-mesenchymal cell surface features. Mammospheres that were forced to adhere generated migratory cells, devoid of RFP, a phenotype which was inhibited by a TGFbeta receptor kinase inhibitor. Further stimulation of RFPlow mammospheres with TGFbeta suppressed the generation of motile cells, but enhanced mammosphere growth. Accordingly, mammary fat-pad-transplanted mammospheres, in the absence of exogenous TGFbeta treatment, established lung metastases with evident MET (RFPhigh cells). In contrast, TGFbeta-treated mammospheres revealed high tumor-initiating capacity, but limited metastatic potential. Thus, the biological context of partial EMT and MET allows TGFbeta to differentiate between pro-stemness and pro-invasive phenotypes.

  View details for DOI 10.1002/1878-0261.13215

  View details for PubMedID 35348275

• Imatinib protects against human beta-cell death via inhibition of mitochondrial respiration and activation of AMPK. Clinical science (London, England : 1979) Elksnis, A., Schiffer, T. A., Palm, F., Wang, Y., Cen, J., Turpaev, K., Ngamjariyawat, A., Younis, S., Huang, S., Shen, Y., Leng, Y., Bergsten, P., Karlsborn, T., Welsh, N., Wang, X. 2021; 135 (19): 2243-2263

  Abstract

  The protein tyrosine kinase inhibitor imatinib is used in the treatment of various malignancies but may also promote beneficial effects in the treatment of diabetes. The aim of the present investigation was to characterize the mechanisms by which imatinib protects insulin producing cells. Treatment of non-obese diabetic (NOD) mice with imatinib resulted in increased beta-cell AMP-activated kinase (AMPK) phosphorylation. Imatinib activated AMPK also in vitro, resulting in decreased ribosomal protein S6 phosphorylation and protection against islet amyloid polypeptide (IAPP)-aggregation, thioredoxin interacting protein (TXNIP) up-regulation and beta-cell death. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) mimicked and compound C counteracted the effect of imatinib on beta-cell survival. Imatinib-induced AMPK activation was preceded by reduced glucose/pyruvate-dependent respiration, increased glycolysis rates, and a lowered ATP/AMP ratio. Imatinib augmented the fractional oxidation of fatty acids/malate, possibly via a direct interaction with the beta-oxidation enzyme enoyl coenzyme A hydratase, short chain, 1, mitochondrial (ECHS1). In non-beta cells, imatinib reduced respiratory chain complex I and II-mediated respiration and acyl-CoA carboxylase (ACC) phosphorylation, suggesting that mitochondrial effects of imatinib are not beta-cell specific. In conclusion, tyrosine kinase inhibitors modestly inhibit mitochondrial respiration, leading to AMPK activation and TXNIP down-regulation, which in turn protects against beta-cell death.

  View details for DOI 10.1042/CS20210604

  View details for PubMedID 34569605

• ZBED6 regulates Igf2 expression partially through its regulation of miR483 expression. Scientific reports Naboulsi, R., Larsson, M., Andersson, L., Younis, S. 2021; 11 (1): 19484

  Abstract

  The expression of Igf2 in mammals shows a complex regulation involving multiple promoters and epigenetic mechanisms. We previously identified a novel regulatory mechanism based on the interaction between the transcriptional factor ZBED6 and Igf2 intron. Disruption of the ZBED6-Igf2 interaction leads to a dramatic up-regulation of IGF2 expression postnatally. In the current study we characterize an additional layer of regulation involving miR483 encoded by another Igf2 intron. We found a highly significant up-regulation of miR483 expression when the ZBED6-Igf2 axis is disrupted in transgenic mice. Furthermore, CRISPR/Cas9 mediated knock-out of miR483 in C2C12 myoblast cells, both wild-type and cells with disrupted ZBED6-Igf2 axis (Igf2dGGCT), resulted in down-regulation of Igf2 expression and a reduced proliferation rate. This was further validated using miR483 mimics and inhibitors. RNA-seq analysis revealed a significant enrichment of genes involved in the PI3K-Akt signaling pathway among genes down-regulated in miR483-/- cells, including Igf2 down-regulation. The opposite pattern was observed in Igf2dGGCT cells, where Igf2 is up-regulated. Our data suggest a positive feedback between miR483 and Igf2 promoter activity, strongly affecting how ZBED6 controls Igf2 expression in various cell types.

