Bingyu Yan

All Publications

• A comprehensive single cell data analysis of lymphoblastoid cells reveals the role of super-enhancers in maintaining EBV latency JOURNAL OF MEDICAL VIROLOGY Yan, B., Wang, C., Chakravorty, S., Zhang, Z., Kadadi, S. D., Zhuang, Y., Sirit, I., Hu, Y., Jung, M., Sahoo, S. S., Wang, L., Shao, K., Anderson, N. L., Trujillo-Ochoa, J. L., Briggs, S. D., Liu, X., Olson, M. R., Afzali, B., Zhao, B., Kazemian, M. 2023; 95 (1): e28362

  Abstract

  We probed the lifecycle of Epstein-Barr virus (EBV) on a cell-by-cell basis using single cell RNA sequencing (scRNA-seq) data from nine publicly available lymphoblastoid cell lines (LCLs). While the majority of LCLs comprised cells containing EBV in the latent phase, two other clusters of cells were clearly evident and were distinguished by distinct expression of host and viral genes. Notably, both were high expressors of EBV LMP1/BNLF2 and BZLF1 compared to another cluster that expressed neither gene. The two novel clusters differed from each other in their expression of EBV lytic genes, including glycoprotein gene GP350. The first cluster, comprising GP350- LMP1hi cells, expressed high levels of HIF1A and was transcriptionally regulated by HIF1-α. Treatment of LCLs with Pevonedistat, a drug that enhances HIF1-α signaling, markedly induced this cluster. The second cluster, containing GP350+ LMP1hi cells, expressed EBV lytic genes. Host genes that are controlled by super-enhancers (SEs), such as transcription factors MYC and IRF4, had the lowest expression in this cluster. Functionally, the expression of genes regulated by MYC and IRF4 in GP350+ LMP1hi cells were lower compared to other cells. Indeed, induction of EBV lytic reactivation in EBV+ AKATA reduced the expression of these SE-regulated genes. Furthermore, CRISPR-mediated perturbation of the MYC or IRF4 SEs in LCLs induced the lytic EBV gene expression, suggesting that host SEs and/or SE target genes are required for maintenance of EBV latency. Collectively, our study revealed EBV-associated heterogeneity among LCLs that may have functional consequence on host and viral biology.

  View details for DOI 10.1002/jmv.28362

  View details for Web of Science ID 000911465200281

  View details for PubMedID 36453088

  View details for PubMedCentralID PMC10027397

• Autocrine vitamin D signaling switches off pro-inflammatory programs of T(H)1 cells NATURE IMMUNOLOGY Chauss, D., Freiwald, T., McGregor, R., Yan, B., Wang, L., Nova-Lamperti, E., Kumar, D., Zhang, Z., Teague, H., West, E. E., Vannella, K. M., Ramos-Benitez, M. J., Bibby, J., Kelly, A., Malik, A., Freeman, A. F., Schwartz, D. M., Portilla, D., Chertow, D. S., John, S., Lavender, P., Kemper, C., Lombardi, G., Mehta, N. N., Cooper, N., Lionakis, M. S., Laurence, A., Kazemian, M., Afzali, B. 2022; 23 (1): 62-+

  Abstract

  The molecular mechanisms governing orderly shutdown and retraction of CD4+ type 1 helper T (TH1) cell responses remain poorly understood. Here we show that complement triggers contraction of TH1 responses by inducing intrinsic expression of the vitamin D (VitD) receptor and the VitD-activating enzyme CYP27B1, permitting T cells to both activate and respond to VitD. VitD then initiated the transition from pro-inflammatory interferon-γ+ TH1 cells to suppressive interleukin-10+ cells. This process was primed by dynamic changes in the epigenetic landscape of CD4+ T cells, generating super-enhancers and recruiting several transcription factors, notably c-JUN, STAT3 and BACH2, which together with VitD receptor shaped the transcriptional response to VitD. Accordingly, VitD did not induce interleukin-10 expression in cells with dysfunctional BACH2 or STAT3. Bronchoalveolar lavage fluid CD4+ T cells of patients with COVID-19 were TH1-skewed and showed de-repression of genes downregulated by VitD, from either lack of substrate (VitD deficiency) and/or abnormal regulation of this system.

