Publications
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2023 |
DEJOSEZ - CELL - 2023 - Click for abstract
Bat pluripotent stem cells reveal unusual entanglement between host and viruses Déjosez M, Marin A, Hughes GM, Morales AE, Godoy-Parejo C, Gray JL, Qin Y, Singh AA, Xu H, Juste J, Ibáñez C, White KM, Rosales R, Francoeur NJ, Sebra RP, Alcock D, Volkert TL, Puechmaille SJ, Pastusiak A, Frost SDW, Hiller M, Young RA, Teeling EC, García-Sastre A, Zwaka TP Cell. 2023 PMID: 36812912 Abstract Bats are distinctive among mammals due to their ability to fly, use laryngeal echolocation, and tolerate viruses. However, there are currently no reliable cellular models for studying bat biology or their response to viral infections. Here, we created induced pluripotent stem cells (iPSCs) from two species of bats: the wild greater horseshoe bat (Rhinolophus ferrumequinum) and the greater mouse-eared bat (Myotis myotis). The iPSCs from both bat species showed similar characteristics and had a gene expression profile resembling that of cells attacked by viruses. They also had a high number of endogenous viral sequences, particularly retroviruses. These results suggest that bats have evolved mechanisms to tolerate a large load of viral sequences and may have a more intertwined relationship with viruses than previously thought. Further study of bat iPSCs and their differentiated progeny will provide insights into bat biology, virus host relationships, and the molecular basis of bats' special traits. |
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2022 |
Pohl - ACS Infectious Diseases - 2022 - Click for abstract
Sec61 Inhibitor Apratoxin S4 Potently Inhibits SARS-CoV-2 and Exhibits Broad-Spectrum Antiviral Activity Pohl MO, Martin-Sancho L, Ratnayake R, White KM, Riva L, Chen QY, Lieber G, Busnadiego I, Yin X, Lin S, Pu Y, Pache L, Rosales R, Déjosez M, Qin Y, De Jesus PD, Beall A, Yoh S, Hale BG, Zwaka TP, Matsunaga N, García-Sastre A, Stertz S, Chanda SK, Luesch H ACS Infectious Diseases. 2022 PMID: 35766385 Abstract There is a pressing need for host-directed therapeutics that elicit broad-spectrum antiviral activities to potentially address current and future viral pandemics. Apratoxin S4 (Apra S4) is a potent Sec61 inhibitor that prevents cotranslational translocation of secretory proteins into the endoplasmic reticulum (ER), leading to anticancer and antiangiogenic activity both in vitro and in vivo. Since Sec61 has been shown to be an essential host factor for viral proteostasis, we tested Apra S4 in cellular models of viral infection, including SARS-CoV-2, influenza A virus, and flaviviruses (Zika, West Nile, and Dengue virus). Apra S4 inhibited viral replication in a concentration-dependent manner and had high potency particularly against SARS-CoV-2 and influenza A virus, with subnanomolar activity in human cells. Characterization studies focused on SARS-CoV-2 revealed that Apra S4 impacted a post-entry stage of the viral life-cycle. Transmission electron microscopy revealed that Apra S4 blocked formation of stacked double-membrane vesicles, the sites of viral replication. Apra S4 reduced dsRNA formation and prevented viral protein production and trafficking of secretory proteins, especially the spike protein. Given the potent and broad-spectrum activity of Apra S4, further preclinical evaluation of Apra S4 and other Sec61 inhibitors as antivirals is warranted. |
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Escalera - Cell Host Microbe - 2022 - Click for abstract
Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission Escalera A, Gonzalez-Reiche AS, Aslam S, Mena I, Laporte M, Pearl RL, Fossati A, Rathnasinghe R, Alshammary H, van de Guchte A, Farrugia K, Qin Y, Bouhaddou M, Kehrer T, Zuliani-Alvarez L, Meekins DA, Balaraman V, McDowell C, Richt JA, Bajic G, Sordillo EM, Dejosez M, Zwaka TP, Krogan NJ, Simon V, Albrecht RA, van Bakel H, García-Sastre A, Aydillo T. Cell Host Microbe. 2022 PMID: 35150638 Abstract SARS-CoV-2 lineages have diverged into highly prevalent variants termed "variants of concern" (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission. |
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Miorin - BioRXIV - 2022 - Click for abstract
The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters Miorin L, Mire CE, Ranjbar S, Hume AJ, Huang J, Crossland NA, White KM, Laporte M, Kehrer T, Haridas V, Moreno E, Nambu A, Jangra S, Cupic A, Dejosez M, Abo KA, Tseng AE, Werder RB, Rathnasinghe R, Mutetwa T, Ramos I, de Aja JS, de Alba Rivas CG, Schotsaert M, Corley RB, Falvo JV, Fernandez-Sesma A, Kim C, Rossignol JF, Wilson AA, Zwaka T, Kotton DN, Mühlberger E, García-Sastre A, Goldfeld AE. BiioRxiv. 2022 PMID: 35169796 Abstract A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs. |
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2021 |
White - Science - 2021 - Click for abstract
Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A White KM, Rosales R, Yildiz S, Kehrer T, Miorin L, Moreno E, Jangra S, Uccellini MB, Rathnasinghe R, Coughlan L, Martinez-Romero C, Batra J, Rojc A, Bouhaddou M, Fabius JM, Obernier K, Dejosez M, Guillén MJ, Losada A, Avilés P, Schotsaert M, Zwaka T, Vignuzzi M, Shokat KM, Krogan NJ, García-Sastre A ScienCe. 2021 PMID: 33495306 Abstract SARS-CoV-2 viral proteins interact with the eukaryotic translation machinery and inhibitors of translation have potent antiviral effects. Here we report that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (IC90 = 0.88 nM) 27.5-fold more potent than remdesivir against SARS-CoV-2 in vitro, with limited toxicity in cell culture. Through the use of a drug resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A. We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19. |
