DOCSLIB.ORG

Stoichiometric and Temporal Requirements of Oct4, Sox2, Klf4, and C-Myc Expression for Efficient Human Ipsc Induction and Differentiation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Stoichiometric and Temporal Requirements of Oct4, Sox2, Klf4, and C-Myc Expression for Efficient Human Ipsc Induction and Differentiation Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation Eirini P. Papapetroua,b, Mark J. Tomishimac,d, Stuart M. Chambersc, Yvonne Micae, Evan Reeda,b, Jayanthi Menona,f, Viviane Tabara,f, Qianxing Mog, Lorenz Studera,c, and Michel Sadelaina,b,1 aCenter for Cell Engineering, bMolecular Pharmacology and Chemistry and cDevelopmental Biology Programs, dSKI Stem Cell Research Facility, eGerstner Sloan-Kettering Graduate School, and Departments of fNeurosurgery and gEpidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065 Communicated by Mark Ptashne, Memorial Sloan–Kettering Cancer Center, New York, NY, May 1, 2009 (received for review April 8, 2009) Human-induced pluripotent stem cells (hiPSCs) are generated from purposes remains controversial (20). Furthermore, the significance somatic cells by ectopic expression of the 4 reprogramming factors of vector silencing for reprogramming and its impact on differen- (RFs) Oct-4, Sox2, Klf4, and c-Myc. To better define the stoichiometric tiation is unclear. Although it has been proposed that silencing of requirements and dynamic expression patterns required for success- factor expression is required for successful reprogramming and ful hiPSC induction, we generated 4 bicistronic lentiviral vectors multilineage differentiation (3, 21, 22), it remains unclear whether encoding the 4 RFs co-expressed with discernable fluorescent pro- vector silencing constitutes a requirement or an epiphenomenon of teins. Using this system, we define the optimal stoichiometry of RF the reprogramming process. expression to be highly sensitive to Oct4 dosage, and we demonstrate Here we present a vector system for iPSC induction that allows the impact that variations in the relative ratios of RF expression exert for simultaneous real-time tracking of expression of the 4 individual on the efficiency of hiPSC induction. Monitoring of expression of each transgenes in single cells during hiPSC induction, maintenance and individual RF in single cells during the course of reprogramming directed differentiation. Using this system, we show that expression revealed that vector silencing follows acquisition of pluripotent cell of the 4 RFs at an optimal stoichiometry is critical for efficient markers. Pronounced lentiviral vector silencing was a characteristic of reprogramming, and we study in detail the kinetics of silencing or successfully reprogrammed hiPSC clones, but lack of complete silenc- the 4 transgenes during the reprogramming process. ing did not hinder hiPSC induction, maintenance, or directed differ- entiation. The vector system described here presents a powerful tool Results for mechanistic studies of reprogramming and the optimization of Reprogramming of Human Fibroblasts with Bicistronic Vectors Coex- hiPSC generation. pressing Each Reprogramming Factor with a Distinct Fluorescent Protein.We constructed lentiviral vectors that co-express each of the fluorescent proteins ͉ lentiviral vectors ͉ silencing ͉ stoichiometry 4 RFs, Oct4, Sox2, Klf4, and c-Myc together with a fluorescent protein linked by a 2A peptide (Fig. 1A). We selected a combination eprogramming of human fibroblasts to a pluripotent embryonic of 4 fluorescent proteins, vexGFP (violet light excited-green fluo- Rstem cell (ESC)-like state has recently been achieved through rescent protein), mCitrine, mCherry, and mCerulean (23, 24), retroviral-mediated gene transfer of the 4 transcription factors which can be separated by fluorescence microscopy and flow Oct-4, Sox-2, Klf-4, and c-Myc (1–3). This combination of factors cytometry, thus enabling parallel monitoring of each reprogram- emerged from an initial screen in mouse fibroblasts based on ming factor expression in real time (Fig. 1B and see below). co-transduction of 24 candidate genes (4). The generation of iPSCs Immunoblots of cells transduced with these vectors revealed correct with this method has now been reported from mouse and human processing of the 2A-linked gene products (Fig. S1 A–D). All 4 ϫ 6 somatic cells using various types of vectors, including gamma- vectors were found to yield similar titers of 1.5–2 10 TU/mL. By retroviral, constitutive, or doxycycline (DOX)-inducible lentiviral co-transduction of human fetal fibroblasts (MRC-5) with these 4 and adenoviral vectors, as