  View details for DOI 10.1038/s41598-021-98777-0

  View details for PubMedID 34593874

• ZBED6 counteracts high-fat diet-induced glucose intolerance by maintaining beta cell area and reducing excess mitochondrial activation. Diabetologia Wang, X., Younis, S., Cen, J., Wang, Y., Krizhanovskii, C., Andersson, L., Welsh, N. 2021

  Abstract

  AIMS/HYPOTHESIS: ZBED6 (zinc finger, BED-type containing 6) is known to regulate muscle mass by suppression of Igf2 gene transcription. In insulin-producing cell lines, ZBED6 maintains proliferative capacity at the expense of differentiation and beta cell function. The aim was to study the impact of Zbed6 knockout on beta cell function and glucose tolerance in C57BL/6 mice.METHODS: Beta cell area and proliferation were determined in Zbed6 knockout mice using immunohistochemical analysis. Muscle and fat distribution were assessed using micro-computed tomography. Islet gene expression was assessed by RNA sequencing. Effects of a high-fat diet were analysed by glucose tolerance and insulin tolerance tests. ZBED6 was overexpressed in EndoC-betaH1 cells and human islet cells using an adenoviral vector. Beta cell cell-cycle analysis, insulin release and mitochondrial function were studied in vitro using propidium iodide staining and flow cytometry, ELISA, the Seahorse technique, and the fluorescent probes JC-1 and MitoSox.RESULTS: Islets from Zbed6 knockout mice showed lowered expression of the cell cycle gene Pttg1, decreased beta cell proliferation and decreased beta cell area, which occurred independently from ZBED6 effects on Igf2 gene expression. Zbed6 knockout mice, but not wild-type mice, developed glucose intolerance when given a high-fat diet. The high-fat diet Zbed6 knockout islets displayed upregulated expression of oxidative phosphorylation genes and genes associated with beta cell differentiation. In vitro, ZBED6 overexpression resulted in increased EndoC-betaH1 cell proliferation and a reduced glucose-stimulated insulin release in human islets. ZBED6 also reduced mitochondrial JC-1 J-aggregate formation, mitochondrial oxygen consumption rates (OCR) and mitochondrial reactive oxygen species (ROS) production, both at basal and palmitate + high glucose-stimulated conditions. ZBED6-induced inhibition of OCR was not rescued by IGF2 addition. ZBED6 reduced levels of the mitochondrial regulator PPAR-gamma related coactivator 1 protein (PRC) and bound its promoter/enhancer region. Knockdown of PRC resulted in a lowered OCR.CONCLUSIONS/INTERPRETATION: It is concluded that ZBED6 is required for normal beta cell replication and also limits excessive beta cell mitochondrial activation in response to an increased functional demand. ZBED6 may act, at least in part, by restricting PRC-mediated mitochondrial activation/ROS production, which may lead to protection against beta cell dysfunction and glucose intolerance in vivo.

  View details for DOI 10.1007/s00125-021-05517-0

  View details for PubMedID 34296320

• Functional differences between TSHR alleles associate with variation in spawning season in Atlantic herring. Communications biology Chen, J., Bi, H., Pettersson, M. E., Sato, D. X., Fuentes-Pardo, A. P., Mo, C., Younis, S., Wallerman, O., Jern, P., Moles, G., Gomez, A., Kleinau, G., Scheerer, P., Andersson, L. 2021; 4 (1): 795

