ISWI ATPase Smarca5 Regulates Differentiation of Thymocytes Undergoing β -Selection

This information is current as Tomas Zikmund, Juraj Kokavec, Tereza Turkova, Filipp of September 28, 2021. Savvulidi, Helena Paszekova, Sona Vodenkova, Radislav Sedlacek, Arthur I. Skoultchi and Tomas Stopka J Immunol 2019; 202:3434-3446; Prepublished online 8 May 2019;

doi: 10.4049/jimmunol.1801684 Downloaded from http://www.jimmunol.org/content/202/12/3434

Supplementary http://www.jimmunol.org/content/suppl/2019/05/07/jimmunol.180168 Material 4.DCSupplemental http://www.jimmunol.org/ References This article cites 60 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/202/12/3434.full#ref-list-1

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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. 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 expression, DNA replication, and gene rearrange- ment. -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 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: 3434–3446.

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 , 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 28, 2021 lic; †Institute of Pathological Physiology, First Faculty of Medicine, Charles University, ‡ silence the group of progenitor-related regulatory 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 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 allowedtoprogressintotheDN4stage(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 The Journal of Immunology 3435

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 higher-order chromatin structure formation of centromeres and IgG-PE/Cy7 (HTK888), anti-Ly6G/Ly6C (GR1, RB6-8C5), anti-Nk1.1 (RSF) (8). Although several members of SWI/SNF (PK136), anti-TCRb (H57-597), anti-TCRb(Va2) (B20.1), and Ter- and CHD family have had their roles established in T cell de- 119 (all from BioLegend). Splenocytes were analyzed as previously described (18). velopment through studies involving gene inactivation mouse models, such a role for the ISWI subfamily has not been deter- BM transplantation mined yet. For BM reconstitution experiments, 1 3 107 BM cells from adult (8-wk- Currently, there is only a limited knowledge of how Smarca5, old) control C57BL/6J Ly5.1 (CD45.1) mice and S5fl/flhCD2iCre Ly5.2 whichishighlyexpressedinlymphocytes (9), participates in (CD45.2) donors were reciprocally transplanted into lethally irradiated fl/fl lymphopoiesis. We previously showed that deletion of the (7.5Gy) adult (8 wk) S5 hCD2iCre Ly5.2 and C57BL/6J Ly5.1 control recipients, respectively. After 1 mo posttransplantation, thymi were tested Smarca5 gene resulted not only in the depletion of myeloer- for the presence of donor-derived cells using flow cytometric analysis. The Downloaded from ythroid precursors but also affected the earliest development of Ab panel included CD45.1, CD45.2, CD44, CD25, CD4, lineage mixture lymphoid progenitors in the mouse fetal liver (10). Additionally, (CD8, B220, Mac-1, Gr-1, Nk1.1, CD11c, and Ter119) and CD45.1, Smarca5 was implicated in the V(D)J cleavage of the poly- CD45.2, CD4, CD5, CD8, and lineage mixture (B220, Mac-1, Gr-1, Nk1.1, nucleosomal substrate in a cell-free system (11). Another re- CD11c, and Ter119) for thymus. port implicated that Smarca5 in the ACF complex represses the BrdU labeling