well as plasmid and transposon/ vectors hiPSC lines were generated that expressed pluripotency transposase transfection systems (1, 2, 5–12). Nonetheless, the markers (Fig. S2A) and could be directed to differentiate into stoichiometric and temporal requirements of factor expression derivatives of all 3 germ layers (Fig. S2B). Microarray-based global during hiPSC induction, maintenance, and differentiation remain gene expression analysis revealed gene expression patterns highly poorly defined. similar to hESCs, but not to the parental human fibroblasts (Fig. Direct reprogramming is a slow and inefficient process, with S2C and Table S1). Furthermore, these hiPSC lines induced estimated efficiencies in human cells ranging from 0.02% to 0.002% teratomas when s.c. injected into immunocompromised mice (Fig. (1, 2, 5). Many on-going efforts aim to identify genetic or chemical 1C). factors that enhance iPSC generation (13–16). A problem faced by these investigations is the lack of a consistent way of reporting Requirements of Factor Expression for Efficient Reprogramming. We reprogramming efficiency, since effects on factor delivery cannot be first took advantage of this traceable vector system to estimate what separated from genuine effects on reprogramming efficiency. Fur- fraction of human fibroblasts co-expressing all 4 factors successfully reprogram (Fig. S2D). We co-transduced human fetal fibroblasts thermore, the lack of proper assessment of factor delivery and CELL BIOLOGY expression obscures the comparison of reprogramming frequencies (MRC-5) with the 4 vectors at equal multiplicity of infection (MOI) across different studies. The low efficiency of direct reprogramming may, at least in part, be accounted for by the requirement for a Author contributions: E.P.P., M.J.T., L.S., and M.S. designed research; E.P.P., M.J.T., S.M.C., stringent stoichiometry of reprogramming factor expression per- Y.M., E.R., J.M., and V.T. performed research; E.P.P., Q.M., and M.S. analyzed data; and missive for successful reprogramming. E.P.P. and M.S. wrote the paper. Several studies have reported silencing of the vector-encoded The authors declare no conflict of interest. reprogramming factors in iPSCs generated with gamma-retroviral 1To whom correspondence should be addressed. E-mail: [email protected]. vectors (1, 17, 18). The silencing of lentiviral vectors in iPSCs is less This article contains supporting information online at www.pnas.org/cgi/content/full/ known (2, 19), and therefore their suitability for reprogramming 0904825106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904825106 PNAS ͉ August 4, 2009 ͉ vol. 106 ͉ no. 31 ͉ 12759–12764 Downloaded by guest on September 30, 2021 Fig. 1. hiPSCs derived from bicistronic vectors co-expressing each RF linked to a fluorescent protein. (A) Schematic representation of the vectors used in this study. LTR, long terminal repeat. (B) hiPSC colony at day 11 after transduction. (C) Hematoxylin and eosin staining of histological sections of a teratoma derived from line iPS-27. From Left to Right: Low power image demonstrating areas of heterogeneous differentiation: neuroectoderm (black arrow), smooth muscle (blank arrow), and primitive myxoid tissue (arrowhead), 4ϫ. Pigmented epithelial tissue compatible with retinal neuroectoderm, 40ϫ. Intestinal like epithelium including goblet cells (endoderm), 40ϫ. Smooth muscle tissue, 20ϫ. Inset demonstrates a high power image of immature mesenchymal tissue, potentially cartilage (40ϫ). and determined the percentage of quadruple transduced cells by the 4 factors. In contrast, changes in the relative ratios of expression flow cytometry 5 days after transduction. To calculate the fre- mediated significant effects on the efficiency of reprogramming. quency of fully reprogrammed cells, we performed immunostaining Increasing relative Oct4 expression resulted in enhanced repro- for Tra-1–81, as described in the Methods, and macroscopically gramming efficiency, whereas increasing the relative ratio of either enumerated positive colonies on day 20 after transduction. Thus, Sox2, Klf4, or c-Myc consistently decreased efficiency of hiPSC through direct quantification of quadruple transduced cells coupled colony generation by more than 5-fold (Fig. 2C Upper). In contrast, with a clonal readout of hiPSC derivation, we were able to accu- relative decrease of Sox2, Klf4 or c-Myc showed little effect, while rately estimate the reprogramming frequency to be 0.4–1% of de relative decrease of Oct4 was detrimental (Fig. 2C Lower). These novo quadruple transduced fetal human fibroblasts (Table S2). data demonstrate that a stoichiometry of equal parts of all 4 vectors We then sought to investigate the effect of the absolute levels of is highly effective, resulting in reprogramming efficiency of de novo expression of the 4 vector-encoded RFs on efficiency of direct quadruple transduced fibroblasts similar to that observed in the reprogramming. Transduction of human fibroblasts at increasing maximally efficient secondary system (5, 7). Most deviations from MOI results in concomitant increase in the percentage of quadru- this 1:1:1:1 stoichiometry
