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ISWI Atpase Smarca5 Regulates Differentiation of Thymocytes Undergoing Β -Selection

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ISWI Atpase Smarca5 Regulates Differentiation of Thymocytes Undergoing Β -Selection ISWI ATPase Smarca5 Regulates Differentiation of Thymocytes Undergoing β -Selection This information is current as Tomas Zikmund, Juraj Kokavec, Tereza Turkova, Filipp of September 25, 2021. Savvulidi, Helena Paszekova, Sona Vodenkova, Radislav Sedlacek, Arthur I. Skoultchi and Tomas Stopka J Immunol published online 8 May 2019 http://www.jimmunol.org/content/early/2019/05/07/jimmun ol.1801684 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/05/07/jimmunol.180168 Material 4.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 8, 2019, doi:10.4049/jimmunol.1801684 The Journal of Immunology ISWI ATPase Smarca5 Regulates Differentiation of Thymocytes Undergoing b-Selection Tomas Zikmund,* Juraj Kokavec,* Tereza Turkova,* Filipp Savvulidi,† Helena Paszekova,* Sona Vodenkova,‡,x Radislav Sedlacek,{ Arthur I. Skoultchi,‖ and Tomas Stopka* Development of lymphoid progenitors requires a coordinated regulation of gene expression, DNA replication, and gene rearrange- ment. Chromatin-remodeling activities directed by SWI/SNF2 superfamily complexes play important roles in these processes. In this study, we used a conditional knockout mouse model to investigate the role of Smarca5, a member of the ISWI subfamily of such complexes, in early lymphocyte development. Smarca5 deficiency results in a developmental block at the DN3 stage of ab thymocytes and pro-B stage of early B cells at which the rearrangement of Ag receptor loci occurs. It also disturbs the devel- opment of committed (CD73+) gd thymocytes. The ab thymocyte block is accompanied by massive apoptotic depletion of b-selected double-negative DN3 cells and premitotic arrest of CD4/CD8 double-positive cells. Although Smarca5-deficient ab Downloaded from T cell precursors that survived apoptosis were able to undergo a successful TCRb rearrangement, they exhibited a highly abnormal mRNA profile, including the persistent expression of CD44 and CD25 markers characteristic of immature cells. We also observed that the p53 pathway became activated in these cells and that a deficiency of p53 partially rescued the defect in thymus cellularity (in contrast to early B cells) of Smarca5-deficient mice. However, the activation of p53 was not primarily responsible for the thymocyte developmental defects observed in the Smarca5 mutants. Our results indicate that Smarca5 plays a key role in the development of thymocytes undergoing b-selection, gd thymocytes, and also B cell progenitors by regulating the http://www.jimmunol.org/ transcription of early differentiation programs. The Journal of Immunology, 2019, 202: 000–000. he production of mature T and B cells is a multistep replication, DNA rearrangement, and repair. Progenitors of T cells process of differentiation from a multipotent progenitor migrate from the bone marrow (BM) into the thymus where they T that requires a coordinated regulation of gene expression, respond to a new environment by initiating a transcriptional pro- gram of T cell specification while proliferating extensively (1). During this process, CD42CD82 double-negative (DN) CD44+ *BIOCEV, First Faculty of Medicine, Charles University, Vestec 25250, Czech Repub- positive early T lineage precursors (immature DN1) permanently by guest on September 25, 2021 lic; †Institute of Pathological Physiology, First Faculty of Medicine, Charles University, ‡ silence the group of progenitor-related regulatory genes leading to Prague 12853, Czech Republic; Institute of Experimental Medicine, Czech Academy of Sciences, Prague 14220, Czech Republic; xThird Faculty of Medicine, Charles the gradual upregulation of CD25 and the downregulation of c-Kit { University, Prague 10000, Czech Republic; Czech Centre for Phenogenomics, In- surface markers and resulting in the commitment completion at stitute of Molecular Genetics, Czech Academy of Sciences, Vestec 25250, Czech + + int Republic; and ‖Department of Cell Biology, Albert Einstein College of Medicine, the end of the DN2 stage (CD44 CD25 c-Kit ) (2). Thymocytes 2 + Bronx 10461, NY at the subsequent DN3 stage (CD44 CD25 ) cease from cycling ORCIDs: 0000-0003-2452-3749 (T.Z.); 0000-0003-0267-4570 (J.K.); 0000-0002- and, importantly, undergo a random