IL2-Ra gene (CD25) via chromatin organizer Satb1 (12). Lastly, http://www.jimmunol.org/ Smarca5 conditional knockout mice and their age- and gender-matched Smarca5 regulates the expression of key ILs (IL-2, IL-3, and IL-5) respective controls were i.p. injected with 1 mg of BrdU in 100 ml in murine EL4 T cell lymphoma (13). Although the role of PBS. After 3 h, the thymocytes were isolated and Ab-stained for flow Smarca5 in lymphopoiesis was previously suggested, the knock- cytometry analysis or cell sorting. BrdU Ag recovery and its detection by out models of Smarca5-interacting partners revealed no alterations fluorescently labeled Ab were performed using the APC BrdU Flow Kit in lymphoid development, including the deletion of Acf1/Baz1a (BD Biosciences). (ACF and CHRAC complexes) (14, 15) or Tip5/Baz2a (NoRC) OP9/N-DLL1 stromal cell cultures (15) genes in mice. Interestingly, it has been shown in vitro that 2 2 2 FACS-sorted DN3 thymocytes (small CD4 CD8 CD25+)andLin Sca1+Kit+ Smarca5 can also remodel nucleosomes alone without being part (LSK) BM cells were cocultured with OP9 stromal cells expressing the of the complexes (16). As several of Smarca5-interacting part- Delta-like ligand (OP9/N-DLL1) in the presence of 1 ng/ml mIL-7 (407- by guest on September 28, 2021 ners are dispensable, studying the requirement of a catalytic ML-005) and 5 ng/ml hrFLT3 ligand (308-FK-005; PeproTech) as previ- subunit of the ISWI complexes by targeting experimentally ously described (19). In BM cell cocultures, the concentration of IL-7 was lowered to 0.1 ng/ml from day 12 to allow differentiation. For CFSE la- Smarca5 during lymphoid development may represent a suc- beling, 3 3 104 DN3 cells were stained with 2.5 mM CFSE following cessful strategy to reveal its function in lymphopoiesis. the manufacturer’s guidelines (CellTrace CFSE Cell Proliferation Kit; We, in this study, focused on deciphering the role of Smarca5 in Invitrogen) and plated onto OP9 stromal cultures. Their proliferation and thymocyte development and studied the molecular consequences survival were analyzed by flow cytometry at 2, 4, 6, and 8 d of cocultures. of conditional Smarca5 deficiency in mice. Our work suggests OP9/N-DLL1 cells were kindly provided by Hiroshi Kawamoto (Kyoto University). that Smarca5 controls early T cell development by guiding early differentiation-coupled transcriptional programs at the DN3 stage Histopathology and the detection of apoptotic cells in and its deficiency results in thymocyte proliferation and survival thymic sections defects through the activation of the DNA damage response. Thymi and spleens were fixed in 4% buffered formaldehyde for 48 h, transferred in 70% ethanol, and paraffin embedded. Sections were obtained Materials and Methods at 3 mm thickness and stained in H&E and Giemsa. Cleaved Caspase-3 was detected by immunohistochemistry using a 1:1000 dilution of Ab Mice (ab52293; Abcam) and visualized on a Zeiss Axio Scan.Z1. Casp3-positive Smarca5flox conditional knockout mice contain loxP1 sites flanking the cells were quantified using the Zen Blue Edition software (ZEISS). exon5oftheSmarca5 gene (17), deletion of which produced a frame- shift mutation (Smarca5del) that disrupts the expression of the Smarca5 RNA sequencing analysis . The murine strain expressing a codon-improved Cre recombinase CD4+ CD8+ (B2202 Gr-12 Nk1.12 CD11c2 CD11b2) thymocytes were (iCre) under hCD2 promoter [B6.Cg-Tg(CD2-cre)4Kio/J] was purchased sorted by FACS, and the total RNA was isolated using TRIzol Reagent from The Jackson Laboratory. The OT-II strain [B6.Cg-Tg(TcraTcrb) fl/fl (Invitrogen). DP cells pooled from 25 to 37 S5 hCD2iCre thymi (each 425Cbn/J] was kindly provided by Dr. T. Brdicka (Institute of Molecular 6 fl/fl 2/2 6 pool of 3 3 10 DP cells), 6 S5 hCD2iCre Trp53 thymi (∼2.5 3 10 Rag1 Genetics, Czech Academy of Sciences). knockout (B6.129S7- DP cells/pool), and single nonpooled control thymi provided sufficient Rag1tm1Mom/J) strain was kindly provided by Dr. T. Brabec (Institute amounts of RNA for RNA sequencing analysis. Strand-specific cDNA li- Trp53 of Molecular Genetics, Czech Academy of Sciences). knockout strain braries were prepared from a minimum of 1.7 mg of each DNase-treated (B6.129S2-Trp53tm1Tyj/J) was kindly provided by Dr. W. Edelmann (AM1906; Ambion DNA-Free Kit) RNA sample using the TruSeq (Albert Einstein College of Medicine). Stranded mRNA LT Kit (Illumina). The RNA libraries were sequenced on Flow cytometry an Illumina HiSeq 2500 instrument in Rapid Run mode with paired-end 100-bp sequencing length. Reads were mapped and aligned to mouse A single cell suspension from thymi and spleens of 4–6-wk-old mice was reference assembly GRCm38.p6, and transcripts were anno- obtained using a Dounce homogenizer. Cells were first preincubated for tated and counted with Ensembl Release 94 (October 2018) using a 10 min on ice with Fc receptor-blocking anti-CD16/32 (clone 93) Ab in HISAT2 aligner (20). The two RNA-seq technical replicates for each PBS-1% biotin-free BSA solution and then stained for 20 min with specific sample were combined. Differential expression analysis of RNA-seq 3436 Smarca5 REQUIREMENT FOR EARLY LYMPHOCYTE DEVELOPMENT data were performed in R Studio (21) using package DEseq2, which uses a (Fig. 1B). However, the deletion of the S5fl allele at DN1 was only median of ratios normalization method that accounts for sequencing depth partial, whereas its complete deletion was observed at the DN3 as well as RNA composition (22). Volcano plots were drawn using the stage. Western blot analysis of the whole thymic cellular extracts ggplot2 (23) package in R suite. Expression levels (transcripts per kb fl/fl million) for Ensembl 94 genes were calculated in R using gene lengths from S5 hCD2iCre mice confirmed a high efficiency of recom- retrieved by EDASeq package (24) and in-house scripts. The shrinkage of bination as we observed a marked reduction of Smarca5 protein log2 fold change values (log2FC) was estimated using DESeq2 lfcShrink levels (Fig. 1C). function using the adaptive t prior shrinkage estimator “apeglm” (25). The S5fl/flhCD2iCre pups were born healthy and at normal Men- RNA-seq data are publicly available at the ArrayExpress database under accession number E-MTAB-7758 (https://www.ebi.ac.uk/arrayexpress/ delian ratios. Following the third month of age, the Smarca5 experiments/E-MTAB-7758/). mutants, however, displayed rectal prolapses, suggesting the dis- turbance of immune functions. Gross dissection of the Smarca5 Quantitative PCR mutant animals revealed severe thymic defects. Thymi from Quantitative measurements of TCRb rearrangement were done on genomic the mutant mice were dramatically reduced in size and weight DNA (DNeasy; QIAGEN) isolated from the FACS-sorted DN subpopu- (Fig. 1D), which was reflected by a 17-fold reduction in cellularity lations. Quantitative PCR was run on 7900HT using Power SYBR Green (Fig. 1E). Histological evaluation revealed an alteration of thymic PCR Master Mix (no. 4367659, Applied Biosystems) with the following primers: Db1_fwd: 59-GTGGTTTCTTCCAGCCCTCAAG-39, Db1_rev: corticomedullary architecture (Fig. 1F) and a higher number of 59-GGCTTCCCATAGAATTGAATCACC-39 and Db2_fwd: 59-CAGGC- cleaved Caspase-3–positive apoptotic death events in the cortex TCTGGGGTAGGCAC-39, Db2_rev: 59-CCTCTTCCAGTTGAATCATT- (Fig. 1G). The quantification of multiple cortical sections from GTGG-39. Primers for the control region were as follows: Apob_ex29_fwd: different animals showed there was a 3-fold increase of apoptotic 59-CTGCCGTGGCCAAAATAAT-39 and Apob_ex29_rev: 59-AATCCT- cells in the cortex of S5fl/flhCD2iCre thymi. These observations Downloaded from GCAGATTGGAGTGG-39. Cycle threshold (ct) values of Db1 and Db2 regions were normalized to ct from Apob control region. indicated a developmental defect in thymocytes, accompanied by an increased cell death within the thymi of S5fl/flhCD2iCre Immunoblotting animals. Freshly isolated thymocytes were collected by centrifugation and resus- To gain insight into the developmental defects of thymocytes, pended in PBS with inhibitors of phosphatases (PhosSTOP; Roche) and we used flow cytometry of thymic cell suspensions of 4–6-wk- proteases (cOmplete ULTRA; Roche). The cell suspension was then diluted fl/fl old S5 hCD2iCre mice. CD4/CD8 immunostaining revealed a http://www.jimmunol.org/ 1:1 by adding a solution of 50 mM Tris-Cl (pH = 8) and 2% NaDodSO4 (SDS) and incubated for 30 min at 97˚C. Protein concentration was de- marked reduction of DP and CD4-SP cell populations with a termined by bicinchoninic acid assay (no. 23228; Pierce Biotechnology). corresponding relative increase in DN thymocytes in the mu- were then resolved on SDS gradient 4–15% polyacrylamide gels tant thymi (Fig. 2A). In absolute counts, the DP cells were (Mini-PROTEAN TGX Stain-Free Gels; Bio-Rad Laboratories) and wet depleted by 60-fold, CD4-SP 90-fold, and CD8-SP cells blotted (1 h at 100 V) onto PVDF membranes (no. 162-0177; Bio-Rad Laboratories). PVDF membranes were blocked for 1 h in 5% nonfat milk 12-fold, whereas the DN thymocyte population was similar to in TBS/0.1%Tween-20 (TBST) and incubated overnight at 4˚C in 3%BSA/ controls (Fig. 2B). Because the Smarca5 allele deletion is TBST 0.1% sodium azide with the following Abs: Smarca5 (1:1000, no. completed by the DN3 stage (Fig. 1B), we analyzed each A301-017A-1; Bethyl Laboratories) and histone H3 (1:1000, no. ab791; subpopulation of DN thymocytes to more precisely determine a 3 Abcam). Membranes were 3 5 min washed in TBST buffer and incu- stage in which the block of development occurs. CD44/CD25 by guest on September 28, 2021 bated with peroxidase-conjugated F(ab9)2 Ab fragment, either donkey anti- rabbit or donkey anti-mouse (1:10,000; Jackson ImmunoResearch), in 5% Ag expression profiles of lineage-negative DN thymocytes nonfat milk in TBST for 1 h. Membranes were 3 3 5 min washed in TBST, revealed a relative increase in DN3 cells to the detriment of and the protein signal was visualized by Pierce SuperSignal West Femto DN4 thymocytes (Fig. 2C). Translated into absolute counts, it Maximum Sensitivity Substrate (Thermo Fisher Scientific) and detected was an almost complete absence of DN4 thymocytes in mu- with the ChemiDoc Imaging System (Bio-Rad Laboratories). tants, whereas the DN3 population was present in comparable numbers to controls (Fig. 2D). Results We also examined the impact of Smarca5 deficiency on gd T cell Smarca5 deficiency disrupts thymocyte development development. Using flow cytometry, we observed that Smarca5- To delineate specific roles of Smarca5 in vivo, we previously deficient animals contain twice as many TCRd+ thymocytes created the Smarca5fl allele containing two loxP1 sites sur- (CD42CD82TCRd+) compared with controls (Fig. 2E, 2F). rounding exon5, the deletion of which results in removing the However, out of these TCRd+ thymocytes, only 18.4% (compared portion of evolutionarily conserved domain and intro- with 42% in controls) were able to adopt the gd fate (Fig. 2E) as ducing a frame-shift mutation (10, 17) (Fig. 1A). To execute indicated by the CD73 surface marker that discriminates TCRd+ the T cell–specific deletion of the Smarca5 gene, we crossed thymocytes committed to the gd lineage (29). Additionally, the Smarca5fl/fl mice with hCD2iCre transgenic mice, in which the expression of surface CD24, which is normally enriched on im- hCD2 promoter and locus control region drive the expression of mature gd thymocytes and downregulated upon maturation into the iCre recombinase (26). Use of the hCD2-iCre transgene to effector cells (30), was reduced in Smarca5 mutants (Fig. 2E). In study thymocyte development was chosen as this model alone contrast to controls, the lower expression of CD24 surface marker caused no defect in thymus development (27) as well as the cel- observed in the mutants compromised a clear separation of the lularity and subset composition of the major lymphoid organs CD73+ population into immature and mature subsets. The mature (including thymus, spleen, and lymph nodes) did not differ be- gd thymocytes (CD24lowCD73+) were further distinguished along tween hCD2-iCre–expressing mice and wild-type (wt) (28). We the expression of the mutually exclusive CD27 and CCR6 sur- initially evaluated the onset of the Smarca5fl gene deletion by face markers into IFN-g(CD27+)–producing or IL-17a(CCR6+)– PCR. Genomic DNA was prepared from FACS-sorted CD4/CD8 producing gd subsets (31, 32). The IFN-g– and IL-17a–producing DN thymocyte subpopulations of Smarca5+/flhCD2iCre mice and gd subsets display a subtle imbalance in favor of an IL-17a– three primer sets amplifying wt, Smarca5-floxed (S5fl), and producing subset in the Smarca5 mutants (Fig. 2E). We conclude recombined-floxed (S5del) allele were used. Consistent with pre- that the commitment to gd lineage and CD24 expression by vious reports that used the hCD2iCre transgene (26), we ob- TCRd+ thymocytes and the development of mature gd subsets are served Cre-mediated recombination of loxP1 sites as early as the impaired in the S5fl/flhCD2iCre mice. Although Smarca5 plays DN1 stage in which the recombined S5del allele was detectable important roles in thymocyte development during the DN3 to DN4 The Journal of Immunology 3437