  View details for DOI 10.1038/s41590-021-01080-3

  View details for Web of Science ID 000717408600001

  View details for PubMedID 34764490

  View details for PubMedCentralID PMC7612139

• TCF-1 controls T-reg cell functions that regulate inflammation, CD8(+) T cell cytotoxicity and severity of colon cancer NATURE IMMUNOLOGY Osman, A., Yan, B., Li, Y., Pavelko, K. D., Quandt, J., Saadalla, A., Singh, M., Kazemian, M., Gounari, F., Khazaie, K. 2021; 22 (9): 1152-+

  Abstract

  The transcription factor TCF-1 is essential for the development and function of regulatory T (Treg) cells; however, its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-sequencing analysis identified effector memory T cells and central memory Treg cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core Treg cell transcriptional signature, but promoted alternative signaling pathways whereby Treg cells became activated and gained gut-homing properties and characteristics of the TH17 subset of helper T cells. TCF-1-deficient Treg cells strongly suppressed T cell proliferation and cytotoxicity, but were compromised in controlling CD4+ T cell polarization and inflammation. In mice with polyposis, Treg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating Treg cells of patients with colorectal cancer showed lower TCF-1 expression and increased TH17 expression signatures compared to adjacent normal tissue and circulating T cells. Thus, Treg cell-specific TCF-1 expression differentially regulates TH17-mediated inflammation and T cell cytotoxicity, and can determine colorectal cancer outcome.

  View details for DOI 10.1038/s41590-021-00987-1

  View details for Web of Science ID 000684510900002

  View details for PubMedID 34385712

  View details for PubMedCentralID PMC8428683

• Host-Virus Chimeric Events in SARS-CoV-2-Infected Cells Are Infrequent and Artifactual JOURNAL OF VIROLOGY Yan, B., Chakravorty, S., Mirabelli, C., Wang, L., Trujillo-Ochoa, J. L., Chauss, D., Kumar, D., Lionakis, M. S., Olson, M. R., Wobus, C. E., Afzali, B., Kazemian, M. 2021; 95 (15): e0029421

  Abstract

  The pathogenic mechanisms underlying severe SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection remain largely unelucidated. High-throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen- and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in transcriptome sequencing (RNA-seq) data from SARS-CoV-2-infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV-2 is a positive-sense RNA virus that replicates in the cytoplasm, it does not have a nuclear phase in its life cycle. Thus, it is biologically unlikely to be in a location where splicing events could result in genome integration. Therefore, we investigated the biological authenticity of HVC events. In contrast to true biological events like mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with coronavirus disease 2019 (COVID-19) and infected cell lines were highly irreproducible. RNA-seq library preparation is inherently error prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spiked-in RNA from an unrelated species, such as the fruit fly, we estimated that ∼1% of RNA-seq reads are artifactually chimeric. In SARS-CoV-2 RNA-seq, we found that the frequency of HVC events was, in fact, not greater than this background "noise." Finally, we developed a novel experimental approach to enrich SARS-CoV-2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV-2-infected cells are extremely rare and are likely artifacts arising from random template switching of reverse transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV-2 fusion to cellular genes and/or integration into human genomes. IMPORTANCE The pathogenic mechanisms underlying SARS-CoV-2, the virus responsible for COVID-19, are not fully understood. In particular, relatively little is known about the reasons some individuals develop life-threatening or persistent COVID-19. Recent studies identified host-virus chimeric (HVC) reads in RNA-sequencing data from SARS-CoV-2-infected cells and suggested that HVC events support potential "human genome invasion" and "integration" by SARS-CoV-2. This suggestion has fueled concerns about the long-term effects of current mRNA vaccines that incorporate elements of the viral genome. SARS-CoV-2 is a positive-sense, single-stranded RNA virus that does not encode a reverse transcriptase and does not include a nuclear phase in its life cycle, so some doubts have rightfully been expressed regarding the authenticity of HVCs and the role played by endogenous retrotransposons in this phenomenon. Thus, it is important to independently authenticate these HVC events. Here, we provide several lines of evidence suggesting that the observed HVC events are likely artifactual.

  View details for DOI 10.1128/JVI.00294-21

  View details for Web of Science ID 000708632900004

  View details for PubMedID 33980601

  View details for PubMedCentralID PMC8274596

• SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation SCIENCE IMMUNOLOGY Yan, B., Freiwald, T., Chauss, D., Wang, L., West, E., Mirabelli, C., Zhang, C. J., Nichols, E., Malik, N., Gregory, R., Bantscheff, M., Ghidelli-Disse, S., Kolev, M., Frum, T., Spence, J. R., Sexton, J. Z., Alysandratos, K. D., Kotton, D. N., Pittaluga, S., Bibby, J., Niyonzima, N., Olson, M. R., Kordasti, S., Portilla, D., Wobus, C. E., Laurence, A., Lionakis, M. S., Kemper, C., Afzali, B., Kazemian, M. 2021; 6 (58)

  Abstract

  Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.