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Zwaka - EMBO Reports - 2021 - Click for abstract
Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development Salewskij K, Gross-Thebing T, Ing-Simmons E, Duethorn B, Rieger B, Fan R, Chen R, Govindasamy N, Brinkmann H, Kremer L, Kuempel-Rink N, Mildner K, Zeuschner D, Stehling M, Dejosez M, Zwaka TP, Schöler HR, Busch KB, Vaquerizas JM, Bedzhov I. EMBo Rep., 2021 PMID: 34515391 Abstract During implantation, the murine embryo transitions from a "quiet" into an active metabolic/proliferative state, which kick-starts the growth and morphogenesis of the post-implantation conceptus. Such transition is also required for embryonic stem cells to be established from mouse blastocysts, but the factors regulating this process are poorly understood. Here, we show that Ronin plays a critical role in the process by enabling active energy production, and the loss of Ronin results in the establishment of a reversible quiescent state in which naïve pluripotency is promoted. In addition, Ronin fine-tunes the expression of genes that encode ribosomal proteins and is required for proper tissue-scale organisation of the pluripotent lineage during the transition from blastocyst to egg cylinder stage. Thus, Ronin function is essential for governing the metabolic capacity so that it can support the pluripotent lineage's high-energy demands for cell proliferation and morphogenesis. |
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Zwaka - Disease Models & Mechanisms - 2021 - Click for abstract
Ronin overexpression induces cerebellar degeneration in a mouse model of ataxia Zwaka, TP, Skowronska M, Richman, R, Dejosez M Disease Models & Mechanisms. 2022 PMID: 34165550 Abstract Spinocerebellar ataxias (SCAs) are a group of genetically heterogeneous inherited neurodegenerative disorders characterized by progressive ataxia and cerebellar degeneration. Here, we used a mouse model to test a possible connection between SCA and Ronin (Thap11), a polyglutamine-containing transcriptional regulator encoded in a region of human chromosome 16q22.1 that has been genetically linked to SCA type 4. We report that transgenic expression of Ronin in mouse cerebellar Purkinje cells leads to detrimental loss of these cells and the development of severe ataxia as early as 10 weeks after birth. Mechanistically, we find that several SCA-causing genes harbor Ronin DNA-binding motifs and are transcriptionally deregulated in transgenic animals. In addition, ectopic expression of Ronin in embryonic stem cells significantly increases the protein level of Ataxin-1, the protein encoded by Atxn1, alterations of which cause SCA type 1. This increase is also seen in the cerebellum of transgenic animals, although the latter was not statistically significant. Hence, our data provide evidence for a link between Ronin and SCAs, and suggest that Ronin may be involved in the development of other neurodegenerative diseases. |
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2020 |
Riva - Nature - 2020 - Click for abstract
Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing Riva .... Dejosez, Zwaka....Chanda Nature. 2020 PMID: 32707573 Abstract The emergence of the novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to require at least 12-18 months, and the typical timeline for approval of a novel antiviral therapeutic can exceed 10 years. Thus, repurposing of known drugs could significantly accelerate the deployment of novel therapies for COVID-19. Towards this end, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. We report the identification of 100 molecules that inhibit viral replication, including 21 known drugs that exhibit dose response relationships. Of these, thirteen were found to harbor effective concentrations likely commensurate with achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4, and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334. Notably, MDL-28170, ONO 5334, and apilimod were found to antagonize viral replication in human iPSC-derived pneumocyte-like cells, and the PIKfyve inhibitor also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, the known pharmacological and human safety profiles of these compounds will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19. |
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Zwaka - BioRXIV - 2020 - Click for abstract
SCA4 locus-associated gene Ronin (Thap11) increases Ataxin-1 protein levels and induces cerebellar degeneration in a mouse model of ataxia Zwaka T, Richman R, Dejosez M BioRxiv doi: https://doi.org/10.1101/2020.03.04.977405 . 2020 Abstract Spinocerebellar ataxias (SCAs) are a group of genetically heterogeneous inherited neurodegenerative disorders characterized by progressive ataxia and cerebellar degeneration. Here, we tested if Ronin (Thap11), a polyglutamine-containing protein encoded in a region on human chromosome 16q22.1 that has been genetically linked to SCA4, can be connected with SCA disease in a mouse model. We report that transgenic expression of Ronin in mouse cerebellar Purkinje cells leads not only histopathologically to detrimental loss of Purkinje cells but also phenotypically to the development of severe ataxia as early as 10-12 weeks after birth. Mechanistically, we find that Ronin is part of a protein complex in the cerebellum that is distinct from the one previously found in embryonic stem cells. Importantly, ectopically expressed Ronin raises the protein level of Ataxin-1 (Atxn1), the causative gene of the most common type of SCA, SCA1. Hence, our data provide evidence for a link between Ronin and SCAs, and also suggest that Ronin may be involved in the development of other neurodegenerative diseases. |