FIGURE 1. Smarca5 is required for thymocyte development and sur- vival. (A) Scheme of Cre-mediated deletion in the Smarca5 gene. Indi- cated are 4–8 (boxes), posi- tions of loxP1 sites (red triangles), and positions of genotyping primers (white arrows). (B) PCR detection of the floxed (S5fl), wt (S5wt), and de- leted (S5del) Smarca5 allele in the DN cell subsets isolated from the S5fl/wthCD2iCre thymus. (C) Immu- noblot showing Smarca5 protein ex- pression in thymi of control (one animal per sample) versus S5fl/flhCD2iCre Downloaded from (a pool of four animals per sample) mice. Histone H3 served as a load- ing control. (D and E) Weight and cellularity of thymi of indicated genotypes. Bars depict the mean 6 SD from four controls and four http://www.jimmunol.org/ S5fl/flhCD2iCre mice. (F) Histology (H&E staining) of thymi from 6-wk-old mice of indicated genotypes showing medulla (M) and cortex (C). (G) Immunohistochemistry of cleaved Caspase-3 in Mayer hematoxylin- stained thymic sections. y-axis: mean number/square millimeter 6 SD of cleaved Casp3-positive cells in the cortex (n = 3/genotype). Signifi- by guest on September 28, 2021 cance in two-tailed t test. *p , 0.05, **p , 0.01.

transition of ab subsets, it also guides the development of the gd stromal components (of S5fl/flhCD2iCre mice) but rather intrinsi- compartment. cally to the thymocytes lacking Smarca5. To further settle the point of whether the developmental defect of thymocytes observed Developmental blockade in Smarca5-deficient thymocytes is in vivo is cell autonomous, we used ex vivo cocultures with a cell autonomous BM-derived stromal OP9/N-DLL1 cell line (19). Sorted pre- As hCD2-iCre transgene initiates deletion also in other murine selection early DN3e cells (small CD42CD82CD25+ thymo- hematopoietic cell subtypes (18), we tested whether the DN3 to cytes) were added on the OP9/N-DLL1 cells to evaluate the DN4 transition defect is cell autonomous to thymocytes or a result formation of DN4 and DP thymocytes in a time course of 8 d of the impaired thymic microenvironment in which they develop. (Fig. 3C). Although control DN3e cells apparently proliferated Using syngeneic transplantation, we assessed the ability of control and progressed into more mature developmental stages under BM (marked by CD45.1 isoform) to repopulate thymocytes in ex vivo conditions, the majority of Smarca5-depleted cells lethally irradiated S5fl/flhCD2iCre mice (marked by CD45.2 were held at the DN3 stage till day 8, and their absolute isoform) and vice versa. At day 35 after transplantation, we numbers remained similar to the starting cocultures (Fig. 3C, observed that engrafted thymocytes from controls (CD45.1) 3D). Thus, the outcome of the ex vivo experiment was highly developed normally to produce CD4/CD8 double and SP cells in reminiscent of the phenotype of S5fl/flhCD2iCre mice. We used the thymic microenvironment of S5fl/flhCD2iCre (CD45.2) ani- yet another approach to test whether the defect of DN3 to DN4 mals (Fig. 3A, 3B). In turn, the engrafted donor S5fl/flhCD2iCre transition could have emerged from a secondary effect, mainly BM-derived thymocytes (CD45.2) recapitulated the develop- because the DN3e thymocytes in the OP9/N-DLL1 cocultures mental defect at DN3 to DN4 transition within the control ac- were sorted from S5fl/flhCD2iCre mice with the potentially ceptor animals (CD45.1). These results suggested that the DN3 impaired thymic microenvironment. We isolated LSK BM he- to DN4 transition became defective independently of the thymic matopoietic progenitors from S5fl/flhCD2iCre or control mice 3438 Smarca5 REQUIREMENT FOR EARLY LYMPHOCYTE DEVELOPMENT Downloaded from

FIGURE 2. Smarca5 is required for thymocyte development at DN3 to DN4 stage. (A and B) Flow cytometry analysis of CD4 and CD8 DN, DP, and SP http://www.jimmunol.org/ cell populations in thymi of 4–6-wk-old control (n = 4) and S5fl/flhCD2iCre (n = 4) mice. Relative (A) and absolute (B) quantitation of thymic fraction sizes are shown. (C and D) Flow cytometry analysis of thymic DN (CD42CD82) cells of indicated genotypes using anti-CD25 and CD44 staining shown as relative (C) or absolute (D) values. Bar graphs depict the mean 6 SD from four controls and four S5fl/flhCD2iCre mice [same animals as in (A) and (B)]. Lineage-positive (B220, Gr-1, CD11b, CD11c, and Nk1.1) cells were excluded from all measurements. (E and F) Flow cytometry analysis of gd T cells in DN (CD42CD82) thymic fraction. (E) Expression profiles of surface markers are shown as a contour plot, and the proportions of cells within each gate are given. (F) Bar graphs depict the mean 6 SD of absolute numbers. Data are representative of three control and three S5fl/flhCD2iCre animals. Significance in two-tailed t test; *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

and kept them differentiating ex vivo on the OP9/N-DLL1 cells. of Smarca5-depleted cells to progress through the various stages by guest on September 28, 2021 Again, both the control as well as S5fl/flhCD2iCre-derived he- of the cell cycle. After a 3 h pulse of BrdU in vivo, we observed matopoietic progenitor cells developed normally into the DN2 that the percentage of proliferating BrdU+ DN3 cells (after ex- stage (day 9); however, from the 16th day of culture, the clusion of postreplicative BrdU+ events) was almost the same in S5fl/flhCD2iCre thymocytes were progressively underrepresented, mutants as in controls (14.8% versus 15.8%), and thus, the G1-to-S and by day 22, the DN3 to DN4 transition defect was revealed progression was not altered after the Smarca5 loss at DN3 stage (Fig. 3E, 3F). Thus, the loss of Smarca5 in developing T cells (Fig. 4C). However, the portion of postreplicative cells at this caused a defect intrinsic to the thymocytes undergoing DN3 to stage, which is defined as strictly diploid BrdU+ events (33), was DN4 transition, as evidenced by the cocultures using the OP9 2.5-fold decreased in mutant DN3 cells. These data indicate that cells, and was not a result of a secondary effect due to impaired Smarca5-depleted DN3 cells normally enter S phase and begin to thymic stromal components or thymic microenvironment. replicate their DNA; however, they are limited in completing the cell cycle to re-emerge as G1 cells. Mutant DP cells were also Smarca5-deficient thymocytes undergo marked apoptosis and impaired because a substantial fraction of the DP cells became proliferation impairment arrested at the G2/M phase (Fig. 4D). Thus, the developmental We next investigated whether reduced cell numbers in defects observed upon the loss of Smarca5 are likely the conse- fl/fl S5 hCD2iCre thymi and ex vivo OP9/N-DLL1 cocultures quence of cell death and impaired cell cycle progression at late S (Figs. 1E, 3D) may be attributed to premature cell death and/or through G2/M phase. This is in contrast to defects in G1/S impaired proliferation. As determined by flow cytometry, the ex checkpoint mechanisms as previously observed in cancer vivo cultivation of purified DN3e thymocytes eventually resulted cell lines upon SMARCA5 knockdown (34). in a gradual increase of Annexin V positivity up to 71% by day 8 that did not exceed 15% in controls (Fig. 4A), indicating that the Smarca5 mutants undergo pre-TCR signaling and the loss of Smarca5 induced apoptosis in developing T cell precursors. TCR rearrangement Similarly, we examined the effect of Smarca5 loss on the prolif- The accumulation of Smarca5-depleted DN3 cells resembles the eration by labeling purified DN3e thymocytes with the intracel- phenotype of mice that have a defect in pre-TCR signaling or lular fluorescent dye (CFSE) immediately before plating them on TCRb locus rearrangement (35). To test whether the induction of OP9/N-DLL1 cells. As seen in Fig. 4B, the analysis of the CFSE pre-TCR signaling was affected by a loss of Smarca5, we per- signal dilution indicated that the DN control thymocytes prolif- formed i.p. injections of anti-CD3ε Ab into S5fl/flhCD2iCre and erated rapidly ex vivo. Conversely, those DN cells that did survive into RAG1 (Rag12/2)–deficient mice. Anti-CD3ε Ab can mimic the ex vivo conditions exhibited a decreased division rate, con- pre-TCR signaling in vivo and stimulates preselection DN3 firming an impaired proliferation of S5fl/flhCD2iCre thymocytes. thymocytes to proliferate and differentiate into the DN4 cells We then used the BrdU incorporation assay to analyze the ability even in the Rag12/2 mice lacking the TCR b-chain expression The Journal of Immunology 3439 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 FIGURE 3. Developmental blockade in Smarca5-deficient thymocytes is cell autonomous. (A) Flow cytometric evaluation of donor-derived thymic populations regenerated after 1 mo following the transplantation of donor BM cells (BMT) into lethally irradiated (7.5Gy) hosts. Donor and host-derived thymocytes were distinguished by the surface expression of distinct variants of marker CD45. Control mice were CD45.1+ (Ly5.1), and S5fl/flhCD2iCre were CD45.2+ (Ly5.2). Data are representative of three BMT experiments. (B) Bar diagram shows mean number 6 SD of donor (CD45.1 = wt; CD45.2 = S5fl/flhCD2iCre) thymocytes 1 mo after BMT. Data are representative of three experiments. Significance in two-tailed t test; *p , 0.05. (C) FACS-sorted DN3e (small CD42CD82CD25+) thymocytes from control and S5fl/flhCD2iCre mice were cocultured with OP9/N-DLL1 stromal cell line. Cells were harvested on days 2, 4, and 8, and expression profiles of CD44/CD25 or CD4/CD8 markers were analyzed by flow cytometry. Data are representative of two experiments. (D) Cumulative growth curve (represented as fold change) of all live CD45+ cells isolated from OP9/N-DLL1 cocultures on days 2, 4, 6, and 8. Cells were the same as in (C). (E) Flow cytometry of purified Lin2Sca1+c-Kit+ (LSK) BM progenitors after 9, 16, and 22 d of cultivation with OP9/N-DLL1 cells. Data are representative of four control and four S5fl/flhCD2iCre animals. (F) Cumulative growth curve (represented as fold change) of cells analyzed in (E).