  View details for DOI 10.1126/sciimmunol.abg0833

  View details for Web of Science ID 000677977300007

  View details for PubMedID 33827897

  View details for PubMedCentralID PMC8139422

• Integrated Pan-Cancer Map of EBV-Associated Neoplasms Reveals Functional Host-Virus Interactions CANCER RESEARCH Chakravorty, S., Yan, B., Wang, C., Wang, L., Quaid, J., Lin, C., Briggs, S. D., Majumder, J., Canaria, D., Chauss, D., Chopra, G., Olson, M. R., Zhao, B., Afzali, B., Kazemian, M. 2019; 79 (23): 6010-6023

  Abstract

  Epstein-Barr virus (EBV) is a complex oncogenic symbiont. The molecular mechanisms governing EBV carcinogenesis remain elusive and the functional interactions between virus and host cells are incompletely defined. Here we present a comprehensive map of the host cell-pathogen interactome in EBV-associated cancers. We systematically analyzed RNA sequencing from >1,000 patients with 15 different cancer types, comparing virus and host factors of EBV+ to EBV- tissues. EBV preferentially integrated at highly accessible regions of the cancer genome, with significant enrichment in super-enhancer architecture. Twelve EBV transcripts, including LMP1 and LMP2, correlated inversely with EBV reactivation signature. Overexpression of these genes significantly suppressed viral reactivation, consistent with a "virostatic" function. In cancer samples, hundreds of novel frequent missense and nonsense variations in virostatic genes were identified, and variant genes failed to regulate their viral and cellular targets in cancer. For example, one-third of patients with EBV+ NK/T-cell lymphoma carried two novel nonsense variants (Q322X, G342X) of LMP1 and both variant proteins failed to restrict viral reactivation, confirming loss of virostatic function. Host cell transcriptional changes in response to EBV infection classified tumors into two molecular subtypes based on patterns of IFN signature genes and immune checkpoint markers, such as PD-L1 and IDO1. Overall, these findings uncover novel points of interaction between a common oncovirus and the human genome and identify novel regulatory nodes and druggable targets for individualized EBV and cancer-specific therapies. SIGNIFICANCE: This study provides a comprehensive map of the host cell-pathogen interactome in EBV+ malignancies.See related commentary by Mbulaiteye and Prokunina-Olsson, p. 5917.

  View details for DOI 10.1158/0008-5472.CAN-19-0615

  View details for Web of Science ID 000500951700012

  View details for PubMedID 31481499

  View details for PubMedCentralID PMC6891172

• DDX5 deficiency drives non-canonical NF-κB activation and NRF2 expression, influencing sorafenib response and hepatocellular carcinoma progression. Cell death & disease Li, Z., Kim, W., Utturkar, S., Yan, B., Lanman, N. A., Elzey, B. D., Kazemian, M., Yeo, Y., Andrisani, O. 2024; 15 (8): 583

  Abstract

  In advanced hepatocellular carcinoma (HCC), RNA helicase DDX5 regulates the Wnt/β-catenin-ferroptosis axis, influencing the efficacy of the multi-tyrosine kinase inhibitor (mTKI) sorafenib. DDX5 inhibits Wnt/β-catenin signaling, preventing sorafenib-induced ferroptosis escape. Sorafenib/mTKIs reduce DDX5 expression, correlating with poor patient survival post-sorafenib treatment. Notably, DDX5-knockout in HCC cells activates Wnt/β-catenin signaling persistently. Herein, we investigate the mechanistic impact of Wnt/β-catenin activation resulting from DDX5 downregulation in the progression and treatment of HCC. RNAseq analyses identified shared genes repressed by DDX5 and upregulated by sorafenib, including Wnt signaling genes, NF-κB-inducing kinase (NIK) essential for non-canonical NF-κB (p52/RelB) activation, and cytoprotective transcription factor NRF2. We demonstrate, Wnt/β-catenin activation induced NIK transcription, leading to non-canonical NF-κB activation, which subsequently mediated NRF2 transcription. Additionally, DDX5 deficiency extended NRF2 protein half-life by inactivating KEAP1 through p62/SQSTM1 stabilization. In a preclinical HCC mouse model, NRF2 knockdown or DDX5 overexpression restricted tumor growth upon sorafenib treatment, via induction of ferroptosis. Importantly, DDX5-knockout HCC cells exhibited elevated expression of Wnt signaling genes, NIK, p52/RelB, and NRF2-regulated genes, regardless of sorafenib treatment. Transcriptomic analyses of HCCs from TCGA and the Stelic Animal Model (STAM) of non-alcoholic steatohepatitis revealed elevated expression of these interconnected pathways in the context of DDX5 downregulation. In conclusion, DDX5 deficiency triggers Wnt/β-catenin signaling, promoting p52/RelB and NRF2 activation, thereby enabling ferroptosis evasion upon sorafenib treatment. Similarly, independent of sorafenib, DDX5 deficiency in liver tumors enhances activation and gene expression of these interconnected pathways, underscoring the clinical relevance of DDX5 deficiency in HCC progression and therapeutic response.