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2019 |
Zwaka - Nature - 10/2019 - Click for abstract
Survival of the fittest cells Nature. 2019. Oct 17; 574:310-312 Cells in the body don’t always play nicely together. Could co-opting their competitive nature help to unlock cutting-edge therapies? |
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Leitner - STEM CELL RESEARCH - 2019 - Click for abstract
Immature mDA Neurons Ameliorate Motor Deficits in a 6-OHDA Parkinson’s Disease Mouse Model and are Functional after Cryopreservation Leitner D, Ramamoorthy M, Dejosez M, Zwaka TP. Stem Cell Research. 2019; in press. Abstract Parkinson’s disease is associated with the loss of dopaminergic neurons in the midbrain. Clinical studies investigating replacement of these neurons with in vitro-generated neurons are currently underway. However, this approach has been limited by difficulties in scaling up on-demand production of midbrain dopaminergic (mDA) neurons from pluripotent stem cells. Cryo-preservation may offer a solution, as it allows for banking of quality controlled mDA neurons. In this study, we tested different freezing conditions and found that optimal cryopreservation of immature human mDA neurons at an early differentiation time point was achieved in STEM-CELLBANKER medium using a controlled freezing program. |
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2018 |
Janssens - Current Protocols In stem cell biology - 2018 - Click for abstract
FACS-Mediated isolation of neruonal cell populations from virus-infected human embryonic stem cell-derived cerebral organoid cultures. Janssens S, Schotsaert M, Manganaro, Dejosez M, Simon, V, García-Sastre A, Zwaka TP Curr Protoc Stem Cell Biol. 2018 Oct:e65 PMID: 30358129 Abstract Organoids - or pluripotent stem cell–derived in vitro‐grown simplified mini organs - have become a tremendously important model to study human organ development and disease. To restrict the noise inherent to the heterogeneous cell mixtures derived from organoid cultures, we developed a new technique of fluorescence‐assisted cell sorting (FACS) of virus‐infected cerebral organoid cultures. This method still includes the advantage of growing cells in a more natural environment than traditional cell culture, but now renders samples suitable for downstream cell type‐specific multi‐omics analyses. The protocol starts from stem cell‐derived mature brain organoids and includes steps for: preparing the culture for viral infection, production of the viral stocks, FACS sample preparation, and gating and sorting implementation. The protocol has been developed for Zika virus infection, but can be extrapolated to other viruses or fluorescent marker expression as illustrated in an alternate protocol using a single‐cycle lentivirus expressing a fluorescent reporter protein. |
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Janssens - mSystems - 2018 - Click for abstract
Zika Virus Alters the DNA Methylation Pattern of Neural Genes in an Organoid Model of the Developing Human Brain. Janssens S, Schotsaert M, Karnik, R, Balasubramaniam V, Dejosez M, Meissner A, García-Sastre A, Zwaka TP MSystems. 2018 FEB;3(1).e00219-17 PMID29435496 Abstract Zika virus (ZIKV) infection during early pregnancy can cause microcephaly and associated defects at birth, but whether it can induce neurologic sequelae that appear later in life remains unclear. Using a model of the developing brain based on embryonic stem cell-derived brain organoids, we studied the impact of ZIKV infection on the DNA methylation pattern across the entire genome in selected neural cell types. The virus unexpectedly alters the DNA methylome of neural progenitors, astrocytes and differentiated neurons at genes that have been implicated in the pathogenesis of a number of brain disorders, most prominently mental retardation and schizophrenia. Our results suggest that ZIKV infection during fetal development could lead to a spectrum of delayed onset neuropsychiatric complications. |
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Dejosez - Stem Cell Research - 2017 - Click for abstract
Generation of hiPSTZ16 (ISMMSi003-A) cell line from normal human forsekin fibroblasts. Dejosez M, Zwaka TP Stem Cell Research. 2017 Dec; 26:44-46 PMID: 29227831 Human foreskin fibroblasts from a commercial source were reprogrammed into induced pluripotent stem cells to establish a clonal stem cell line, hiPSTZ16 (ISMMSi003-A). These cells show a normal karyotype and full differentiation potential in teratoma assays. The described cells provide a useful resource in combination with other iPS cell lines generated from normal human foreskin fibroblasts to study source- and reprogramming method-independent effects in downstream applications. |
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2017 |
Zakirova - PLOS Genetics - 2018 - Click for abstract