(36). We observed that the anti-CD3ε Ab stimulated the down- Next, we analyzed the intracellular expression and rear- regulation of surface CD25 molecules on Rag12/2 as well as on rangement of the TCR b-chain, another prerequisite for the Smarca5-deficient DN3 thymocytes (Fig. 5A), which suggested DN3 to DN4 transition. Analysis of intracellular (i)TCRb ex- that pre-TCR signaling pathway was not disrupted after Smarca5 pression together with membrane-bound CD28 has been shown loss. However, we noted that compared with highly proliferating as a tool to distinguish between preselection and b-selected Rag12/2 DN cells, the S5fl/flhCD2iCre DN cells were almost DN3 cells that have successfully rearranged their TCRb locus completely absent at day 2 of treatment (Fig. 5B), further con- (39). Using this approach, we observed that mutant DN3 thy- firming the poor survival of differentiating and proliferating mocytes contain a significantly reduced fraction of iTCRb+/ Smarca5-deficient DN cells. We next examined the expression CD28+ b-selected cells compared with controls (Fig. 5C). Con- of CD2 and CD5 on the DN and DP cells to assess their upreg- versely, the genomic DNA analysis of the Smarca5 mutant DN ulation upon pre-TCR signaling (37, 38). Data from flow cytom- thymocytes by quantitative PCR showed that the recombination etry show that b-selected DN cells that survived Smarca5 loss are rate of Db1-Jb1andDb2-Jb2 gene segments was not altered still capable of upregulating the expression of CD2 and CD5 (Supplemental Fig. 1C). As determined by RNA-seq analysis of molecules (Supplemental Fig. 1A). Additionally, at the DP stage, DP cells (see further), the expression pattern of the constant the expression of these molecules was almost identical com- (Trbc) and variable (Trbv) gene segments was similar to controls pared with controls (Supplemental Fig. 1B). In summary, the (Supplemental Fig. 1D), indicating that Smarca5 deficiency did ability of S5fl/flhCD2iCre DN cells to upregulate CD2 and CD5 not abolish the TCRb locus rearrangement. To test whether the indicated that Smarca5 deficiency in thymocytes does not defective formation of the b-selected DN3 cells was a result of perturb the pre-TCR signaling. impaired TCRb rearrangement, we crossed S5fl/flhCD2iCre mice 3440 Smarca5 REQUIREMENT FOR EARLY LYMPHOCYTE DEVELOPMENT

FIGURE 4. Smarca5-deficient thymocytes induce apoptosis and block proliferation. (A) Purified preselection DN3e thymocytes (from two animals of each genotype) were cocultured with OP9/N-DLL1 cells, and the mean fraction 6 SD of CD45+ cells that became Annexin V positive was assessed by flow cytometry following 2, 4, 6, and 8 d. Histograms (blank = control mice; gray, filled histogram = S5fl/flhCD2iCre mice) show results of the experiments at day 2 and 8. (B) Histograms of control and S5fl/flhCD2iCre DN thymocytes stained with CFSE and cocultured with OP9/N-DLL1 cells. CFSE signal dilution was analyzed by flow cytometry on days 2, 4, 6, and 8. Data are representative of four control and three S5fl/flhCD2iCre animals. (C) Flow cytometry analysis of control and S5fl/flhCD2iCre DN3 population cell cycle Downloaded from progression using BrdU/7-aminoactinomycin D (7AAD) double staining. Black rectangles depict all gated BrdU+ cells. Red trapezoid indicates BrdU+ diploid postreplicative cells that accomplished the mitosis. (D)Flow cytometry for BrdU and 7-AAD in DP cells. Rectangles show the proportion of G2/M frac- http://www.jimmunol.org/ tion. Data are representative of at least three individual animals of each genotype.

with TCRb/a transgenic mice (OT-II). Expression of the fully and upregulate the surface TCRb expression during their devel- rearranged TCRb/a construct in OT-II background is able to sup- opment with an exception at the DN4 stage that almost lacks press V(D)J recombination at endogenous loci and also “rescue” fraction of TCRb-positive cells. by guest on September 28, 2021 thymocyte development in mice lacking essential factors for the TCRb/a rearrangement (40, 41). Analysis of S5fl/flhCD2iCre Smarca5 deficiency altered the developmental program of b OT-II animals revealed that expression of the transgenic TCR post -selection stages b/a-chains failed to rescue the Smarca5 knockout phenotype. It has been shown that during differentiation from DN to DP The absolute and relative counts of DN and DP subpopula- stage, the ACF complex containing Smarca5 and Acf1 represses, tions remained similar to the S5fl/flhCD2iCre mice (Fig. 5D– in cooperation with Satb1, the Il2ra (CD25) gene (12, 42). In- F). Particular exceptions were mature SP thymocytes, where deed, flow cytometry indicated that DP cells of S5fl/flhCD2iCre the CD4 SP cells whose 3-fold increase to the detriment of the mice inappropriately express both CD44 and CD25, the markers CD8 SP cells likely reflected the positive selection of thy- of earlier developmental stages. Whereas the CD25 molecule mocytes toward CD4 lineage that normally occurs in the OT-II becomes partially downregulated, the CD44 remains upregu- strain (Fig. 5D). Thus, rather the poor survival of b-selected lated in DP cells, implicating the dysregulation of early expression DN cells than defects in pre-TCR signaling or TCRb locus rear- programs (Fig. 6A). Our previous studies suggested that Smarca5 rangement could best explain the phenotype of S5fl/flhCD2iCre participates in the regulation of gene expression programs asso- mice. ciated with survival and differentiation of lens, cerebellum, and As the surface expression of TCRb in DP thymocytes is es- hematopoietic progenitor cells (10, 17, 43). To gain a global view sential for the production of SP subpopulations, we focused on on the gene expression programs dysregulated by Smarca5 loss in stages beyond DN3 to decipher how Smarca5 deficiency influ- b-selected thymocytes, we purified DP cells from S5fl/flhCD2iCre enced the TCRb expression. Normally, the surface expression of mice and used RNA-seq to compare the gene expression profiles TCRb is low or none in the DN3 stage, becomes induced at the with those from control DP cells. Of .21.500 expressed genes, a DN4 stage, and further upregulated in SP thymocytes. We ex- total of 3.318 transcripts were differentially expressed with false amined the level of surface TCRb expression on individual de- discovery rate (FDR) ,0.05 and BaseMean value .10. From velopmental stages of thymocytes and mature peripheral T cells in these, 1.503 mRNAs were (,2-fold) downregulated and 1.815 the spleen. We observed that surface TCRb expression was de- mRNAs were (.2-fold) upregulated. analysis of tectable in all developmental stages from a subfraction of DN3 the differential expression using gene set enrichment analysis (low expression) to mature SP populations (high expression) in (GSEA) (44) showed the enrichment of mRNAs involved in both Smarca5-deficient as well as control thymocytes (Fig. 5G, expected categories such as apoptosis and the p53 pathway; Supplemental Fig. 1E). However, the fraction of cells with up- however, most of them were either immunologic or lymphocyte regulated TCRb expression was reduced within each analyzed associated (Fig. 6B). By dividing the differentially expressed Smarca5-deficient thymic subpopulation compared with controls. genes into previously published mRNA clusters with similar Taken together, Smarca5-deficient thymocytes are able to express behaviors during thymocyte differentiation (45), we observed The Journal of Immunology 3441 Downloaded from http://www.jimmunol.org/