  View details for DOI 10.1038/s41419-024-06977-z

  View details for PubMedID 39122708

  View details for PubMedCentralID PMC11315975

• RNA helicase DDX5 modulates sorafenib sensitivity in hepatocellular carcinoma via the Wnt/β-catenin-ferroptosis axis. Cell death & disease Li, Z., Caron de Fromentel, C., Kim, W., Wang, W. H., Sun, J., Yan, B., Utturkar, S., Lanman, N. A., Elzey, B. D., Yeo, Y., Zhang, H., Kazemian, M., Levrero, M., Andrisani, O. 2023; 14 (11): 786

  Abstract

  Reduced expression of the RNA helicase DDX5 associated with increased hepatocellular carcinoma (HCC) tumor grade and poor patient survival following treatment with sorafenib. While immunotherapy is the first-line treatment for HCC, sorafenib and other multi-tyrosine kinase inhibitors (mTKIs) are widely used when immunotherapy is contra-indicated or fails. Herein, we elucidate the role of DDX5 in sensitizing HCC to sorafenib, offering new therapeutic strategies. Treatment of various human HCC cell lines with sorafenib/mTKIs downregulated DDX5 in vitro and in preclinical HCC models. Conversely, DDX5 overexpression reduced the viability of sorafenib-treated cells via ferroptosis, suggesting a role for DDX5 in sorafenib sensitivity. RNAseq of wild-type vs. DDX5-knockdown cells treated with or without sorafenib identified a set of common genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps with Wnt signaling genes, including Disheveled-1 (DVL1), an indispensable Wnt activator and prognostic indicator of poor survival for sorafenib-treated patients. DDX5-knockout (DDX5KO) HCC cells exhibited DVL1 induction, Wnt/β-catenin pathway activation, and ferroptosis upon inhibition of canonical Wnt signaling. Consistently, xenograft HCC tumors exhibited reduced growth by inhibition of Wnt/β-catenin signaling via induction of ferroptosis. Significantly, overexpression of DDX5 in HCC xenografts repressed DVL1 expression and increased ferroptosis, resulting in reduced tumor growth by sorafenib. We conclude that DDX5 downregulation by sorafenib mediates adaptive resistance by activating Wnt/β-catenin signaling, leading to ferroptosis escape. Conversely, overexpression of DDX5 in vivo enhances the anti-tumor efficacy of sorafenib by suppressing Wnt/β-catenin activation and induction of ferroptosis. Thus, DDX5 overexpression in combination with mTKIs is a promising therapeutic strategy for HCC.

  View details for DOI 10.1038/s41419-023-06302-0

  View details for PubMedID 38036507

  View details for PubMedCentralID 7523244

• Distinct use of super-enhancer elements controls cell type-specific CD25 transcription and function. Science immunology Spolski, R., Li, P., Chandra, V., Shin, B., Goel, S., Sakamoto, K., Liu, C., Oh, J., Ren, M., Enomoto, Y., West, E. E., Christensen, S. M., Wan, E. C., Ge, M., Lin, J. X., Yan, B., Kazemian, M., Yu, Z. X., Nagao, K., Vijayanand, P., Rothenberg, E. V., Leonard, W. J. 2023; 8 (89): eadi8217

  Abstract

  The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.

  View details for DOI 10.1126/sciimmunol.adi8217

  View details for PubMedID 37922339

• Tox induces T cell IL-10 production in a BATF-dependent manner. Frontiers in immunology Canaria, D. A., Rodriguez, J. A., Wang, L., Yeo, F. J., Yan, B., Wang, M., Campbell, C., Kazemian, M., Olson, M. R. 2023; 14: 1275423

  Abstract

  Tox is a member of the high mobility group (HMG)-Box transcription factors and plays important roles in thymic T cell development. Outside of the thymus, however, Tox is also highly expressed by CD8 and CD4 T cells in various states of activation and in settings of cancer and autoimmune disease. In CD4 T cells, Tox has been primarily studied in T follicular helper (TFH) cells where it, along with Tox2, promotes TFH differentiation by regulating key TFH-associated genes and suppressing CD4 cytotoxic T cell differentiation. However, the role of Tox in other T helper (Th) cell subtypes is less clear. Here, we show that Tox is expressed in several physiologically-activated Th subtypes and its ectopic expression enhances the in vitro differentiation of Th2 and T regulatory (Treg) cells. Tox overexpression in unpolarized Th cells also induced the expression of several genes involved in cell activation (Pdcd1), cellular trafficking (Ccl3, Ccl4, Xcl1) and suppressing inflammation (Il10) across multiple Th subtypes. We found that Tox binds the regulatory regions of these genes along with the transcription factors BATF, IRF4, and JunB and that Tox-induced expression of IL-10, but not PD-1, is BATF-dependent. Based on these data, we propose a model where Tox regulates Th cell chemotactic genes involved in facilitating dendritic cell-T cell interactions and aids in the resolution or prevention of inflammation through the production of IL-10.