Load more

Recommended publications
  • AIRE Is a Critical Spindle-Associated Protein in Embryonic Stem Cells Bin Gu1, Jean-Philippe Lambert2, Katie Cockburn1, Anne-Claude Gingras2,3, Janet Rossant1,3*
    RESEARCH ARTICLE AIRE is a critical spindle-associated protein in embryonic stem cells Bin Gu1, Jean-Philippe Lambert2, Katie Cockburn1, Anne-Claude Gingras2,3, Janet Rossant1,3* 1Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada; 2Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital, Toronto, Canada; 3Department of Molecular Genetics, University of Toronto, Toronto, Canada Abstract Embryonic stem (ES) cells go though embryo-like cell cycles regulated by specialized molecular mechanisms. However, it is not known whether there are ES cell-specific mechanisms regulating mitotic fidelity. Here we showed that Autoimmune Regulator (Aire), a transcription coordinator involved in immune tolerance processes, is a critical spindle-associated protein in mouse ES(mES) cells. BioID analysis showed that AIRE associates with spindle-associated proteins in mES cells. Loss of function analysis revealed that Aire was important for centrosome number regulation and spindle pole integrity specifically in mES cells. We also identified the c-terminal LESLL motif as a critical motif for AIRE’s mitotic function. Combined maternal and zygotic knockout further revealed Aire’s critical functions for spindle assembly in preimplantation embryos. These results uncovered a previously unappreciated function for Aire and provide new insights into the biology of stem cell proliferation and potential new angles to understand fertility defects in humans carrying Aire mutations. DOI: 10.7554/eLife.28131.001 *For correspondence: janet. [email protected] Introduction Competing interests: The Self-renewal capability, defined as the ability of cells to proliferate while sustaining differentiation authors declare that no potential, is one of the defining features of stem cells (Martello and Smith, 2014).
    [Show full text]
  • The GATA2 Transcription Factor Negatively Regulates the Proliferation of Neuronal Progenitors
    RESEARCH ARTICLE 2155 Development 133, 2155-2165 (2006) doi:10.1242/dev.02377 The GATA2 transcription factor negatively regulates the proliferation of neuronal progenitors Abeer El Wakil*, Cédric Francius*,†, Annie Wolff, Jocelyne Pleau-Varet† and Jeannette Nardelli†,§ Postmitotic neurons are produced from a pool of cycling progenitors in an orderly fashion that requires proper spatial and temporal coordination of proliferation, fate determination, differentiation and morphogenesis. This probably relies on complex interplay between mechanisms that control cell cycle, specification and differentiation. In this respect, we have studied the possible implication of GATA2, a transcription factor that is involved in several neuronal specification pathways, in the control of the proliferation of neural progenitors in the embryonic spinal cord. Using gain- and loss-of-function manipulations, we have shown that Gata2 can drive neural progenitors out of the cycle and, to some extent, into differentiation. This correlates with the control of cyclin D1 transcription and of the expression of the p27/Kip1 protein. Interestingly, this functional aspect is not only associated with silencing of the Notch pathway but also appears to be independent of proneural function. Consistently, GATA2 also controls the proliferation capacity of mouse embryonic neuroepithelial cells in culture. Indeed, Gata2 inactivation enhances the proliferation rate in these cells. By contrast, GATA2 overexpression is sufficient to force such cells and neuroblastoma cells to stop dividing but not to drive either type of cell into differentiation. Furthermore, a non-cell autonomous effect of Gata2 expression was observed in vivo as well as in vitro. Hence, our data have provided evidence for the ability of Gata2 to inhibit the proliferation of neural progenitors, and they further suggest that, in this regard, Gata2 can operate independently of neuronal differentiation.