Mutations in THAP1/DYT6 Reveal that Diverse Dystonia Genes Disrupt similar neuronal pathways ad functions. Zakirova Z, Fanutza, T, Bonet, J, Readhead, B, Zhang, W, Yi, Z, Beauvais, G, ZWAKA, TP, Ozelius, LJ, Blitzer, RD, Gonzalez-Alegre, P, Ehrlich, ME PLOS Genetics. 2018 Jan; 14(1):E1007169 PMID: 29364887 Abstract Dystonia is characterized by involuntary muscle contractions. Its many forms are genetically, phenotypically and etiologically diverse and it is unknown whether their pathogenesis converges on shared pathways. Mutations in THAP1 [THAP (Thanatos-associated protein) domain containing, apoptosis associated protein 1], a ubiquitously expressed transcription factor with DNA binding and protein-interaction domains, cause dystonia, DYT6. There is a unique, neuronal 50-kDa Thap1-like immunoreactive species, and Thap1 levels are auto-regulated on the mRNA level. However, THAP1 downstream targets in neurons, and the mechanism via which it causes dystonia are largely unknown. We used RNA-Seq to assay the in vivo effect of a heterozygote Thap1 C54Y or ΔExon2 allele on the gene transcription signatures in neonatal mouse striatum and cerebellum. Enriched pathways and gene ontology terms include eIF2α Signaling, Mitochondrial Dysfunction, Neuron Projection Development, Axonal Guidance Signaling, and Synaptic LongTerm Depression, which are dysregulated in a genotype and tissue-dependent manner. Electrophysiological and neurite outgrowth assays were consistent with those enrichments, and the plasticity defects were partially corrected by salubrinal. Notably, several of these pathways were recently implicated in other forms of inherited dystonia, including DYT1. We conclude that dysfunction of these pathways may represent a point of convergence in the pathophysiology of several forms of inherited dystonia. |
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Zwaka - Developmental Cell - 2017 - Click for abstract
Status Anxiety among Pluripotent Stem Cells? Zwaka TP Developmental Cell. 2017 SEP;42:555-556 PMID: 28950094 Abundant cell death marks early embryonic development. New work reported in Developmental Cell from Diaz-Diaz and colleagues (2017) proposes that this death results from cell competition arising from differences in cellular differentiation status, thus providing a physiological mechanism for controlling the make-up of the pluripotent stem cell population. |
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Fujita - Cell Reports - 2017 - Click for abstract
Ronin governs heart development by controlling core gene Expression programs. Fujita F, Freire P, Coarfa C, Benham A, Gunaratne, P, Schneider MD, Dejosez M, Zwaka TP Cell Reports. 2017 NOv; 21:1562-1573 PMID: 29117561 Abstract Ronin (THAP11), an idiosyncratic DNA-binding protein that evolved from a primordial DNA transposon by molecular domestication, recognizes a hyperconserved promoter sequence through which it controls a variety of developmentally and metabolically essential genes in pluripotent stem cells. However, it remains unclear whether Ronin or related THAP proteins perform similar functions elsewhere in development. Here, we present evidence indicating that Ronin functions within the nascent heart as it arises from the mesoderm and forms a four-chambered organ. We show that Ronin is vital for cardiogenesis during midgestation through its control of a core set of critical genes. The activity of Ronin coincided with the recruitment of its cofactor, Hcf-1, and the elevation of H3K4me3 levels at specific target genes, suggesting the involvement of an epigenetic mechanism. On the strength of these findings, we propose that Ronin activity during cardiogenesis may offer a template that could be used to understand how important gene programs are sustained across different cell types within a developing organ, such as the heart. |
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Zwaka - Nature - 2017 - Click for abstract
Stem Cells: The cost of perpetual youth. Zwaka TP NAture. 2017 Aug;548:165-166 PMID: 28746313 The ability to become nearly any cell type is restricted to eggs, sperm and primitive stem cells in very early embryos. Two studies reveal that maintaining this pluripotent state in vitro comes at a cost. |
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Seifert - Stem Cell Research - 2017 - Click for abstract
Ronin influences the DNA damage response in pluripotent stem cells. Seifert BA, Dejosez M, Zwaka TP. Stem Cell Res. 2017 Aug;23:98-104 PMID: 28715716 Abstract Early mammalian embryonic cells must maintain a particularly robust DNA repairsystem, as mutations at this developmental point have detrimental consequences for the organism. How the repair system can be tuned to fulfill such elevated requirements is largely unknown, but it may involve transcriptional regulation. Ronin (Thap11) is a transcriptional regulator responsible for vital programs in pluripotent cells. Here, we report that this protein also modulates the DNA damage response of such cells. We show that conditional Ronin knockout sensitizes embryonic stem cells (ESCs) to UV-C-induced DNA damage in association with Atr pathway activation and G2/M arrest. Ronin binds to and regulates the genes encoding several DNA repair factors, including Gtf2h4and Rad18, providing a potential mechanism for this phenotype. Our results suggest that the unique DNA repair requirements of the early embryo are not met by a static system, but rather via highly regulated processes. |
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Aguilo - Stem Cell Reports - 2017 - Click for abstract