  View details for DOI 10.3389/fimmu.2023.1275423

  View details for PubMedID 38054003

• IL-1 beta promotes IL-9-producing Th cell differentiation in IL-2-limiting conditions through the inhibition of BCL6 FRONTIERS IN IMMUNOLOGY Canaria, D., Clare, M. G., Yan, B., Campbell, C. B., Ismaio, Z. A., Anderson, N. L., Park, S., Dent, A. L., Kazemian, M., Olson, M. R. 2022; 13: 1032618

  Abstract

  IL-9-producing CD4+ T helper cells, termed Th9 cells, differentiate from naïve precursor cells in response to a combination of cytokine and cell surface receptor signals that are elevated in inflamed tissues. After differentiation, Th9 cells accumulate in these tissues where they exacerbate allergic and intestinal disease or enhance anti-parasite and anti-tumor immunity. Previous work indicates that the differentiation of Th9 cells requires the inflammatory cytokines IL-4 and TGF-β and is also dependent of the T cell growth factor IL-2. While the roles of IL-4 and TGF-β-mediated signaling are relatively well understood, how IL-2 signaling contributes to Th9 cell differentiation outside of directly inducing the Il9 locus remains less clear. We show here that murine Th9 cells that differentiate in IL-2-limiting conditions exhibit reduced IL-9 production, diminished NF-kB activation and a reduced NF-kB-associated transcriptional signature, suggesting that IL-2 signaling is required for optimal NF-kB activation in Th9 cells. Interestingly, both IL-9 production and the NF-kB transcriptional signature could be rescued by addition of the NF-kB-activating cytokine IL-1β to IL-2-limiting cultures. IL-1β was unique among NF-kB-activating factors in its ability to rescue Th9 differentiation as IL-2 deprived Th9 cells selectively induced IL-1R expression and IL-1β/IL-1R1 signaling enhanced the sensitivity of Th9 cells to limiting amounts of IL-2 by suppressing expression of the Th9 inhibitory factor BCL6. These data shed new light on the intertwined nature of IL-2 and NF-kB signaling pathways in differentiating Th cells and elucidate the potential mechanisms that promote Th9 inflammatory function in IL-2-limiting conditions.

  View details for DOI 10.3389/fimmu.2022.1032618

  View details for Web of Science ID 000885197400001

  View details for PubMedID 36389679

  View details for PubMedCentralID PMC9663844

• Reply to Grigoriev et al., "Sequences of SARS-CoV-2 'Hybrids' with the Human Genome: Signs of Non-coding RNA?" JOURNAL OF VIROLOGY Yan, B., Chakravorty, S., Mirabelli, C., Wang, L., Trujillo-Ochoa, J. L., Chauss, D., Kumar, D., Lionakis, M. S., Olson, M. R., Wobus, C. E., Afzali, B., Kazemian, M. 2022; 96 (2): e0169021

  View details for DOI 10.1128/JVI.01690-21

  View details for Web of Science ID 000766751500003

  View details for PubMedID 34705544

  View details for PubMedCentralID PMC8791293

• Mitochondrial C5aR1 activity in macrophages controls IL-1 beta production underlying sterile inflammation SCIENCE IMMUNOLOGY Niyonzima, N., Rahman, J., Kunz, N., West, E. E., Freiwald, T., Desai, J., Merle, N. S., Gidon, A., Sporsheim, B., Lionakis, M. S., Evensen, K., Lindberg, B., Skagen, K., Skjelland, M., Singh, P., Haug, M., Ruseva, M. M., Kolev, M., Bibby, J., Marshall, O., O'Brien, B., Deeks, N., Afzali, B., Clark, R. J., Woodruff, T. M., Pryor, M., Yang, Z., Remaley, A. T., Mollnes, T. E., Hewitt, S. M., Yan, B., Kazemian, M., Kiss, M. G., Binder, C. J., Halvorsen, B., Espevik, T., Kemper, C. 2021; 6 (66): eabf2489

  Abstract

  While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the “complosome,” functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also nonredundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and generate autocrine C5a via formation of an intracellular C5 convertase. Cholesterol crystal sensing by macrophages induced C5aR1 signaling on mitochondrial membranes, which shifted ATP production via reverse electron chain flux toward reactive oxygen species generation and anaerobic glycolysis to favor IL-1β production, both at the transcriptional level and processing of pro–IL-1β. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1β produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell-autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate the unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.