    [Show full text]
  • Long Noncoding RNA Lncpgcr Mediated by TCF7L2 Regulates Primordial Germ Cell Formation in Chickens
    animals Article Long Noncoding RNA LncPGCR Mediated by TCF7L2 Regulates Primordial Germ Cell Formation in Chickens Jingyi Jiang 1, Chen Chen 1, Shaoze Cheng 1, Xia Yuan 1, Jing Jin 1, Chen Zhang 1, Xiaolin Sun 1 , Jiuzhou Song 2 , Qisheng Zuo 1, Yani Zhang 1, Guohong Chen 1 and Bichun Li 1,* 1 Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu, China; [email protected] (J.J.); [email protected] (C.C.); [email protected] (S.C.); [email protected] (X.Y.); [email protected] (J.J.); [email protected] (C.Z.); [email protected] (X.S.); [email protected] (Q.Z.); [email protected] (Y.Z.); [email protected] (G.C.) 2 Animal & Avian Sciences, University of Maryland, College Park, MD 20741, USA; [email protected] * Correspondence: [email protected] Simple Summary: The potential of primordial germ cells (PGCs) for multidirectional differentiation, together with their unique regeneration ability, makes them one of the most promising seed cells in clinical medicine and tissue engineering research. However, not enough PGCs can be obtained to meet the demand, which limits their application. We defined a novel long noncoding RNA (lncRNA) mediated by epigenetics, which could activate the miR-6577-5p/Btrc pathway to promote the formation of PGCs. The technical system we have established is a useful tool to obtain sufficient PGCs for scientific research. Our study offers great theoretical and practical value in the production of transgenic animals or genomic imprinting in poultry.
    [Show full text]
  • NIH Public Access Author Manuscript Curr Opin Immunol
    NIH Public Access Author Manuscript Curr Opin Immunol. Author manuscript; available in PMC 2012 April 1. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Curr Opin Immunol. 2011 April ; 23(2): 198±206. doi:10.1016/j.coi.2010.11.007. Aire and T cell Development Mark S. Andersona,* and Maureen A. Sub,* a Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, CA b Inflammatory Diseases Institute and Department of Pediatrics, University of North Carolina, Chapel Hill, Chapel Hill, NC Abstract In the thymus, developing T cells that react against self-antigens with high affinity are deleted in the process of negative selection. An essential component of this process is the display of self- antigens, including those whose expression are usually restricted to specific tissues, to developing T cells within the thymus. The Autoimmune Regulator (Aire) gene plays a critical role in the expression of tissue specific self-antigens within the thymus, and disruption of Aire function results in spontaneous autoimmunity in both humans and mice. Recent advances have been made in our understanding of how Aire influences the expression of thousands of tissue-specific antigens in the thymus. Additional roles of Aire, including roles in chemokine and cytokine expression, have also been revealed. Factors important in the differentiation of Aire-expressing medullary thymic epithelial cells have been defined. Finally, the identity of antigen presenting cells in negative selection, including the role of medullary thymic epithelial cells in displaying tissue specific antigens to T cells, has also been clarified.