rearrangement of gene 5329-1812 (T.T.); 0000-0002-6479-6592 (H.P.); 0000-0003-0315-5668 (S.V.); segments at the TCRb locus and commence the expression of 0000-0001-7236-6894 (T.S.). components related to the b-selection program. Upon the suc- Received for publication January 2, 2019. Accepted for publication April 15, 2019. cessful rearrangement that yields functional pre-TCR complexes, The Stopka Laboratory at the BIOCEV was supported by Charles University thymocytes proliferate rapidly, become rescued from the p53- Grant Agency GAUK 534212, Czech Science Foundation GACR 18-01687S and 19-03586S, Czech Health Research Council AZV 16-27790A, Charles University regulated cell cycle arrest and apoptosis (3), and then are 2 2 UNCE/MED/016, KONTAKT LH15170, Czech Ministry of Education, Youth and allowed to progress into the DN4 stage (CD44 CD25 ). This Sport (MEYS) LM2015040 and NPU II LQ1604, Operational Programmes of MEYS transient population hence upregulates the expression of CD4 OP RDI CZ.1.05/2.1.00/19.0395 and CZ.1.05/1.1.00/02.0109, and institutional programs funded by Charles University PROGRES Q26 and SVV 260374/2017. S.V. and CD8 to become double-positive (DP) cells and initiates support: GACR 19-10543S and AZV 17-30920A. A.I.S. support: National Institutes TCRa locus rearrangement. DP cells with productive TCRab are of Health GM116143 and DK096266. positively and negatively selected so that only those with The sequences presented in this article have been submitted to ArrayExpress (https:// “proven” TCR can undergo differentiation into CD4 or CD8 www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-7758/) under accession number E-MTAB-7758. single-positive (SP) cells (4). Address correspondence and reprint requests to Dr. Tomas Stopka, BIOCEV, First Eukaryotic cells evolved numerous epigenetic regulatory Faculty of Medicine of the Charles University, Prumyslova 595, Vestec 25250, Czech mechanisms of gene expression, DNA replication, and repair to Republic. E-mail address: [email protected] accomplish the T cell development. During early T cell differ- The online version of this article contains supplemental material. entiation, NURD and SWI/SNF chromatin-remodeling complexes Abbreviations used in this article: BM, bone marrow; DN, double-negative; DP, were shown to play important roles in both activating as well double-positive; DSB, double-strand break; FDR, false discovery rate; GSEA, gene as silencing the gene transcription (5, 6). The SWI/SNF-related set enrichment analysis; iCre, codon-improved Cre recombinase; log2FC, log2 fold change value; LSK, Lin2Sca1+Kit+; RNA-seq, RNA sequencing; Smarca5, matrix-associated actin-dependent regulator of chromatin sub- SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily family A member 5 (Smarca5) represents a widely expressed and A member 5; SP, single-positive; wt, wild-type. conserved chromatin-remodeling factor required for the early Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 development in mouse and lower organisms (7). Smarca5 is an www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801684 2 Smarca5 REQUIREMENT FOR EARLY LYMPHOCYTE DEVELOPMENT ATPase from the ISWI subfamily that functions as a molecular primary Abs. Biotinylated primary Abs were revealed with streptavidin- motor for nuclear complexes that assemble and slide basic conjugated fluorescent dyes (SAv-PE/Cy7, SAv-APC, and SAv-APC/Cy7). chromatin subunits, nucleosomes. Smarca5-containing com- Labeled cells were analyzed on BD FACSCanto II (BD Biosciences) or CytoFLEX (Beckman Coulter) flow cytometers, and data analysis was plexes have diverse nuclear functions: guiding the transcription performed using FlowJo software. The clones of mAbs were as follows: of ribosomal (in NoRC and B-WICH complexes) and some anti-CCR6 (29-2L17), anti-CD3ε (145-2C11), anti-CD4 (GK1.5), anti- coding genes (within the ACF or RSF complexes), participating CD5 (53-7.3), anti-CD8 (53-6.7), anti-CD11c (N418), anti-CD24 in regularly spacing the nucleosomal array before and after DNA (M1/69), anti-CD25 (PC61), anti-CD27 (LG.3A10), anti-CD28 (E18), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD71 replication, facilitating the recruitment of DNA repair machinery (RI7217), anti-CD73 (TY/11.8), anti-CD117 (c-Kit, 2B8), anti-B220 (CHRAC and WICH complexes), and finally, orchestrating (RA3-6B2), anti-CD11b (Mac1, M1/70), anti-gd T cell (GL3), hamster
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