Thap1: Role in Mouse Embryonic stem cell Survival and Differentiation. Aguilo F, Zakirova Z, Nolan K, Wagner R, Sharma R, Hogan M, Wei C, Sun Y, Walsh MJ, Kelley K, Zhang W, Ozelius LJ, Gonzalez-Alegre P, Zwaka TP, Ehrlich ME. Stem Cell Reports: 2017 May 19;67711(17):30186-30188 PMID: 28579396 Abstract THAP1 (THAP [Thanatos-associated protein] domain-containing, apoptosis-associated protein 1) is a ubiquitously expressed member of a family of transcription factors with highly conserved DNA-binding and protein-interacting regions. Mutations in THAP1 cause dystonia, DYT6, a neurologic movement disorder. THAP1 downstream targets and the mechanism via which it causes dystonia are largely unknown. Here, we show that wild-type THAP1 regulates embryonic stem cell (ESC) potential, survival, and proliferation. Our findings identify THAP1 as an essential factor underlying mouse ESC survival and to some extent, differentiation, particularly neuroectodermal. Loss of THAP1 or replacement with a disease-causing mutation results in an enhanced rate of cell death, prolongs Nanog, Prdm14, and/or Rex1 expression upon differentiation, and results in failure to upregulate ectodermal genes. ChIP-Seq reveals that these activities are likely due in part to indirect regulation of gene expression. |
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2016 |
Poche - Cell Reports - 2016 - Click for abstract
RONIN Is an Essential Transcriptional Regulator of Genes Required for Mitochondrial Function in the Developing Retina. Poché RA, Zhang M, Rueda EM, Tong X, McElwee ML, Wong L, Hsu CW, Dejosez M, Burns AR, Fox DA, Martin JF, Zwaka TP, Dickinson ME. Cell Rep. 2016 Feb 23;14(7):1684-97 PMID: 26876175 Abstract A fundamental principle governing organ size and function is the fine balance between cell proliferation and cell differentiation. Here, we identify RONIN (THAP11) as a key transcriptional regulator of retinal progenitor cell (RPC) proliferation. RPC-specific loss of Ronin results in a phenotype strikingly similar to that resulting from the G1- to S-phase arrest and photoreceptor degeneration observed in the Cyclin D1 null mutants. However, we determined that, rather than regulating canonical cell-cycle genes, RONIN regulates a cohort of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Coincidentally, with premature cell-cycle exit, Ronin mutants exhibited deficient ETC activity, reduced ATP levels, and increased oxidative stress that we ascribe to specific loss of subunits within complexes I, III, and IV. These data implicate RONIN as a positive regulator of mitochondrial gene expression that coordinates mitochondrial activity and cell-cycle progression. |
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Skinner - Elife - 2016 - Click for abstract
Single-cell analysis of transcription kinetics across the cell cycle. Skinner SO, Xu H, Nagarkar-Jaiswal S, Freire PR, Zwaka TP, Golding I. Elife. 2016 Jan 29;5. PMID: 26824388 Abstract Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-interest, researchers commonly compare the distribution of mRNA copy-number to the prediction of a theoretical model. However, the reliability of this procedure is limited because the measured mRNA numbers represent integration over the mRNA lifetime, contribution from multiple gene copies, and mixing of cells from different cell-cycle phases. We address these limitations by simultaneously quantifying nascent and mature mRNA in individual cells, and incorporating cell-cycle effects in the analysis of mRNA statistics. We demonstrate our approach on Oct4 and Nanog in mouse embryonic stem cells. Both genes follow similar two-state kinetics. However, Nanog exhibits slower ON/OFF switching, resulting in increased cell-to-cell variability in mRNA levels. Early in the cell cycle, the two copies of each gene exhibit independent activity. After gene replication, the probability of each gene copy to be active diminishes, resulting in dosage compensation. |
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2015 |
Clark - Zwaka - Cell reports - 2015 - Click for abstract
Editorial overview: Cell reprogramming, regeneration and repair: Reprogramming: the eternal circle. Clark AT, Zwaka TP. Curr Opin Genet Dev. 2015 Oct;34:v-vi. doi: 10.1016/j.gde.2015.09.006. Epub 2015 Oct 20. PMID: 26476785 No abstract available. |
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Zheng - Cell reports - 2015 - Click for abstract
Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation. Zheng ZY, Tian L, Bu W, Fan C, Gao X, Wang H, Liao YH, Li Y, Lewis MT, Edwards D, Zwaka TP, Hilsenbeck SG, Medina D, Perou CM, Creighton CJ, Zhang XH, Chang EC. CELL REP. 2015 JUL 21;12(3):511-24. PMID:26166574 Abstract Basal-like breast cancers (BLBCs) are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines; e.g., IL8. Expression levels of these chemokines and N-RAS in tumors correlate with outcome. N-Ras, but not K-Ras, induces IL-8 by binding and activating the cytoplasmic pool of JAK2; IL-8 then acts on both the cancer cells and stromal fibroblasts. Thus, BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells. |
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Bisson - Developmental Biology - 2015 - Click for abstract