FIGURE 5. Smarca5 is not required for pre-TCR signaling and the TCR rearrangement. (A) Flow cytometry analysis of differentiation markers on thymocytes isolated 1 and 2 d after i.p. injection of Rag12/2 and S5fl/flhCD2iCre mice with anti-CD3 (50 mg/mice). Contour plots showing DN (CD42CD82) cells. Data are representative of five (day 0), two (day 1), and three (day 2) Rag12/2 or S5fl/flhCD2iCre animals. (B) Mean of absolute numbers 6 SD of cells isolated from the thymus of mice used in (A). (C) Contour plots showing the expression of CD28 and intracellular TCRb (iTCRb)in DN3 thymocytes of indicated genotypes. Data are representative of three experiments. (D) Flow cytometry of DN, DP, and SP fractions using CD4 and CD8 markers in thymic suspensions from 4- to 6-wk-old S5fl/fl OT-II+/2 control, S5fl/flhCD2iCre, and S5fl/flhCD2iCre OT-II+/2 mice. The relative population sizes are indicated. DN thymocytes were analyzed for CD25 and CD44 marker expressions. All B220, Gr-1, Mac-1, CD11c, and Nk1.1 lineage-positive cells were excluded from the analysis. Data are representative of more than three experiments. (E and F) Mean of absolute numbers 6 SD of thymic sub- by guest on September 28, 2021 populations as in (D). Two-tailed t test; *p , 0.05. (G) Surface expression of TCRb protein on CD4 and CD8 T cells of control and S5fl/flhCD2iCre animals analyzed by flow cytometry. As an isotype control to H57-597 clone (anti-TCRb), fluorescently labeled Armenian Hamster IgG was used (dark histograms). Data are representative of three experiments. that the group of upregulated genes in the mutant DP cells greatly disorders the developmental programming of T cell overlapped to those mRNAs that peaked in expression at the progenitors. early (DN1–DN2) or pre–b-selection DN3a stage (Fig. 6C). Such genes were, for example, receptors (Il7r, Ctla4, Ptcra, Ly6a Smarca5 is required for pro–B/pre–B transition of and also Il2ra/Cd25 and Cd44), signaling molecules (Dtx1, B cell progenitors Hes1, Notch1, Lfng, Rab44), and transcription factors (Irf7, Additional analysis of S5fl/flhCD2iCre mice revealed also a dramatic Tcf7l2, Spib) (Fig. 6D). Accordingly, the group of downregulated reduction of the spleen cellularity (Fig. 7A). Besides the reduction genes (total 97) fall into the category of genes that are gradually of splenic T cells and NKT cells, which both developed in the expressed by b-selected cells during the transition into DP, such thymus from the CD4+CD8+ DP precursors (46), we also observed as transcription factors (Klf7, Ets2, Ikzf3, Bcl6), surface mole- a marked depletion of B lymphocytes, whereas the numbers of cules (Plxnd1, Slamf1, Cd81), signaling (Themis), and others myeloid cells were not significantly altered (Fig. 7B, 7C). Indeed, (Tdrd5, Cacna1e). To evaluate whether the mutant DP cells more immunohistochemistry of mutant spleens showed a prominent closely resembled pre– or post–b-selected cells, we used a follicular hypoplasia affecting both T cell as well as B cell zones previously published transcriptome analysis of microarray data (Fig. 7D), suggesting also a defect in the B cell development of of wt C57BL/6J DN3a and DP stage cells (45) and created two S5fl/flhCD2iCre mice. To investigate a stage at which the devel- sets of the 200 most upregulated and downregulated genes in wt opmental defect occurred, the early B cell progenitor populations DN3a compared with wt DP stage (Supplemental Fig. 2A). from BM were analyzed. Flow cytometry analysis revealed an Hence, the GSEA analysis of mRNAs dysregulated upon almost complete loss of pre-B cells (B220+CD432) in mutants, Smarca5 deficiency revealed a strong enrichment of upregulated whereas the proportion of pro-B cells (B220+CD43+) was virtually (normalized enrichment score = 2.37, FDR ,0.001) and down- unperturbed (Fig. 7E). Closer examination of the pro-B population regulated (normalized enrichment score = 22.53, FDR ,0.001) showed a developmental arrest between early preselected pre–B-I mRNAs of the wt DN3a stage (Fig. 6E). This finding indicates cells (CD117+) and pre–B-II (CD25+) cells that underwent pro- that although Smarca5-deficient cells express post–b-selection ductive IgH gene loci rearrangement (47). Taken together, surface markers (e.g., CD4, CD8, CD5, CD2) as normal DP Smarca5 deficiency affects development of early B220+CD43+ cells, RNA-seq data reveals that they retain a transcriptional pro-B cells in BM, implicating the requirement of Smarca5 for program of pre–b-selection DN cells. Thus, Smarca5 ablation both early T as well as B lymphocyte development. 3442 Smarca5 REQUIREMENT FOR EARLY LYMPHOCYTE DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Smarca5 deficiency altered the developmental program of post–b-selection stages. (A) Flow cytometry of thymus. Dot plot shows the analysis of CD25 and CD44 marker expressions on the surface of CD4/CD8 DN (black dots) and overlapping DP (red dots) thymocytes. (B) The list of 11 most significantly enriched Hallmark gene sets in Molecular Signatures Database v6.2 by GSEA. (C) Heat maps of upregulated genes (log2FC scale) in S5fl/fl hCD2iCre mutant DP cells versus control S5fl/fl DP thymocytes. The c-Kit and Il7r gene clusters were defined previously using microarray analysis of thymocyte development (45). Gene expression profile heatmaps represent three individual experiments from sorted DP cells, each pooled from 25 to 37 fl/fl S5 hCD2iCre animals and normalized to controls (n = 3). (D) Data from RNA-seq presented as Volcano plot. The x-axis represents log2 fold change in the fl/fl expression of mutant DP cells as compared with the control (S5 ) DP cells. The y-axis represents adjusted p value (padj). The horizontal dashed line is equal to padj = 0.05. Genes (as green dots) are those that are normally downregulated in DN3a cells and become upregulated in DP cells during development and vice versa; the violet gene dots are upregulated in DN3a and, during development, become downregulated [based on microarray analysis of thymocytes

(45)]. Numbers in upper corners indicate the numbers of differentially expressed genes between mutant and control of each gene set within log2FC of ,21 or .1. (E) Enrichment analysis of differentially expressed genes in S5fl/flhCD2iCre DP thymocytes versus control DP cells [same as in (D)] was performed on the two gene sets containing 200 most upregulated (left plot) and downregulated (right plot) transcripts in wt DN3a compared with wt DP stage thymocytes [according to (45)]. For the complete list of genes pertaining to each of the gene sets, see Supplemental Fig. 2A. Positive (left plot) GSEA enrichment score curve indicates that the genes comprising the leading edge of the GSEA plot (mostly DN3a abundant transcripts) are positively correlated with mutant S5fl/flhCD2iCre DP cells. Similarly, for downregulated genes, the GSEA indicated a correlation between S5fl/flhCD2iCre DP cells and normal DN3a cells (lower plot). NES, normalized enrichment score. The Journal of Immunology 3443

FIGURE 7. Smarca5-deficient B cell progenitors are arrested at pro- B/pre-B transition. (A) Bar diagrams show the mean number 6 SD of CD45+ cells or (B) peripheral lymphocytes or (C) myeloid cells in spleens of control (n =5)and S5fl/flhCD2iCre (n = 5) mice. Two- tailed t test; *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. (D) Immunohistochemistry for CD3 (T cell marker) and Pax5 (B cell marker) in the spleen of 6-wk-old control or S5fl/flhCD2iCre mice. Data are representative of three ex- periments. CA, central arterioles. (E) Flow cytometry analysis of early B cell subpopulation in the BM of control or S5fl/flhCD2iCre mice. Left plots show B220 and CD43 staining Downloaded from of all Ter119-negative cells in BM. Right plots show CD117 and CD25 staining of CD43+B220+ (pro-B) cells gated in upper plots. Data are representative of six control and nine S5fl/flhCD2iCre animals. http://www.jimmunol.org/

p53 guides survival in the Smarca5-depleted thymocytes of H3S10phos positive (mitotic) cells as compared with single undergoing b-selection Smarca5 knockout (Fig. 8H). However, the G2/M blockade in the It has been previously noted that deletion of Smarca5 gene in DPs of the Smarca5 and Trp53 double knockout mice persisted hematopoietic progenitors induces the expression of p53 tran- (Supplemental Fig. 2C). scriptional targets (10). Our current RNA-seq data suggested To test whether the p53 loss could also recover the development of early B cell progenitors, BM cells from S5fl/fl hCD2iCre mice