  View details for DOI 10.1126/sciimmunol.abf2489

  View details for Web of Science ID 000744651900010

  View details for PubMedID 34932384

  View details for PubMedCentralID PMC8902698

• STAT5 Represses a STAT3-Independent Th17-like Program during Th9 Cell Differentiation JOURNAL OF IMMUNOLOGY Canaria, D., Yan, B., Clare, M. G., Zhang, Z., Taylor, G. A., Boone, D. L., Kazemian, M., Olson, M. R. 2021; 207 (5): 1265-1274

  Abstract

  IL-9-producing Th cells, termed Th9 cells, contribute to immunity against parasites and cancers but have detrimental roles in allergic disease and colitis. Th9 cells differentiate in response to IL-4 and TGF-β, but these signals are insufficient to drive Th9 differentiation in the absence of IL-2. IL-2-induced STAT5 activation is required for chromatin accessibility within Il9 enhancer and promoter regions and directly transactivates the Il9 locus. STAT5 also suppresses gene expression during Th9 cell development, but these roles are less well defined. In this study, we demonstrate that human allergy-associated Th9 cells exhibited a signature of STAT5-mediated gene repression that is associated with the silencing of a Th17-like transcriptional signature. In murine Th9 cell differentiation, blockade of IL-2/STAT5 signaling induced the expression of IL-17 and the Th17-associated transcription factor Rorγt. However, IL-2-deprived Th9 cells did not exhibit a significant Th17- or STAT3-associated transcriptional signature. Consistent with these observations, differentiation of IL-17-producing cells under these conditions was STAT3-independent but did require Rorγt and BATF. Furthermore, ectopic expression of Rorγt and BATF partially rescued IL-17 production in STAT3-deficient Th17 cells, highlighting the importance of these factors in this process. Although STAT3 was not required for the differentiation of IL-17-producing cells under IL-2-deprived Th9 conditions, their prolonged survival was STAT3-dependent, potentially explaining why STAT3-independent IL-17 production is not commonly observed in vivo. Together, our data suggest that IL-2/STAT5 signaling plays an important role in controlling the balance of a Th9 versus a Th17-like differentiation program in vitro and in allergic disease.

  View details for DOI 10.4049/jimmunol.2100165

  View details for Web of Science ID 000731636500005

  View details for PubMedID 34348976

  View details for PubMedCentralID PMC8387395

• The C3-like molecule CD109 controls Th1 versus Th17 induction in CD4+T cells Merle, N. S., Kolev, M., Rahman, J., West, E., Yan, B., Kazemian, M., Afzali, B., Kemper, C. AMER ASSOC IMMUNOLOGISTS. 2021

  View details for Web of Science ID 000713665800282

• Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and artifactual. bioRxiv : the preprint server for biology Yan, B., Chakravorty, S., Mirabelli, C., Wang, L., Trujillo-Ochoa, J. L., Chauss, D., Kumar, D., Lionakis, M. S., Olson, M. R., Wobus, C. E., Afzali, B., Kazemian, M. 2021

  Abstract

  Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV2 is a positive sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle, it is biologically unlikely to be in a location where splicing events could result in genome integration. Here, we investigated the biological authenticity of HVC events. In contrast to true biological events such as mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and infected cell lines, were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ~1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events was, in fact, not greater than this background "noise". Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes.

  View details for DOI 10.1101/2021.02.17.431704

  View details for PubMedID 33619483

• Epstein-Barr Virus Episome Physically Interacts with Active Regions of the Host Genome in Lymphoblastoid Cells JOURNAL OF VIROLOGY Wang, L., Laing, J., Yan, B., Zhou, H., Ke, L., Wang, C., Narita, Y., Zhang, Z., Olson, M. R., Afzali, B., Zhao, B., Kazemian, M. 2020; 94 (24)

  Abstract

  The Epstein-Barr virus (EBV) episome is known to interact with the three-dimensional structure of the human genome in infected cells. However, the exact locations of these interactions and their potential functional consequences remain unclear. Recently, high-resolution chromatin conformation capture (Hi-C) assays in lymphoblastoid cells have become available, enabling us to precisely map the contacts between the EBV episome(s) and the human host genome. Using available Hi-C data at a 10-kb resolution, we have identified 15,000 reproducible contacts between EBV episome(s) and the human genome. These contacts are highly enriched in chromatin regions denoted by typical or super enhancers and active markers, including histone H3K27ac and H3K4me1. Additionally, these contacts are highly enriched at loci bound by host transcription factors that regulate B cell growth (e.g., IKZF1 and RUNX3), factors that enhance cell proliferation (e.g., HDGF), or factors that promote viral replication (e.g., NBS1 and NFIC). EBV contacts show nearly 2-fold enrichment in host regions bound by EBV nuclear antigen 2 (EBNA2) and EBNA3 transcription factors. Circular chromosome conformation capture followed by sequencing (4C-seq) using the EBV origin of plasmid replication (oriP) as a "bait" in lymphoblastoid cells further confirmed contacts with active chromatin regions. Collectively, our analysis supports interactions between EBV episome(s) and active regions of the human genome in lymphoblastoid cells.IMPORTANCE EBV is associated with ∼200,000 cancers each year. In vitro, EBV can transform primary human B lymphocytes into immortalized cell lines. EBV-encoded proteins, along with noncoding RNAs and microRNAs, hijack cellular proteins and pathways to control cell growth. EBV nuclear proteins usurp normal transcriptional programs to activate the expression of key oncogenes, including MYC, to provide a proliferation signal. EBV nuclear antigens also repress CDKN2A to suppress senescence. EBV membrane protein activates NF-κB to provide survival signals. EBV genomes are maintained by EBNA1, which tethers EBV episomes to the host chromosomes during mitosis. However, little is known about where EBV episomes are located in interphase cells. In interphase cells, EBV promoters drive the expression of latency genes, while oriP functions as an enhancer for these promoters. In this study, integrative analyses of published lymphoblastoid cell line (LCL) Hi-C data and our 4C-seq experiments position EBV episomes to host genomes with active epigenetic marks. These contact points were significantly enriched for super enhancers. The close proximity of EBV episomes and the super enhancers that are enriched for transcription cofactors or mediators in lymphoblasts may benefit EBV gene expression, suggesting a novel mechanism of transcriptional activation.