Wnt5a and Wnt11 inhibit the canonical Wnt pathway and promote cardiac progenitor development via the Caspase-dependent degradation of AKT. Bisson JA, Mills B, Paul Helt JC, Zwaka TP, Cohen ED. Dev Biol. 2015 Feb 1. PMID: 25482987 Abstract How is it that Oct4, a transcription factor that controls pluripotency in stem cells, also controls lineage specification? A recent study investigating common Oct4 targets in vertebrate species indicates an evolutionarily conserved role in mediating cell adhesion. This finding may help decipher Oct4's versatility in governing stem cell behaviors. Copyright @ 2013 Elsevier Ltd. All rights reserved. Comment on A conserved Oct4/POUV-dependent network links adhesion and migration to progenitor maintenance. [Curr Biol. 2013] |
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Zwaka - Stem Cell Research - 2015 - Click for abstract
New Editor-in-Chief. Zwaka TP Stem Cell Rearch. January 2015 PMID: 25576466 No abstract available |
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2014 |
Wang - Stem Cells - 2014 - Click for abstract
GCNF-dependent activation of cyclin D1 expression via repression of Mir302a during ES cell differentiation. Wang H, Wang X, Archer TK, Zwaka TP, Cooney AJ. Stem Cells. 2014 Feb 28. PMID: 24578347 Abstract Cyclin D1 plays an important role in the regulation of cellular proliferation and its expression is activated during gastrulation in the mouse, however, it remains unknown how cyclin D1 expression is regulated during early embryonic development. Here we define the role of germ cell nuclear factor (GCNF) in the activation of cyclin D1 expression during embryonic stem (ES) cell differentiation as a model of early development. During our study of GCNF knock out (GCNF-/- ) ES cells, we discovered that loss of GCNF leads to the repression of cyclin D1 activation during ES cell differentiation. This was determined to be an indirect effect of de-regulation Mir302a, which is a cyclin D1 suppressor via binding to the 3'UTR of cyclin D1 mRNA. Moreover, we showed that Mir302 is a target gene of GCNF that inhibits Mir302 expression by binding to a DR0 element within its promoter. Inhibition of Mir302a using Mir302 inhibitor during differentiation of GCNF-/- ES cells restored cyclin D1 expression. Similarly over-expression of GCNF during differentiation of GCNF-/- ES cells rescued the inhibition of Mir302a expression and the activation of cyclin D1. These results reveal that GCNF plays a key role in regulating activation of cyclin D1 expression via inhibition of Mir302a. |
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2013 |
Wagner - Current Biology - 2013 - Click for abstract
Sketch for a theory of Oct4. Wagner R.T. and Zwaka T.P. Current Biol. PMID: 24262829 Abstract How is it that Oct4, a transcription factor that controls pluripotency in stem cells, also controls lineage specification? A recent study investigating common Oct4 targets in vertebrate species indicates an evolutionarily conserved role in mediating cell adhesion. This finding may help decipher Oct4's versatility in governing stem cell behaviors. Copyright @ 2013 Elsevier Ltd. All rights reserved. Comment on A conserved Oct4/POUV-dependent network links adhesion and migration to progenitor maintenance. [Curr Biol. 2013] |
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Dejosez - Science - 2013 - Click for abstract
Safeguards for Cell Cooperation in Mouse Embryogenesis Shown by Genome-Wide Cheater Screen. Dejosez, M, Ura, H, Brandt, VL and Zwaka TP Science. 2013 Sep 16. PMID: 24030493 Abstract Ensuring cooperation among formerly autonomous cells has been a central challenge in the evolution of multicellular organisms. One solution is monoclonality, but this option still leaves room for exploitative behavior, as it does not eliminate genetic and epigenetic variability. We therefore hypothesized that embryonic development must be protected by robust regulatory mechanisms that prevent aberrant clones from superseding wild-type cells. Using a genome-wide screen in murine induced pluripotent stem cells, we identified a network of genes (centered on p53, topoisomerase 1, and olfactory receptors) whose down-regulation caused the cells to replace wild-type cells in vitro and in the mouse embryo-without perturbing normal development. These genes thus appear to fulfill an unexpected role in fostering cell cooperation. |
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Wang - Stem Cells - 2013 - Click for abstract
Epigenetic Re-programming of the Germ Cell Nuclear Factor Gene is Required for Proper Differentiation of Induced Pluripotent Cells. Wang H, Wang X, Xu X, Zwaka TP, Cooney AJ. Stem Cells. 2013 Mar 14. PMID: 23495137 Abstract Somatic cells have been reprogrammed into induced pluripotent stem (iPS) cells that recapitulate the pluripotent nature of embryonic stem (ES) cells. Reduced pluripotency and variable differentiation capacities have hampered progress with this technology for applications in regeneration medicine. We have previously shown that Germ Cell Nuclear Factor (Gcnf) is required for the repression of pluripotency genes during ES cell differentiation and embryonic development. Here we report that iPS cell lines, in which the Gcnf gene was properly re-programmed, allowing expression of Gcnf, repress pluripotency genes during subsequent differentiation. In contrast, iPS clones in which the Gcnf gene was not re-programmed maintained pluripotency gene expression during differentiation and did not differentiate properly either in vivo or in vitro. These mal-reprogrammed cells re-capitulated the phenotype of Gcnf knock out (Gcnf-/- ) ES cells. Re-introduction of Gcnf into either the Gcnf negative iPS cells or the Gcnf-/- ES cells, rescued repression of Oct4 during differentiation. Our findings establish a key role for Gcnf as a regulator of iPS cell pluripotency gene expression. It also demonstrates that reactivation of the Gcnf gene may serve as a marker to distinguish completely re-programmed iPS cells from incompletely pluripotent cells, which would make therapeutic use of iPS cells safer and more practical as it would reduce the oncogenic potential of iPS cells. |