that increased cell death and impaired cell cycle progression of by guest on September 28, 2021 with or without Trp532/2 loci were analyzed for the expression of thymocytes coincide with the activationofthep53program B220 and CD43 molecules. However, unlike the partial rescue (Fig. 8A). To test the biological significance and require- observed in the thymocyte compartment, the data from flow ment of the Trp53 gene for the Smarca5 mutant phenotype cytometry show that the p53 loss failed to rescue the survival in developing lymphocytes, we used an additional mouse strain and/or maturation at pro-B (B220+CD43+) to pre-B stage homozygous for the Trp53 null allele (48) to create the + 2 2 2 (B220 CD43 ) transition (Fig. 8I). To conclude this part, the in- S5fl/flhCD2iCre Trp53 / mice. Interestingly, the Smarca5 dele- troduction of the Trp53 knockout allele into the Smarca5-deficient tion slightly prolonged the survival of the Trp53 knockout mice strain significantly improved the proliferation and/or survival of (Fig. 8B). We observed that the introduction of the Trp53 thymocytes but not of early B cells. Importantly, the rescue ex- knockout allele improved thymic cellularity of the Smarca5- periment was unable to restore the dysregulated differentiation deficient mice (Fig. 8C). Flow cytometry analysis revealed a pathways in both B and T lineages. proportional increase (from 26.3 to 55.1%) as well as absolute cell number expansion (4-fold) of the double knockout DP population Discussion (Fig. 8D, 8E), whereas the absolute numbers of DN cells were Although the SWI/SNF and CHD chromatin-remodeling factors unchanged, indicating a DN to DP transition rescue (Fig. 8E, 8F). have been implicated in the regulation of lymphocyte progenitor- We performed RNA-seq of samples derived from the double specific transcription and differentiation (6, 49), the role of ISWI knockout DP cells and compared gene expression profiles with proteins in the development of early T and B cells has not been previous data. Expression analysis confirmed that the p53 targets, addressed. This study brings yet unknown evidence that the especially those that are associated with the induction of apoptosis ISWI ATPase Smarca5 regulates early lymphocyte develop- in response to DNA damage such as p21/Cdkn1a, Noxa/Pmaip1, ment by promoting stage-specific gene expression and, second- and Bax, were upregulated specifically in the Smarca5-deficient arily, cell survival and proliferation. Interestingly, the role of 2/2 DP cells, whereas upon the introduction of the Trp53 allele, Smarca5-containing remodeling complexes was previously im- their expression became normalized (Fig. 8G). Although the plicated in the DNA double-strand break (DSB) repair in human thymic cellularity in double knockouts was partially recov- immortalized cell lines (50, 51). These reports showed that ered, we still observed markedly dysregulated expression of SMARCA5israpidlyrecruitedtoDSBs,andtheknockdownof the mRNAs connected to normal thymocyte development SMARCA5 sensitizes cells to DNA damage. SMARCA5 protein (Supplemental Fig. 2B). Thus, the activation of p53 targets was was shown recruited to the sites of DSB by histone deacetylase rather a modifier of the severity of the phenotype and not con- Sirtuin 6 (SIRT6) (51). Additional pathways and interaction tributory to differentiation defects observed in S5fl/flhCD2iCre partners of Smarca5 participating at the sites of DNA damage mice. Indeed, flow cytometry analysis showed that Smarca5 and were also established (52, 53). Thus, Smarca5 seemed to be a Trp53 double knockout DP cells contained up to a 3.5-fold excess suitable candidate for testing its in vivo role in lymphocytes, in 3444 Smarca5 REQUIREMENT FOR EARLY LYMPHOCYTE DEVELOPMENT Downloaded from http://www.jimmunol.org/

A

FIGURE 8. Trp53 codeletion rescues survival but not differentiation of Smarca5-deficient thymocytes. ( ) GSEA showing a representative enrichment by guest on September 28, 2021 plot of genes involved in p53 pathways and networks comparing DP thymocytes sorted from S5fl/flhCD2iCre (three individual pools, see Fig. 6C) mice versus DP thymocytes isolated from S5fl/fl controls (n = 3). The negative GSEA enrichment score curve indicates that the genes comprising the leading edge of the GSEA plot are positively correlated with mutant S5fl/flhCD2iCre DP cells. (B) Kaplan–Meier survival curve of controls (dashed line, n = 125) and Smarca5/Trp53 double mutants (n = 115). Log-rank (Mantel–Cox) test used; *p , 0.05. (C) Bars depict the mean of absolute numbers 6 SD of thymocytes from S5fl/flhCD2iCre (n = 4) and S5fl/flhCD2iCre Trp532/2 (n = 5) mice. (D) Flow cytometric plots showing distributions of thymic CD4/CD8 positive and negative developmental stages of indicated genotypes. The CD4/CD8 DN cells were further distinguished using CD25 and CD44 surface markers (lower graphs). Lineage-positive (B220, Gr-1, Mac-1, Nk1.1, CD11c, Ter119) cells were excluded from all measurements. Data are representative of four S5fl/flhCD2iCre and five S5fl/flhCD2iCre Trp532/2 animals. (E and F) Mean of absolute numbers 6 SD of thymic subpopulations as in (D). Two-tailed t test; *p , 0.05. (G) Heat map showing the expression of genes that were differentially regulated in S5fl/flhCD2iCre mice but normally expressed in S5fl/flhCD2iCre 2/2 2/2 Trp53 animals compared with controls and Trp53 mice. The expression is normalized to controls; log2 scale. The last column represents gene expression profiles of pooled DP cells sorted from six S5fl/flhCD2iCre Trp532/2 animals. (H) Absolute counts of mitotic phospho-histone H3 (Ser10) positive DP cells in S5fl/flhCD2iCre and S5fl/flhCD2iCre Trp532/2 double knockouts. Two-tailed t test; ****p , 0.0001. (I) Relative numbers of early B cell subpopulations in BM of indicated genotypes. Data are normalized to all CD45+ cells and represent the mean of at least three animals. Two-tailed t test; *p , 0.05, ****p , 0.0001. which the developmentally programmed DSBs occur. Indeed, control DP cells (Supplemental Fig. 1D), indicating that the our data showed that S5fl/flhCD2iCre mice initially exhibited a relative use of the different gene segments during TCRb rear- marked reduction of those early progenitors that productively rangement was not affected in the mutants. In addition, the rearranged Ag receptor loci, the TCRb expressing DN3 thymo- S5fl/flhCD2iCre Trp532/2 mice displayed a comparable life span cytes (Fig. 5C) and B220+CD43+CD25+ early B cells (Fig. 7E). as S5fl/fl Trp532/2 mice (Fig. 8B), which is contrasting to the Coincidently, the upregulation of p53 target genes (Fig. 8G) and mouse knockout models of genes employed directly in the NHEJ the partial recovery of thymus cellularity in the S5fl/flhCD2iCre that upon codeleting with the Trp53, accelerated tumorigenesis Trp532/2 mice (Fig. 8C) could also be interpreted as Smarca5 with shortened animal survival (54). Thus, the Smarca5 deletion- being involved in the DSB repair during the Ag receptor gene mediated maturation defect is not primarily mediated via the rearrangement. However, further experiments challenged this disrupted repair of developmentally programmed DSBs. view. The developmental defect, at least in thymocytes, could not Generally, except in the SP CD8 and CD4 T cells that expressed a be attributed mainly to the DSBs repair aberration, as the OT-II lower level of surface TCRb (Fig. 5G, Supplemental Fig. 1E), the transgene was unable to rescue the DN3 to DN4 transition defect iTCRb expression and proximal pre-TCR signaling seem to be of Smarca5-deficient cells (Fig. 5D). Notably, the data from RNA- preserved in the Smarca5-deficient mice as S5fl/flhCD2iCre DN3 seq showed that the expression pattern of the constant (Trbc) and cells gave rise (albeit with a very low rate) to some DP-like cells variable (Trbv) gene segments was similar compared with the and could be normally stimulated by anti-CD3 Ab. The DN3 stage The Journal of Immunology 3445 and all the following developmental stages were considerably Smarca5 as a transcriptional activator and repressor was shown in altered in the S5fl/flhCD2iCre mice. Once Smarca5 was inacti- studies using murine EL4 T lymphoma cell line upon small in- vated, b-selected DN3, DN4, DP, and also pre-B cells lost their terfering RNA–mediated depletion. Although Smarca5 partici- ability to accumulate and became depleted. Smarca5 loss leads to pates in repression of IL genes, including Il2, Il5, Il13, Il17a, and a marked increase in the number of cells undergoing apoptosis in activation of Il3 after stimulation with PMA and ionomycin (Fig. 1G). Our data indicate that this was not caused by the in- (13), we have not observed this during the transition into the DP duction of a generalized apoptotic response as resting DN3e cells stage as those ILs are expressed mainly by mature T cells. Per- displayed very low level (up to 5%) of cell deaths even after 6 d turbation of Smarca5 functions may have also affected other of ex vivo cultivation (data not shown). This result, together with profiles during the transition from pre– to post–b-selected DN3 the observation that Smarca5-deficient DN3 stage lacks post- thymocytes by dysregulating genes related to proliferation, replicative cells (Fig. 4C), rather suggested that the disruption of metabolism, and b-selection (45). To conclude, Smarca5 repre- Smarca5 function triggers apoptosis of highly proliferating cells sents an important transcriptional regulator that participates in- and especially those that have already entered the S phase. Others dispensably during early T cell development. To further address reported that depletion of Smarca5 in murine lens (using Le-Cre the role of Smarca5 in regulating T cell promoters, we are cur- system) results in a reduction of BrdU and Ki67 positive pre- rently preparing a transgenic mouse line with tagged Smarca5 sumptive lens epithelial cells, leading to the lens developmental protein. defect (43). Also, the early deletion of Smarca5 in cerebellar progenitors (using the Nestin-Cre system) resulted in a lower Acknowledgments number of BrdU-positive Purkinje cells and of granule neuron We thank Christian Lanctoˆt and Vladimı´r Divoky´ for helpful discussion, Downloaded from progenitors at E17.5, possibly because of massive cell death (17). Ivan Kanchev for histopathology reports, Emanuel Necas and Martin Moreover, the defective S phase progression in the DN3 stage Molı´k for CD45.1 mice and assistance with the BM transplantations, Dana  could also explain the formation of G2/M-arrested S5fl/fl hCD2iCre Mikulenkova´ and Ilona Jiraskova´ for cytospin preparations, Ludek Sefc for DP cells (Fig. 4D). In the erythroid cell compartment, the help with flow cytometry analysis, Jan Kubovciak for help with RNA-seq  Smarca5 loss caused the emergence of tetraploid cells perma- analysis, and Martin Drobis for style and writing.