  View details for DOI 10.1128/JVI.01390-20

  View details for Web of Science ID 000595868300006

  View details for PubMedID 32999023

  View details for PubMedCentralID PMC7925191

• An autocrine Vitamin D-driven Th1 shutdown program can be exploited for COVID-19. bioRxiv : the preprint server for biology McGregor, R., Chauss, D., Freiwald, T., Yan, B., Wang, L., Nova-Lamperti, E., Zhang, Z., Teague, H., West, E. E., Bibby, J., Kelly, A., Malik, A., Freeman, A. F., Schwartz, D., Portilla, D., John, S., Lavender, P., Lionakis, M. S., Mehta, N. N., Kemper, C., Cooper, N., Lombardi, G., Laurence, A., Kazemian, M., Afzali, B. 2020

  Abstract

  Pro-inflammatory immune responses are necessary for effective pathogen clearance, but cause severe tissue damage if not shut down in a timely manner 1,2 . Excessive complement and IFN-gamma-associated responses are known drivers of immunopathogenesis 3 and are among the most highly induced immune programs in hyper-inflammatory SARS-CoV2 lung infection 4 . The molecular mechanisms that govern orderly shutdown and retraction of these responses remain poorly understood. Here, we show that complement triggers contraction of IFN-gamma producing CD4 + T helper (Th) 1 cell responses by inducing expression of the vitamin D (VitD) receptor (VDR) and CYP27B1, the enzyme that activates VitD, permitting T cells to both activate and respond to VitD. VitD then initiates the transition from pro-inflammatory IFN-gamma + Th1 cells to suppressive IL-10 + Th1 cells. This process is primed by dynamic changes in the epigenetic landscape of CD4 + T cells, generating superenhancers and recruiting c-JUN and BACH2, a key immunoregulatory transcription factor 5-7 . Accordingly, cells in psoriatic skin treated with VitD increased BACH2 expression, and BACH2 haplo-insufficient CD4 + T cells were defective in IL-10 production. As proof-of-concept, we show that CD4 + T cells in the bronchoalveolar lavage fluid (BALF) of patients with COVID-19 are Th1-skewed and that VDR is among the top regulators of genes induced by SARS-CoV2. Importantly, genes normally down-regulated by VitD were de-repressed in CD4 + BALF T cells of COVID-19, indicating that the VitD-driven shutdown program is impaired in this setting. The active metabolite of VitD, alfacalcidol, and cortico-steroids were among the top predicted pharmaceuticals that could normalize SARS-CoV2 induced genes. These data indicate that adjunct therapy with VitD in the context of other immunomodulatory drugs may be a beneficial strategy to dampen hyperinflammation in severe COVID-19.

  View details for DOI 10.1101/2020.07.18.210161

  View details for PubMedID 32743590

• SARS-CoV2 drives JAK1/2-dependent local and systemic complement hyper-activation. Research square Yan, B., Freiwald, T., Chauss, D., Wang, L., West, E., Bibby, J., Olson, M., Kordasti, S., Portilla, D., Laurence, A., Lionakis, M. S., Kemper, C., Afzali, B., Kazemian, M. 2020

  Abstract

  Patients with coronavirus disease 2019 (COVID-19) present with a range of devastating acute clinical manifestations affecting the lungs, liver, kidneys and gut. The best-characterized entry receptor for the disease-causing virus SARS-CoV2, angiotensin converting enzyme (ACE) 2, is highly expressed in these tissues. However, the pathways that underlie the disease are still poorly understood. Here we show that the complement system is unexpectedly one of the intracellular pathways most highly induced by SARS-CoV2 infection in lung epithelial and liver cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Modelling the regulome of host genes induced by COVID-19 and the drugs that could normalize these genes both implicated the JAK1/2-STAT1 signaling system downstream of type I interferon receptors, and NF-kB. Ruxolitinib, a JAK1/2 inhibitor and the top predicted pharmaceutical candidate, normalized interferon signature genes, IL-6 (the best characterized severity marker in COVID-19) and all complement genes induced by SARS-CoV2, but did not affect NF-kB-regulated genes. We predict that combination therapy with JAK inhibitors and other agents with the potential to normalize NF-kB-signaling, such as anti-viral agents, may serve as an effective clinical strategy.