    [Show full text]
  • Crosstalk Between SOX2 and TGF-Β Signaling Regulates EGFR-TKI Tolerance and Lung Cancer Dissemination
    Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/0008-5472.CAN-19-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Crosstalk between SOX2 and TGF-β signaling regulates EGFR-TKI tolerance and lung cancer dissemination Ming-Han Kuo1,10, An-Chun Lee1,10, Shih-Hsin Hsiao2,10, Sey-En Lin3,4, Yu-Fan Chiu1, Li-Hao Yang1, Chia-Cherng Yu5, Shih-Hwa Chiou6,7, Hsien-Neng Huang8, Jen-Chung Ko9*, Yu-Ting Chou1* 1Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan; 2Division of Pulmonary Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan; 3Department of Pathology, Taipei Medical University Hospital, Taipei, Taiwan; 4Department of Pathology, Taipei Municipal Wan Fang Hospital, Taipei, Taiwan; 5Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan; 6Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan; 7Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan; 8Department of pathology, National Taiwan University Hospital, Hsin-Chu Branch, Hsinchu, Taiwan; 9Department of Internal Medicine, National Taiwan University Hospital, Hsin-Chu Branch, Hsinchu, Taiwan;10Co-first authors *Corresponding authors Jen-Chung Ko, National Taiwan University Hospital, Hsin-Chu Branch, No. 25, Lane 442, Sec. 1, Jingguo Rd., Hsinchu 30013, Taiwan. E-mail: [email protected] Yu-Ting Chou, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan. E-mail: [email protected] Running title: Interplay of SOX2 and TGF- on EGFR–TKI tolerance Conflicts of interest The authors disclose no potential conflicts of interest. This work was supported by National Tsing Hua University and Ministry of Science and Technology, Executive Yuan, Taiwan.
    [Show full text]
  • A Sox2 Distal Enhancer Cluster Regulates Embryonic Stem Cell Differentiation Potential
    Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential Harry Y. Zhou,1,3 Yulia Katsman,1,3 Navroop K. Dhaliwal,1 Scott Davidson,1 Neil N. Macpherson,1 Moorthy Sakthidevi,1 Felicia Collura,1 and Jennifer A. Mitchell1,2 1Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada; 2Center for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Ontario M5S 3G5, Canada The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers—SRR18, SRR107, and SRR111—that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/ Cas9 system and F1 ES cells (Mus musculus129 3 Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies.
    [Show full text]
  • O-Glcnac Modification of Oncogenic Transcription Factor Sox2 Promotes Protein Stability and Regulates Self-Renewal in Pancreatic Cancer
    bioRxiv preprint doi: https://doi.org/10.1101/345223; this version posted June 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. O-GlcNAc modification of oncogenic transcription factor Sox2 promotes protein stability and regulates self-renewal in pancreatic cancer Nikita S Sharma1, Vineet K Gupta1, Patricia Dauer2, Kousik Kesh1, Roey Hadad1, Bhuwan Giri1, Anjali Chandra5, Vikas Dudeja1,4, Chad Slawson3, Santanu Banerjee1,4, Selwyn M Vickers6, Ashok Saluja1, 4 and Sulagna Banerjee1, 4* 1Department of Surgery, University of Miami, Miami, FL. 2Department of Pharmacology, University of Minnesota, Minneapolis Minnesota. 3University of Kansas Medical Center, Kansas City, KS. 4Sylvester Comprehensive Cancer Center, Miami, FL. 5 Department of Psychology, Harvard University. 6 School of Medicine Dean’s Office, University of Alabama at Birmingham. * Corresponding Author Address of Correspondence: 1550 NW 10th Ave Papanicolau Building, Suite 109B Miami, FL 33136 [email protected] (305)-243-8242 Running Title: O-GlcNAc modified Sox2 mediates self-renewal in PDAC Keywords: OGT, O-GlcNAc, Sox2, Pancreatic cancer, metabolism, self-renewal Financial Support This study was funded by NIH grants R01-CA170946 and R01-CA124723 (to AKS); NIH grant R01-CA184274 (to SB); Katherine and Robert Goodale foundation support (to AKS), Minneamrita Therapeutics LLC (to AKS). The authors would also like to acknowledge Oncogenomics Core at the Sylvester Cancer Center, University of Miami for their help with the RNA seq transcriptomics study. bioRxiv preprint doi: https://doi.org/10.1101/345223; this version posted June 12, 2018.