  Abstract

  The underlying molecular mechanisms that determine long day versus short day breeders remain unknown in any organism. Atlantic herring provides a unique opportunity to examine the molecular mechanisms involved in reproduction timing, because both spring and autumn spawners exist within the same species. Although our previous whole genome comparisons revealed a strong association of TSHR alleles with spawning seasons, the functional consequences of these variants remain unknown. Here we examined the functional significance of six candidate TSHR mutations strongly associated with herring reproductive seasonality. We show that the L471M missense mutation in the spring-allele causes enhanced cAMP signaling. The best candidate non-coding mutation is a 5.2kb retrotransposon insertion upstream of the TSHR transcription start site, near an open chromatin region, which is likely to affect TSHR expression. The insertion occurred prior to the split between Pacific and Atlantic herring and was lost in the autumn-allele. Our study shows that strongly associated coding and non-coding variants at the TSHR locus may both contribute to the regulation of seasonal reproduction in herring.

  View details for DOI 10.1038/s42003-021-02307-7

  View details for PubMedID 34172814

• Brain transcriptomics of wild and domestic rabbits suggests that changes in dopamine signalling and ciliary function contributed to evolution of tameness. Genome biology and evolution Sato, D. X., Rafati, N., Ring, H., Younis, S., Feng, C., Blanco-Aguiar, J. A., Rubin, C. J., Villafurte, R., Hallböök, F., Carneiro, M., Andersson, L. 2020

  Abstract

  Domestication has resulted in immense phenotypic changes in animals despite their relatively short evolutionary history. The European rabbit is one of the most recently domesticated animals, but exhibits distinct morphological, physiological and behavioural differences from their wild conspecifics. A previous study revealed that sequence variants with striking allele frequency differences between wild and domestic rabbits were enriched in conserved non-coding regions, in the vicinity of genes involved in nervous system development. This suggests that a large proportion of the genetic changes targeted by selection during domestication might affect gene regulation. Here, we generated RNA-sequencing data for four brain regions (amygdala, hypothalamus, hippocampus and parietal/temporal cortex) sampled at birth and revealed hundreds of differentially expressed genes (DEGs) between wild and domestic rabbits. DEGs in amygdala were significantly enriched for genes associated with dopaminergic function and all 12 DEGs in this category showed higher expression in domestic rabbits. DEGs in hippocampus were enriched for genes associated with ciliary function, all 21 genes in this category showed lower expression in domestic rabbits. These results indicate an important role of dopamine signalling and ciliary function in the evolution of tameness during rabbit domestication. Our study shows that gene expression in specific pathways has been profoundly altered during domestication, but that the majority of genes showing differential expression in this study have not been the direct targets of selection.

  View details for DOI 10.1093/gbe/evaa158

  View details for PubMedID 32835359

• The importance of the ZBED6-IGF2 axis for metabolic regulation in mouse myoblast cells FASEB JOURNAL Younis, S., Naboulsi, R., Wang, X., Cao, X., Larsson, M., Sargsyan, E., Bergsten, P., Welsh, N., Andersson, L. 2020

  Abstract

  The transcription factor ZBED6 acts as a repressor of Igf2 and affects directly or indirectly the transcriptional regulation of thousands of genes. Here, we use gene editing in mouse C2C12 myoblasts and show that ZBED6 regulates Igf2 exclusively through its binding site 5'-GGCTCG-3' in intron 1 of Igf2. Deletion of this motif (Igf2ΔGGCT ) or complete ablation of Zbed6 leads to ~20-fold upregulation of the IGF2 protein. Quantitative proteomics revealed an activation of Ras signaling pathway in both Zbed6-/- and Igf2ΔGGCT myoblasts, and a significant enrichment of mitochondrial membrane proteins among proteins showing altered expression in Zbed6-/- myoblasts. Both Zbed6-/- and Igf2ΔGGCT myoblasts showed a faster growth rate and developed myotube hypertrophy. These cells exhibited an increased O2 consumption rate, due to IGF2 upregulation. Transcriptome analysis revealed ~30% overlap between differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with an enrichment of upregulated genes involved in muscle development. In contrast, ZBED6-overexpression in myoblasts led to cell apoptosis, cell cycle arrest, reduced mitochondrial activities, and ceased myoblast differentiation. The similarities in growth and differentiation phenotypes observed in Zbed6-/- and Igf2ΔGGCT myoblasts demonstrates that ZBED6 affects mitochondrial activity and myogenesis largely through its regulation of IGF2 expression. This study adds new insights how the ZBED6-Igf2 axis affects muscle metabolism.