  View details for DOI 10.21203/rs.3.rs-33390/v1

  View details for PubMedID 32702726

• Restoration of RNA helicase DDX5 suppresses hepatitis B virus (HBV) biosynthesis and Wnt signaling in HBV-related hepatocellular carcinoma THERANOSTICS Mani, S., Yan, B., Cui, Z., Sun, J., Utturkar, S., Foca, A., Fares, N., Durantel, D., Lanman, N., Merle, P., Kazemian, M., Andrisani, O. 2020; 10 (24): 10957-10972

  Abstract

  Rationale: RNA helicase DDX5 is downregulated during hepatitis B virus (HBV) replication, and poor prognosis HBV-related hepatocellular carcinoma (HCC). The aim of this study is to determine the mechanism and significance of DDX5 downregulation for HBV-driven HCC, and identify biologics to prevent DDX5 downregulation. Methods: Molecular approaches including immunoblotting, qRT-PCR, luciferase transfections, hepatosphere assays, Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), and RNA-seq were used with cellular models of HBV replication, HBV infection, and HBV-related liver tumors, as well as bioinformatic analyses of liver cancer cells from two independent cohorts. Results: We demonstrate that HBV infection induces expression of the proto-oncogenic miR17~92 and miR106b~25 clusters which target the downregulation of DDX5. Increased expression of these miRNAs is also detected in HBV-driven HCCs exhibiting reduced DDX5 mRNA. Stable DDX5 knockdown (DDX5KD) in HBV replicating hepatocytes increased viral replication, and resulted in hepatosphere formation, drug resistance, Wnt activation, and pluripotency gene expression. ATAC-seq of DDX5KD compared to DDX5 wild-type (WT) cells identified accessible chromatin regions enriched in regulation of Wnt signaling genes. RNA-seq analysis comparing WT versus DDX5KD cells identified enhanced expression of multiple genes involved in Wnt pathway. Additionally, expression of Disheveled, DVL1, a key regulator of Wnt pathway activation, was significantly higher in liver cancer cells with low DDX5 expression, from two independent cohorts. Importantly, inhibitors (antagomirs) to miR17~92 and miR106b~25 restored DDX5 levels, reduced DVL1 expression, and suppressed both Wnt activation and viral replication. Conclusion: DDX5 is a negative regulator of Wnt signaling and hepatocyte reprogramming in HCCs. Restoration of DDX5 levels by miR17~92 / miR106b~25 antagomirs in HBV-infected patients can be explored as both antitumor and antiviral strategy.

  View details for DOI 10.7150/thno.49629

  View details for Web of Science ID 000573667200008

  View details for PubMedID 33042264

  View details for PubMedCentralID PMC7532671

• A potent Cas9-derived gene activator for plant and mammalian cells NATURE PLANTS Li, Z., Zhang, D., Xiong, X., Yan, B., Xie, W., Sheen, J., Li, J. 2017; 3 (12): 930-936

  Abstract

  Overexpression of complementary DNA represents the most commonly used gain-of-function approach for interrogating gene functions and for manipulating biological traits. However, this approach is challenging and inefficient for multigene expression due to increased labour for cloning, limited vector capacity, requirement of multiple promoters and terminators, and variable transgene expression levels. Synthetic transcriptional activators provide a promising alternative strategy for gene activation by tethering an autonomous transcription activation domain (TAD) to an intended gene promoter at the endogenous genomic locus through a programmable DNA-binding module. Among the known custom DNA-binding modules, the nuclease-dead Streptococcus pyogenes Cas9 (dCas9) protein, which recognizes a specific DNA target through base pairing between a synthetic guide RNA and DNA, outperforms zinc-finger proteins and transcription activator-like effectors, both of which target through protein-DNA interactions 1 . Recently, three potent dCas9-based transcriptional activation systems, namely VPR, SAM and SunTag, have been developed for animal cells 2-6 . However, an efficient dCas9-based transcriptional activation platform is still lacking for plant cells 7-9 . Here, we developed a new potent dCas9-TAD, named dCas9-TV, through plant cell-based screens. dCas9-TV confers far stronger transcriptional activation of single or multiple target genes than the routinely used dCas9-VP64 activator in both plant and mammalian cells.

  View details for DOI 10.1038/s41477-017-0046-0

  View details for Web of Science ID 000419067900013

  View details for PubMedID 29158545

  View details for PubMedCentralID PMC5894343