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2012 |
Bao - Cell Stem Cell - 2012 - Click for abstract
The germ cell determinant Blimp1 is not required for derivation of pluripotent stem cells. Bao S, Leitch HG, Gillich A, Nichols J, Tang F, Kim S, Lee C, Zwaka T, Li X, Surani MA. Cell Stem Cell. 2012 Jul 6;11(1):110-7. doi: 10.1016/j.stem.2012.02.023. PMID: 22770244 Abstract Blimp1 (Prdm1), the key determinant of primordial germ cells (PGCs), plays a combinatorial role with Prdm14 during PGC specification from postimplantation epiblast cells. They together initiate epigenetic reprogramming in early germ cells toward an underlying pluripotent state, which is equivalent to embryonic stem cells (ESCs). Whereas Prdm14 alone can promote reprogramming and is important for the propagation of the pluripotent state, it is not known whether Blimp1 is similarly involved. By using a genetic approach, we demonstrate that Blimp1 is dispensable for the derivation and maintenance of ESCs and postimplantation epiblast stem cells (epiSCs). Notably, Blimp1 is also dispensable for reprogramming epiSCs to ESCs. Thus, although Blimp1 is obligatory for PGC specification, it is not required for the reversion of epiSCs to ESCs and for their maintenance thereafter. This study suggests that reprogramming, including that of somatic cells to ESCs, may not entail an obligatory route through a Blimp1-positive PGC-like state. Comment in Pluripotency takes off without Blimp1. [Cell Stem Cell. 2012] |
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Dejosez - Annual Review of Biochemistry - 2012 - Click for abstract
Pluripotency and nuclear reprogramming. Dejosez M, Zwaka TP. Annu Rev Biochem. 2012;81:737-65. doi: 10.1146/annurev-biochem-052709 104948. Epub 2012 Mar 20. Review. PMID: 22443931 Abstract Pluripotency is a "blank" cellular state characteristic of specific cells within the early embryo (e.g., epiblast cells) and of certain cells propagated in vitro (e.g., embryonic stem cells, ESCs). The terms pluripotent cell and stem cell are often used interchangeably to describe cells capable of differentiating into multiple cell types. In this review, we discuss the prevailing molecular and functional definitions of pluripotency and the working parameters employed to describe this state, both in the context of cells residing within the early embryo and cells propagated in vitro. |
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Jain - PLos Biology - 2012 - Click for abstract
p53 regulates cell cycle and microRNAs to promote differentiation of human embryonic stem cells. Jain AK, Allton K, Iacovino M, Mahen E, Milczarek RJ, Zwaka TP, Kyba M, Barton MC. PLoS Biol. 2012;10(2):e1001268. doi: 10.1371/journal.pbio.1001268. Epub 2012 Feb 28. PMID: 22389628 Abstract Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G(1) phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state. |
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2011 |
Chu - Current Biology - 2011 - Click for abstract
Blimp1 expression predicts embryonic stem cell development in vitro. Chu LF, Surani MA, Jaenisch R, Zwaka TP. Curr Biol. 2011 Oct 25;21(20):1759-65. doi: 10.1016/j.cub.2011.09.010. Epub 2011 Oct 13. PMID: 22000107 Abstract Despite recent critical insights into the pluripotent state of embryonic stem cells (ESCs), there is little agreement over the inaugural and subsequent steps leading to its generation [1-4]. Here we show that inner cell mass (ICM)-generated cells expressing Blimp1, a key transcriptional repressor of the somatic program during germ cell specification [5, 6], emerge on day 2 of blastocyst culture. Single-cell gene expression profiling indicated that many of these Blimp1-positive cells coexpress other genes typically associated with early germ cell specification. When genetically traced in vitro, these cells acquired properties normally associated with primordial germ cells. Importantly, fate-mapping experiments revealed that ESCs commonly arise from Blimp1-positive precursors; indeed, prospective sorting of such cells from ICM outgrowths increased the rate of ESC derivation more than 9-fold. Finally, using genetic ablation or distinct small molecules [7, 8], we show that epiblast cells can become ESCs without first acquiring Blimp1 positivity. Our findings suggest that the germ cell-like state is facultative for the stabilization of pluripotency in vitro. Thus, the association of Blimp1 expression with ESC development furthers understanding of how the pluripotent state of these cells is established in vitro and suggests a means to enhance the generation of new stem cell lines from blastocysts. Copyright @ 2011 Elsevier Ltd. All rights reserved. Comment in Embryonic stem cells: testing the germ-cell theory. [Curr Biol. 2011] |
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Gu - Journal of Biological Chemistry - 2011 - Click for abstract