    [Show full text]
  • In Thymic Epithelial Differentiation Aire
    The Journal of Immunology Aire-Dependent Alterations in Medullary Thymic Epithelium Indicate a Role for Aire in Thymic Epithelial Differentiation1 Geoffrey O. Gillard,2* James Dooley,† Matthew Erickson,† Leena Peltonen,§ and Andrew G. Farr3*†‡ The prevalent view of thymic epithelial differentiation and Aire activity holds that Aire functions in terminally differentiated medullary thymic epithelial cells (MTECs) to derepress the expression of structural tissue-restricted Ags, including pancreatic endocrine hormones. An alternative view of these processes has proposed that Aire functions to regulate the differentiation of immature thymic epithelial cells, thereby affecting tissue-restricted Ag expression and negative selection. In this study, we dem- onstrate that Aire impacts several aspects of murine MTECs and provide support for this second model. Expression of transcrip- tion factors associated with developmental plasticity of progenitor cells, Nanog, Oct4, and Sox2, by MTECs was Aire dependent. Similarly, the transcription factors that regulate pancreatic development and the expression of pancreatic hormones are also expressed by wild-type MTECs in an Aire-dependent manner. The altered transcriptional profiles in Aire-deficient MTECs were accompanied by changes in the organization and composition of the medullary epithelial compartment, including a reduction in the medullary compartment defined by keratin (K) 14 expression, altered patterns of K5 and K8 expression, and more prominent epithelial cysts. These findings implicate Aire in the
    [Show full text]
  • GATA2 Regulates Mast Cell Identity and Responsiveness to Antigenic Stimulation by Promoting Chromatin Remodeling at Super- Enhancers
    ARTICLE https://doi.org/10.1038/s41467-020-20766-0 OPEN GATA2 regulates mast cell identity and responsiveness to antigenic stimulation by promoting chromatin remodeling at super- enhancers Yapeng Li1, Junfeng Gao 1, Mohammad Kamran1, Laura Harmacek2, Thomas Danhorn 2, Sonia M. Leach1,2, ✉ Brian P. O’Connor2, James R. Hagman 1,3 & Hua Huang 1,3 1234567890():,; Mast cells are critical effectors of allergic inflammation and protection against parasitic infections. We previously demonstrated that transcription factors GATA2 and MITF are the mast cell lineage-determining factors. However, it is unclear whether these lineage- determining factors regulate chromatin accessibility at mast cell enhancer regions. In this study, we demonstrate that GATA2 promotes chromatin accessibility at the super-enhancers of mast cell identity genes and primes both typical and super-enhancers at genes that respond to antigenic stimulation. We find that the number and densities of GATA2- but not MITF-bound sites at the super-enhancers are several folds higher than that at the typical enhancers. Our studies reveal that GATA2 promotes robust gene transcription to maintain mast cell identity and respond to antigenic stimulation by binding to super-enhancer regions with dense GATA2 binding sites available at key mast cell genes. 1 Department of Immunology and Genomic Medicine, National Jewish Health, Denver, CO 80206, USA. 2 Center for Genes, Environment and Health, National Jewish Health, Denver, CO 80206, USA. 3 Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, ✉ CO 80045, USA. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:494 | https://doi.org/10.1038/s41467-020-20766-0 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-20766-0 ast cells (MCs) are critical effectors in immunity that at key MC genes.