  View details for DOI 10.1096/fj.201901321R

  View details for Web of Science ID 000541805900001

  View details for PubMedID 32557799

• ZBED6 negatively regulates insulin production, neuronal differentiation, and cell aggregation in MIN6 cells FASEB JOURNAL Wang, X., Jiang, L., Wallerman, O., Younis, S., Yu, Q., Klaesson, A., Tengholm, A., Welsh, N., Andersson, L. 2019; 33 (1): 88–100

  Abstract

  Zinc finger BED domain containing protein 6 ( Zbed6) has evolved from a domesticated DNA transposon and encodes a transcription factor unique to placental mammals. The aim of the present study was to investigate further the role of ZBED6 in insulin-producing cells, using mouse MIN6 cells, and to evaluate the effects of Zbed6 knockdown on basal β-cell functions, such as morphology, transcriptional regulation, insulin content, and release. Zbed6-silenced cells and controls were characterized with a range of methods, including RNA sequencing, chromatin immunoprecipitation sequencing, insulin content and release, subplasma membrane Ca2+ measurements, cAMP determination, and morphologic studies. More than 700 genes showed differential expression in response to Zbed6 knockdown, which was paralleled by increased capacity to generate cAMP, as well as by augmented subplasmalemmal calcium concentration and insulin secretion in response to glucose stimulation. We identified >4000 putative ZBED6-binding sites in the MIN6 genome, with an enrichment of ZBED6 sites at upregulated genes, such as the β-cell transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene homolog A and Nk6 homeobox 1. We also observed altered morphology/growth patterns, as indicated by increased cell clustering, and in the appearance of axon-like Neurofilament, medium polypeptide and tubulin β 3, class III-positive protrusions. We conclude that ZBED6 acts as a transcriptional regulator in MIN6 cells and that its activity suppresses insulin production, cell aggregation, and neuronal-like differentiation.-Wang, X., Jiang, L., Wallerman, O., Younis, S., Yu, Q., Klaesson, A., Tengholm, A., Welsh, N., Andersson, L. ZBED6 negatively regulates insulin production, neuronal differentiation, and cell aggregation in MIN6 cells.

  View details for DOI 10.1096/fj.201600835R

  View details for Web of Science ID 000457401500007

  View details for PubMedID 29957057

• Multiple nuclear-replicating viruses require the stress-induced protein ZC3H11A for efficient growth PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Younis, S., Kamel, W., Falkeborn, T., Wang, H., Yu, D., Daniels, R., Essand, M., Hinkula, J., Akusjarvi, G., Andersson, L. 2018; 115 (16): E3808–E3816

  Abstract

  The zinc finger CCCH-type containing 11A (ZC3H11A) gene encodes a well-conserved zinc finger protein that may function in mRNA export as it has been shown to associate with the transcription export (TREX) complex in proteomic screens. Here, we report that ZC3H11A is a stress-induced nuclear protein with RNA-binding capacity that localizes to nuclear splicing speckles. During an adenovirus infection, the ZC3H11A protein and splicing factor SRSF2 relocalize to nuclear regions where viral DNA replication and transcription take place. Knockout (KO) of ZC3H11A in HeLa cells demonstrated that several nuclear-replicating viruses are dependent on ZC3H11A for efficient growth (HIV, influenza virus, herpes simplex virus, and adenovirus), whereas cytoplasmic replicating viruses are not (vaccinia virus and Semliki Forest virus). High-throughput sequencing of ZC3H11A-cross-linked RNA showed that ZC3H11A binds to short purine-rich ribonucleotide stretches in cellular and adenoviral transcripts. We show that the RNA-binding property of ZC3H11A is crucial for its function and localization. In ZC3H11A KO cells, the adenovirus fiber mRNA accumulates in the cell nucleus. Our results suggest that ZC3H11A is important for maintaining nuclear export of mRNAs during stress and that several nuclear-replicating viruses take advantage of this mechanism to facilitate their replication.