nently exiting the cell cycle in populations of highly proliferating http://www.jimmunol.org/ proerythroblasts-to-basophilic erythroblasts (10). This, along with Disclosures a substantial number of apoptotic events, was interpreted mainly The authors have no financial conflicts of interest. as a consequence of the activation of the DNA damage-associated p53 program. Indeed, stressed replicating cells activate their References replication checkpoint to delay S phase progression and G2/M 1. Rothenberg, E. V., J. E. Moore, and M. A. Yui. 2008. Launching the T-cell- transition (55). The p53 and its downstream molecules are lineage developmental programme. Nat. Rev. Immunol. 8: 9–21. then required to maintain a G2 or tetraploid G1 arrest, which 2. Zhang, J. A., A. Mortazavi, B. A. Williams, B. J. Wold, and E. V. Rothenberg. 2012. Dynamic transformations of genome-wide epigenetic marking and tran- afterward promotes cell senescence (56–58). However, although scriptional control establish T cell identity. Cell 149: 467–482. S5fl/flhCD2iCre DP cells also upregulated some proapoptotic 3. Haks, M. C., P. Krimpenfort, J. H. van den Brakel, and A. M. Kruisbeek. 1999. by guest on September 28, 2021 (Noxa, Bax, Puma) and cell cycle regulating (Cdkn1a/p21) p53 Pre-TCR signaling and inactivation of p53 induces crucial cell survival pathways in pre-T cells. Immunity 11: 91–101. target genes, the rescue of phenotype was incomplete, and the 4. Shah, D. K., and J. C. Zu´n˜iga-Pflu¨cker. 2014. An overview of the intrathymic tetraploid events were still present in the Smarca5 and Trp53 intricacies of T cell development. J. Immunol. 192: 4017–4023. double knockouts (Supplemental Fig. 2C), indicating that the cell 5. Winandy, S. 2005. Regulation of chromatin structure during thymic T cell de- velopment. J. Cell. Biochem. 95: 466–477. cycle arrest and induction of apoptosis were predominantly p53 6. Dege, C., and J. Hagman. 2014. 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Mouse BAZ1A (ACF1) is dispensable for double-strand Transcriptome analysis by RNA-seq confirmed a considerable break repair but is essential for averting improper gene expression during number of ectopically expressed transcripts associated with spermatogenesis. PLoS Genet. 9: e1003945. b-selection and also genes that were not activated and remained 15. Koscielny, G., G. Yaikhom, V. Iyer, T. F. Meehan, H. Morgan, J. Atienza- downregulated during the transition into the DP stage (Fig. 6D), Herrero, A. Blake, C. K. Chen, R. Easty, A. Di Fenza, et al. 2014. The Inter- national Mouse Phenotyping Consortium Web Portal, a unified point of access suggesting that the absence of Smarca5 disables a crucial com- for knockout mice and related phenotyping data. Nucleic Acids Res. 42(Database ponent of the b-selection transcription machinery. The function of issue): D802–D809. 3446 Smarca5 REQUIREMENT FOR EARLY LYMPHOCYTE DEVELOPMENT

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A DN DN C Dβ1XJβ1 Dβ2XJβ2 125 125 Norm. count Norm. count

100 100 ± SD 75 ± SD 75 CD2 CD5

B DP 50 50

DP alleles alleles β 25 β 25 TCR TCR % of unrecombined Norm. count Norm. count 0 % of unrecombined 0

DN1 DN2 DN3 DN4 DN1 DN2 DN3 DN4 S5fl/fl CD2 CD5 S5fl/flhCD2iCre S5fl/fl S5fl/fl hCD2iCre C Obrázek: Vložit do supplementary - zjistit co je ta negativní populace

fl/fl flox/flox flox/flox D S5 E Thymocytes: Smarca5 Smarca5 hCD2-iCre Trbv1 fl/fl DP DP Trbv12-1 S5 hCD2iCre DN1 DN2 DN3 DN4 o Mode

T iTCRβ iTCRβ Trbv12-2 99.0% 97.7% Trbv12-3 Trbv13-1 Normalized

Trbv13-2 Norm. count Trbv13-3 Trbv14 Trbv15 Trbv16 Tcrβ Trbv17 S5fl/fl Trbv19 fl/fl Trbv2 S5 hCD2iCre Trbv20 Smarca5 isotype control Trbv21 Trbv23 Trbv24 0 50 100 150 DP CD4 CD8 Trbv26 TPM value Trbv29 Trbc1 Trbv3

Trbc2 Norm. count Trbv30 Trbv31 0 5000 10000 15000 Trbv4 TPM value Trbv5 Trbv6 Tcrβ 0 500 1000 1500 2000 TPM value

Supplemental Figure 1 Flow cytometry analysis of CD2 (A) and CD5 (B) molecules on CD4 and CD8 double negative (DN) and double positive (DP) cell populations in thymi of control and S5fl/flhCD2iCre mice. Similar results were obtained in at least 3 repeat experiments. (C) Quantitative detection of the D-J recombination of the Tcrb gene. qPCR determines relative loss of the intact allele after the TCR recombination. Graphs show relative proportion ± SD of non-recombinated Tcrb gene (measured here as presence of intact Dβ1-Jβ1 and Dβ2-Jβ2 segments, respectively) in individual DN populations. Data are representative at least of three control and three S5fl/flhCD2iCre animals. (D) RNA-seq analysis of Tcrb gene rearrangement associated transcripts expressed in control (white bars) and S5fl/flhCD2iCre (black bars) DP stage thymocytes. The transcript abundance of Smarca5, Tcrb constant regions (both shown in rectangle) and all expressed Tcrb variable regions with BaseMean value higher than 10 were quantified as transcripts per kilobase million (TPM). (E) Surface expression of Tcrβ on individual thymic subpopulations of control and S5fl/flhCD2iCre animals analyzed by flow cytometry. As an isotype control to H57-597 clone (ani-Tcrβ) was used fluorescently labeled Armenian Hamster IgG (dark histograms). Data are representative of three experiments. B - Log Adjusted P A 10 50 −5.0 0 Log S5 t w c in w Genes upr do Genes 1 corresponds gated within 2013). DN3a is the DN3a Supplemental ( o ompar A 180 86 56 t DN3ac wnr DP 2 fl/fl equal Ankrd6 Supplemental Fig.2 ) Fmnl2 FoldChange Cacna1e t DN3a Smoc1 expression The egula Tdrd5 e

thymic and cells hCD2-iCre d Numbers log t egula ompar to top Rasgrp4 o DP t ed in 2 DP FC p as adj −2.5 to t 200 ed Cd81