    [Show full text]
  • FOS Rescues Neuronal Differentiation of Sox2-Deleted Neural Stem Cells by Genome-Wide Regulation of Common SOX2 and AP1(FOS-JUN) Target Genes
    cells Article FOS Rescues Neuronal Differentiation of Sox2-Deleted Neural Stem Cells by Genome-Wide Regulation of Common SOX2 and AP1(FOS-JUN) Target Genes Miriam Pagin 1,† , Mattias Pernebrink 2,3,†, Mattia Pitasi 1, Federica Malighetti 1, Chew-Yee Ngan 4, Sergio Ottolenghi 1, Giulio Pavesi 5, Claudio Cantù 2,3,* and Silvia K. Nicolis 1,* 1 Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy; [email protected] (M.P.); [email protected] (M.P.); [email protected] (F.M.); [email protected] (S.O.) 2 Wallenberg Centre for Molecular Medicine, Linköping University, SE-581 83 Linköping, Sweden; [email protected] 3 Department of Biomedical and Clinical Sciences, Division of Molecular Medicine and Virology, Faculty of Medicine and Health Sciences, Linköping University, SE-581 83 Linköping, Sweden 4 The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA; [email protected] 5 Department of Biosciences, University of Milano, Via Celoria 26, 20134 Milano, Italy; [email protected] * Correspondence: [email protected] (C.C.); [email protected] (S.K.N.) † Joint first authors. Abstract: The transcription factor SOX2 is important for brain development and for neural stem Citation: Pagin, M.; Pernebrink, M.; cells (NSC) maintenance. Sox2-deleted (Sox2-del) NSC from neonatal mouse brain are lost after Pitasi, M.; Malighetti, F.; Ngan, C.-Y.; few passages in culture. Two highly expressed genes, Fos and Socs3, are strongly downregulated in Ottolenghi, S.; Pavesi, G.; Cantù, C.; Sox2-del NSC; we previously showed that Fos or Socs3 overexpression by lentiviral transduction fully Nicolis, S.K.
    [Show full text]
  • Sox2 Is Essential for Oligodendroglial Proliferation and Differentiation During Postnatal Brain Myelination and CNS Remyelination
    1802 • The Journal of Neuroscience, February 14, 2018 • 38(7):1802–1820 Development/Plasticity/Repair Sox2 Is Essential for Oligodendroglial Proliferation and Differentiation during Postnatal Brain Myelination and CNS Remyelination X Sheng Zhang,1,2* Xiaoqing Zhu,1,2,3* Xuehong Gui,1 Christopher Croteau,1 Lanying Song,1,2 Jie Xu,1 XAijun Wang,1,4 Peter Bannerman,1,5 and XFuzheng Guo1,2 1Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children, Northern California, California 95817, 2Department of Neurology, 3Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China, 4Department of Surgery, and 5Department of Cell Biology and Human Anatomy, School of Medicine, University of California–Davis, California 95817 In the CNS, myelination and remyelination depend on the successful progression and maturation of oligodendroglial lineage cells, including proliferation and differentiation of oligodendroglial progenitor cells (OPCs). Previous studies have reported that Sox2 transiently regulates oligodendrocyte (OL) differentiation in the embryonic and perinatal spinal cord and appears dispensable for myelination in the postnatal spinal cord. However, the role of Sox2 in OL development in the brain has yet to be defined. We now report that Sox2 is an essential positive regulator of developmental myelination in the postnatal murine brain of both sexes. Stage-specific paradigms of genetic disruption demonstrated that Sox2 regulated brain myelination by coordinating upstream OPC population supply and downstream OL differentiation. Transcriptomic anal- yses further supported a crucial role of Sox2 in brain developmental myelination. Consistently, oligodendroglial Sox2-deficient mice developed severe tremors and ataxia, typical phenotypes indicative of hypomyelination, and displayed severe impairment of motor function and promi- nent deficits of brain OL differentiation and myelination persisting into the later CNS developmental stages.
    [Show full text]
  • 2244.Full.Pdf
    Cutting Edge: The Transcription Factor Sox2 Regulates AID Expression in Class-Switched B Cells This information is current as Lauren J. DiMenna, Wei-Feng Yen, Laura Nicolas, Rahul of September 25, 2021. Sharma, Zara N. Saldanha and Jayanta Chaudhuri J Immunol 2017; 198:2244-2248; Prepublished online 10 February 2017; doi: 10.4049/jimmunol.1502266 http://www.jimmunol.org/content/198/6/2244 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/02/09/jimmunol.150226 Material 6.DCSupplemental http://www.jimmunol.org/ References This article cites 35 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/198/6/2244.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology Cutting Edge: The Transcription Factor Sox2 Regulates AID Expression in Class-Switched B Cells Lauren J.
    [Show full text]