  View details for DOI 10.1073/pnas.1722333115

  View details for Web of Science ID 000430191900026

  View details for PubMedID 29610341

  View details for PubMedCentralID PMC5910864

• The ZBED6-IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Younis, S., Schonke, M., Massart, J., Hjortebjerg, R., Sundstrom, E., Gustafson, U., Bjornholm, M., Krook, A., Frystyk, J., Zierath, J. R., Andersson, L. 2018; 115 (9): E2048–E2057

  Abstract

  A single nucleotide substitution in the third intron of insulin-like growth factor 2 (IGF2) is associated with increased muscle mass and reduced subcutaneous fat in domestic pigs. This mutation disrupts the binding of the ZBED6 transcription factor and leads to a threefold up-regulation of IGF2 expression in pig skeletal muscle. Here, we investigated the biological significance of ZBED6-IGF2 interaction in the growth of placental mammals using two mouse models, ZBED6 knock-out (Zbed6-/-) and Igf2 knock-in mice that carry the pig IGF2 mutation. These transgenic mice exhibit markedly higher serum IGF2 concentrations, higher growth rate, increased lean mass, and larger heart, kidney, and liver; no significant changes were observed for white adipose tissues. The changes in body and lean mass were most pronounced in female mice. The phenotypic changes were concomitant with a remarkable up-regulation of Igf2 expression in adult tissues. Transcriptome analysis of skeletal muscle identified differential expression of genes belonging to the extracellular region category. Expression analysis using fetal muscles indicated a minor role of ZBED6 in regulating Igf2 expression prenatally. Furthermore, transcriptome analysis of the adult skeletal muscle revealed that this elevated expression of Igf2 was derived from the P1 and P2 promoters. The results revealed very similar phenotypic effects in the Zbed6 knock-out mouse and in the Igf2 knock-in mouse, showing that the effect of ZBED6 on growth of muscle and internal organs is mediated through the binding site in the Igf2 gene. The results explain why this ZBED6 binding site is extremely well conserved among placental mammals.

  View details for DOI 10.1073/pnas.1719278115

  View details for Web of Science ID 000426152500018

  View details for PubMedID 29440408

  View details for PubMedCentralID PMC5834713

• Transcriptional modulator ZBED6 affects cell cycle and growth of human colorectal cancer cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ali, M., Younis, S., Wallerman, O., Gupta, R., Andersson, L., Sjoblom, T. 2015; 112 (25): 7743–48

  Abstract

  The transcription factor ZBED6 (zinc finger, BED-type containing 6) is a repressor of IGF2 whose action impacts development, cell proliferation, and growth in placental mammals. In human colorectal cancers, IGF2 overexpression is mutually exclusive with somatic mutations in PI3K signaling components, providing genetic evidence for a role in the PI3K pathway. To understand the role of ZBED6 in tumorigenesis, we engineered and validated somatic cell ZBED6 knock-outs in the human colorectal cancer cell lines RKO and HCT116. Ablation of ZBED6 affected the cell cycle and led to increased growth rate in RKO cells but reduced growth in HCT116 cells. This striking difference was reflected in the transcriptome analyses, which revealed enrichment of cell-cycle-related processes among differentially expressed genes in both cell lines, but the direction of change often differed between the cell lines. ChIP sequencing analyses displayed enrichment of ZBED6 binding at genes up-regulated in ZBED6-knockout clones, consistent with the view that ZBED6 modulates gene expression primarily by repressing transcription. Ten differentially expressed genes were identified as putative direct gene targets, and their down-regulation by ZBED6 was validated experimentally. Eight of these genes were linked to the Wnt, Hippo, TGF-β, EGF receptor, or PI3K pathways, all involved in colorectal cancer development. The results of this study show that the effect of ZBED6 on tumor development depends on the genetic background and the transcriptional state of its target genes.