e suspension. thymocytes

compared of d = in the Figur

in 0.05). Unc93b1, T T Snor T Snor Rpl36a, Rgs14, Plekha7, Nsg2, My L L His Hes1, His Gm5481, Gm10459, Eph Dnajb4, Cd5, Ccnd2, A Ankr A130050O07Rik, 1700048O20Rik, upregulated S5 <-1 y cr fng span32, r tp10a, G2/M a s g-V1, Gm9821 corners t, v14-1, t1h2br t1h2be, c, T fl/fl x3, T Ptms , d1b, d15b, d27, span32 Macf1, Cd52, e Myl10, rp53 Lg hCD2iCre or Olfr1537, Hmg -1 2 Ern1, Cd53, Rmnd5a, als3bp, Colored Rpl38, A T Dusp6, >1. P T to Snor Ape arrested cr Camk4 , tp2b1, Gm6525, T Smarca5 Usmg5, Slamf1 ube1, olr1b, Ets2 Snor cs2, His r (45). Gm10644, g-V4, Cd6, a Genotypes His -/- Plxnd1 Map4, wild-type ND3, indicate v14d-3-dv8, E Klf7 x1, Cd59a, t1h4f d1c, ( tv6, 0.0 DP t1h2b C Ikzf3 I730030J21Rik, Llph, Rpl39, d16a, and P3h4, T E2f2, B230359F08Rik, Cd81, Arhg Pp T ) Rnu2-10, ( xk, 4930427A07Rik, Abc T Ptcra Ndr W Up cr B Snor F Kbtbd1 cf7l2 dots T Flow , Map4k5, Gm6756, t1, ads2, cells df g-V6, vs. His ) Ube2v1, rp53 L f top Cdh1, a1, Spc25, , g1, ap26, OC547349, Itgb4 Phlda1, Data y4, Gm10785, Rpl41, E2f8, 1 P His d32a, dif Cd8a, t1h4h, Do B2m Prss57 qlc3, DP Nkg7, indicate Adam12, 1 T Yb F T 200 are population. t1h2bg S5 cytometric r -/- am169b, ferentially g H2-T22 wn R a Cdk6, Arhg Ndrg1 x3, tp1,

from v14n-2, Sqr Medag ock1, vs

E Snor stage Ublcp1, Gm7120, Rpl5, Pr Cd8b1, Ph ct2, fl/fl Hmmr indicated Idi1, Nlr Zfp36l2, Rab44 downregulated dl, dx3, T xr2, e T Gm16489, , L 2.5 rp53 g Bcl2l1, Cdkn1a, Unchanged f10l, d47, Nln c5, y6a, tp2, Egr1, St6g T R 4930567H12Rik, His RNA-seq Adgr Rpl9, , Ifitm2, genes Fkbp5, or thymocytes Pik3cb, T rp53 Me Pr , Umad1, Not Cdh24, r Id3, t1h2bh, c, a Snrpg Ass1, ss57, L al1, Tif Gm8995, -/- v16d-dv11, Zfp991 e5, y6c1, f2a, expressed Snx10 plots Data Bcl6, Rrm2, Rnf135 Ephb6,

Rp ch1, DP a, Clpb, Ikz Il2ra Igbp1, L on T -/- Frmd4a, Ak normally s18, T Plcl1, y6c1 cr Gm4884, Mfhas1, A , P Cd44 mem173, f3, Cdkn1b, Sn

Usp33, tp2b4, Not L sme1, cells. a-V22.1, Bub1, ap12, DP in wtDN3acells y6c2, are L His presented the showing Sg x30, Cor y6a Il21r Rp Er Gnl3, ch3, Il17rb, Plcl2, t1h2bj, ol2a, transcripts c1, Spib (from representative sa, o2a, T cells Mpzl2 top. Ctla4 Gas5, Gm1 Spa 5430410E06Rik, r Hes1 C920008G01Rik, 5.0 P Als2cl, L , A Mki67, genes The W Cdh1 a y6d, t Il4r Npm1, Gpr162, tp8b4, Cenpe, v3-3, Esc cr Rr T Gp apl, T Plekh Skil, downregulated H2-Bl ts2, dr Gm4737 Clec12a mem176b, Cpa3, 1 a, Il2r as2, 1603 a, 127 DP x1, 248 o2, d5, His Gfr Y L 12 expression a Rabl2, Wisp3, y Itgb2, as Ankr a, Spib, T -axis Mns1, z2, Sla, microarray between f1, r Nsdhl, A t1h2bk, Gria3, a1,

Rrp1b, T a Cenp Esrp2, Il7r W112010, fractions Cpne2, V drp, Gr v3n-3, Mc Plxnd1, identified olcano d44, Gid4, Slam amd3, Sr , Jup, R Ir Xrr represents oln2, Mr1, f gn, epin1, T T , C Odc1, gm1, Grn, nk2, mem180, at Chd1, Rsu1, E a1, 5830411N06Rik, Cacna1e, Ankr T f1, His Cp ts2, Kif11, Srm, Gm12191,

CD71 r least Smar a P Gsn, Na Mif of t1a, Zfp36 v4d-4, Gs t1h2bl, olg T B2m, plot. Slc37a3, Itg P op2a, Rn designated d50, analysis 82.7% P3 F13a1, v2, (green) ark7, Rtp4, t Ck Ss , am, , CD71 o1, y3, Kif14, Milr1, Pp Crip1, H2-Q2, in tr2, of ap2l, 14.6% P4 ca5 Nc T Bri3bp, Camk4, The t An T mem50b, Gs adjusted S5 T K c7, Rpl12, FSC-A microarrays P r three apg His op2b, Scin, cnk5, St6g a Gm2a, arp8, x tp1, Slc6a19,

F v7-4, Kif15, mutants a5, Cnr2, Mir675, Ctla4, Pr as, t1h2bm, fl/fl and X-axis , His or of 0.2% c1, Ndc80, alnac4, Bs Scn4b, Adam Gs Ar Casc5, K Rpl13a, T Flna, P experiments.

t1h1a, P5 T upregulated r thymocytes as delc1, dgfrb, t1, Gm32211, Pvr a r ap2, Kif5b, Colq, forward tz1, Cy T a v12-1, DP n p-value v7d-4, Slc7a11, Mp Bs P3-P5 , fr th4, ts17, Rasa2, Fmnl2, Ne represents Suclg2, His and s Gzma, Sema4a, Ccna2, t2, Arhg f1b, His P Khdc1a, 21.4% P3 zl2, Csg Rpl30, Klf7, comparing t ebp1, hCD2 t1h2bn, D8Ert o2, T Camkv t1h2ba, T r 55.2% P4 Adgr alnact2, controls a r T Mrp e Gm4759, Rasgrp1, a v12-2, scatter pi1,

f11, Nf are S5 H2-D1, Slc Sup Ldlr Gimap8, (horizontal v9d-3, Ccr4, Phg d82e, Rpl31, a g1, by Sigmar1, Khdc1b, s18b, o3a1, (violet) , t T t6, ad3, fl/fl c3, 19.4% -iCre Capn5, Arn shown. His r dh, His gv2, log T Mingueneau Ccr9, Ahr Dek, Susd1, r T Nfkbia,

H2-K1, P5 Dtx1, t1h2bp, a tl, Rb1, (FSC-A) r t1h2bb, Msmo1, of Lipa, Gm5111, Rpl31-p Pik3cd, v12n-1, a Smoc1, adult Glcci1, 2 , DP v9d-4, T

Ar fold Khdc1c, span31, Akr1c13, Cblb, Depdc1a, Cd2, each Smim5, si, in Rcb E S Lmnb1, S5 P5 dashed e H2-L y Nhsl1, 25.5% P3 hCD2 Aspm, tl1, f2k, Plac8, wild-type wild-type change s12, His tb2, Cd28, His T T Gm10210, Ccdc109b, Mybbp1a, Snor rib2, r fl/fl gene Gm5424, fraction a 55.8% P4 , Tbc1d5, Kit, T t1h2bq, of t1h2bc, v12n-2, Sm Epc Hdac4, uba4a, Rdm1, Aldh2, Rpl35, Nipbl, T Dgk d15a, Lrig1, et Pld4, A Cd4, DP- T y Ldlr line am, rio tl2, d2, -iCre rp53 set al. 13.7% of d, , ,

P5 DP -/-

Pmaip1/Noxa log2 Fold Change Cdkn1a Sac3d1 Rnf19b Phlda3 Rps27l Epha2 Zmat3 Mast4 Fuca1 Dyrk3 Vwce Padi4 Bbc3 Enc1 Ninj1 Rrad Plk2 Aen Bax Fas S5 fl/fl F ACS-sorted DP T rp53

-/- Thymocytes hCD2-iCre S5 fl/fl

hCD2-iCre S5 T rp53 fl/fl -/-

-2 -1 0 1 2