Caspase-3 cleavage links delta-catenin to the novel nuclear protein ZIFCAT. Gu D, Tonthat NK, Lee M, Ji H, Bhat KP, Hollingsworth F, Aldape KD, Schumacher MA, Zwaka TP, McCrea PD. J Biol Chem. 2011 Jul 1;286(26):23178-88. doi: 10.1074/jbc.M110.167544. Epub 2011 May 11. PMID: 21561870 Abstract δ-Catenin is an Armadillo protein of the p120-catenin subfamily capable of modulating cadherin stability, small GTPase activity, and nuclear transcription. From yeast two-hybrid screening of a human embryonic stem cell cDNA library, we identified δ-catenin as a potential interacting partner of the caspase-3 protease, which plays essential roles in apoptotic as well as non-apoptotic processes. Interaction of δ-catenin with caspase-3 was confirmed using cleavage assays conducted in vitro, in Xenopus apoptotic extracts, and in cell line chemically induced contexts. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo repeat 6 of δ-catenin, was identified through peptide sequencing. Cleavage thus generates an amino-terminal (residues 1-816) and carboxyl-terminal (residues 817-1314) fragment, each containing about half of the central Armadillo domain. We found that cleavage of δ-catenin both abolishes its association with cadherins and impairs its ability to modulate small GTPases. Interestingly, 817-1314 possesses a conserved putative nuclear localization signal that may facilitate the nuclear targeting of δ-catenin in defined contexts. To probe for novel nuclear roles of δ-catenin, we performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating interaction with an uncharacterized KRAB family zinc finger protein, ZIFCAT. Our results indicate that ZIFCAT is nuclear and suggest that it may associate with DNA as a transcriptional repressor. We further determined that other p120 subfamily catenins are similarly cleaved by caspase-3 and likewise bind ZIFCAT. Our findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120-catenin subfamily members, facilitating the coordinate modulation of cadherins, small GTPases, and nuclear functions. |
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2010 |
Zwaka - PNAS - 2010 - Click for abstract
Unraveling the score of the enhancer symphony. Zwaka TP. Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21240-1. doi: 10.1073/pnas.1016297108. Epub 2010 Dec 6. No PMID: 21135244 text
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Zwaka - Molecular Therapy - 2010 - Click for abstract
The stem cell "tunnel" effect. Zwaka TP. Mol Ther. 2010 Dec;18(12):2043-4. doi: 10.1038/mt.2010.245. No abstract available. PMID: 21119690 Comment on Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors. [Mol Ther. 2010] |
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Zwaka - Nature - 2010 - Click for abstract
Stem cells: Troublesome memories. Zwaka TP. Nature. 2010 Sep 16;467(7313):280-1. doi: 10.1038/467280a. No abstract available. PMID: 20844526 Comment on Comprehensive methylome map of lineage commitment from haematopoietic progenitors. [Nature. 2010] Epigenetic memory in induced pluripotent stem cells. [Nature. 2010] |
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Dejosez - Genes and Development - 2010 - Click for abstract
Ronin/Hcf-1 binds to a hyperconserved enhancer element and regulates genes involved in the growth of embryonic stem cells. Dejosez M, Levine SS, Frampton GM, Whyte WA, Stratton SA, Barton MC, Gunaratne PH, Young RA, Zwaka TP. Genes Dev. 2010 Jul 15;24(14):1479-84. doi: 10.1101/gad.1935210. Epub 2010 Jun 25. PMID: 20581084 Abstract Self-renewing embryonic stem (ES) cells have an exceptional need for timely biomass production, yet the transcriptional control mechanisms responsible for meeting this requirement are largely unknown. We report here that Ronin (Thap11), which is essential for the self-renewal of ES cells, binds with its transcriptional coregulator, Hcf-1, to a highly conserved enhancer element that previously lacked a recognized binding factor. The subset of genes bound by Ronin/Hcf-1 function primarily in transcription initiation, mRNA splicing, and cell metabolism; genes involved in cell signaling and cell development are conspicuously underrepresented in this target gene repertoire. Although Ronin/Hcf-1 represses the expression of some target genes, its activity at promoter sites more often leads to the up-regulation of genes essential to protein biosynthesis and energy production. We propose that Ronin/Hcf-1 controls a genetic program that contributes to the unimpeded growth of ES cells. |
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Zwaka - Molecular Theraypy - 2010 - Click for abstract
Stem cell vaccination against cancer: fighting fire with fire? Zwaka TP. Mol Ther. 2010 Jan;18(1):8-9. doi: 10.1038/mt.2009.287. No abstract available. PMID: 20048741 A recent report1 suggests yet another application for embryonic stem (ES) cell technology: using such cells to immunize against cancer. However, before this novel strategy can prove useful, several issues need to be resolved. ES cells derived from preimplantation embryos2 and induced pluripotent stem (iPS) cells obtained by transient overexpression of specific transcription factors in somatic cells3,4,5 are frequently said to hold the potential to revolutionize biomedical research by providing new substrates for biological studies designed to understand human diseases, by offering cellular transplantation and replacement therapies, and by serving as a platform for pioneering drug development and screening efforts. However, as the history of biomedical research has made clear, the most triumphant breakthroughs have often emerged from areas initially not considered promising by most researchers. |
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