  View details for DOI 10.1073/pnas.1509193112

  View details for Web of Science ID 000356731300067

  View details for PubMedID 26056301

  View details for PubMedCentralID PMC4485122

• Rabbit genome analysis reveals a polygenic basis for phenotypic change during domestication SCIENCE Carneiro, M., Rubin, C., Di Palma, F., Albert, F. W., Alfoeldi, J., Barrio, A., Pielberg, G., Rafati, N., Sayyab, S., Turner-Maier, J., Younis, S., Afonso, S., Aken, B., Alves, J. M., Barrell, D., Bolet, G., Boucher, S., Burbano, H. A., Campos, R., Chang, J. L., Duranthon, V., Fontanesi, L., Garreau, H., Heiman, D., Johnson, J., Mage, R. G., Peng, Z., Queney, G., Rogel-Gaillard, C., Ruffier, M., Searle, S., Villafuerte, R., Xiong, A., Young, S., Forsberg-Nilsson, K., Good, J. M., Lander, E. S., Ferrand, N., Lindblad-Toh, K., Andersson, L. 2014; 345 (6200): 1074–79

  Abstract

  The genetic changes underlying the initial steps of animal domestication are still poorly understood. We generated a high-quality reference genome for the rabbit and compared it to resequencing data from populations of wild and domestic rabbits. We identified more than 100 selective sweeps specific to domestic rabbits but only a relatively small number of fixed (or nearly fixed) single-nucleotide polymorphisms (SNPs) for derived alleles. SNPs with marked allele frequency differences between wild and domestic rabbits were enriched for conserved noncoding sites. Enrichment analyses suggest that genes affecting brain and neuronal development have often been targeted during domestication. We propose that because of a truly complex genetic background, tame behavior in rabbits and other domestic animals evolved by shifts in allele frequencies at many loci, rather than by critical changes at only a few domestication loci.

  View details for DOI 10.1126/science.1253714

  View details for Web of Science ID 000340870900046

  View details for PubMedID 25170157

  View details for PubMedCentralID PMC5421586

• ZBED6 Modulates the Transcription of Myogenic Genes in Mouse Myoblast Cells PLOS ONE Jiang, L., Wallerman, O., Younis, S., Rubin, C., Gilbert, E. R., Sundstrom, E., Ghazal, A., Zhang, X., Wang, L., Mikkelsen, T. S., Andersson, G., Andersson, L. 2014; 9 (4): e94187

  Abstract

  ZBED6 is a recently discovered transcription factor, unique to placental mammals, that has evolved from a domesticated DNA transposon. It acts as a repressor at the IGF2 locus. Here we show that ZBED6 acts as a transcriptional modulator in mouse myoblast cells, where more than 700 genes were differentially expressed after Zbed6-silencing. The most significantly enriched GO term was muscle protein and contractile fiber, which was consistent with increased myotube formation. Twenty small nucleolar RNAs all showed increased expression after Zbed6-silencing. The co-localization of histone marks and ZBED6 binding sites and the effect of Zbed6-silencing on distribution of histone marks was evaluated by ChIP-seq analysis. There was a strong association between ZBED6 binding sites and the H3K4me3, H3K4me2 and H3K27ac modifications, which are usually found at active promoters, but no association with the repressive mark H3K27me3. Zbed6-silencing led to increased enrichment of active marks at myogenic genes, in agreement with the RNA-seq findings. We propose that ZBED6 preferentially binds to active promoters and modulates transcriptional activity without recruiting repressive histone modifications.

  View details for DOI 10.1371/journal.pone.0094187

  View details for Web of Science ID 000334160900101

  View details for PubMedID 24714595

  View details for PubMedCentralID PMC3979763