Transcription Factor Zinc Finger and BTB Domain 1 Is Essential for Lymphocyte Development

This information is current as Divya Punwani, Karen Simon, Youngnim Choi, Amalia of September 28, 2021. Dutra, Diana Gonzalez-Espinosa, Evgenia Pak, Martin Naradikian, Chang-Hwa Song, Jenny Zhang, David M. Bodine and Jennifer M. Puck J Immunol published online 29 June 2012

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 29, 2012, doi:10.4049/jimmunol.1200623 The Journal of Immunology

Transcription Factor Zinc Finger and BTB Domain 1 Is Essential for Lymphocyte Development

Divya Punwani,*,1 Karen Simon,†,1 Youngnim Choi,‡ Amalia Dutra,† Diana Gonzalez-Espinosa,* Evgenia Pak,† Martin Naradikian,*,x Chang-Hwa Song,*,{ Jenny Zhang,* David M. Bodine,† and Jennifer M. Puck*

Absent T lymphocytes were unexpectedly found in homozygotes of a transgenic mouse from an unrelated project. T cell develop- ment did not progress beyond double-negative stage 1 thymocytes, resulting in a hypocellular, vestigial thymus. B cells were present, but NK cell number and B cell isotype switching were reduced. Transplantation of wild-type hematopoietic cells corrected the defect, which was traced to a deletion involving five contiguous at the transgene insertion site on 12C3. Com- plementation using bacterial artificial chromosome transgenesis implicated zinc finger BTB-POZ domain 1 (Zbtb1) in the immunodeficiency, confirming its role in T cell development and suggesting involvement in B and NK cell differentiation. Targeted Downloaded from disruption of Zbtb1 recapitulated the T2B+NK2 SCID phenotype of the original transgenic animal. Knockouts for Zbtb1 had expanded populations of bone marrow hematopoietic stem cells and also multipotent and early lymphoid lineages, suggesting a differentiation bottleneck for common lymphoid progenitors. Expression of mRNA encoding Zbtb1, a predicted transcription repressor, was greatest in hematopoietic stem cells, thymocytes, and pre-B cells, highlighting its essential role in lymphoid devel- opment. The Journal of Immunology, 2012, 189: 000–000. http://www.jimmunol.org/

evelopment of lymphocytes from hematopoietic stem DN1–DN4, after which they undergo TCR rearrangement and cells (HSCs) is incompletely understood. Current models become CD4+CD8+ double-positive (DP) cells (5). DP cells un- D suggest that T, B, and NK cell precursors are predomi- dergo selection, with surviving self-tolerant CD8+ or CD4+ single- nantly derived from common lymphoid progenitors (CLPs) in bone positive, naive T cells emerging from the thymus to populate marrow (BM) (1). CLPs can differentiate into all-lymphoid pro- peripheral lymphoid organs. The zinc finger BTB-POZ (Zbtb) genitors (ALPs), which retain full lymphoid differentiation po- transcription factor 7 (Zbtb7/Th-POK/cKrox) is crucial for tential, forming both T cell progenitors that seed the thymus and switching a fraction of DP cells from the default CD8+ pathway B cell-biased lymphoid progenitors that become B cells (2). to form CD4+ cells (6, 7). Identification of additional signaling by guest on September 28, 2021 Notch-1 signals are required to redirect common lymphoid pro- factors at developmental branch points is required to understand genitors from the default B lineage to the T lineage (3, 4). After lymphoid lineage commitment and differentiation in both mice migration to the thymus, T lineage precursors proceed through and humans. distinct CD42CD82 (double-negative, or DN) thymic stages, Human SCID syndromes, defined by profound defects in both cellular and humoral immunity, comprise experiments of nature that have revealed many nonredundant, critical functions in human *Department of Pediatrics, University of California San Francisco, San Francisco, CA 91413; †National Research Institute, National Institutes of lymphoid developmental pathways. Human SCID is caused by Health, Bethesda, MD 20892; ‡Department of Oromaxillofacial Infection and Immu- defects in a wide variety of genes encoding necessary and nity, School of Dentistry, Seoul National University, Seoul 110-744, South Korea; x { specific for lymphocyte differentiation, proliferation, and activation University of Pennsylvania, Philadelphia, Pennsylvania, PA 19104; and Department of Microbiology, College of Medicine, Chungnam National University, Daejeon 301- (8–11). About two-thirds of SCID patients lack T cells but have 2 2 747, South Korea B cells that fail to make specific Abs (T B+ phenotype); T B+ 1D.P. and K.S. contributed equally to this work and are listed in alphabetical order. SCID can be caused by defects in the X-linked common g-chain Received for publication February 23, 2012. Accepted for publication May 31, 2012. receptor for IL-2, -7, -9, -15, and -21; the IL-7 receptor a-chain; This work was supported in part by National Institutes of Health Grant R01 intracellular cytokine signal transducers JAK3 or STAT5; protein AI078248 (to J.M.P.) and by the Division of Intramural Research, National Human tyrosine phosphatase receptor CD45; or components of the CD3 Genome Research Institute, National Institutes of Health. receptor. Patients with these forms of SCID are in contrast to those D.P., K.S., Y.C., M.N., C.-H.S., J.Z., D.G.-E., E.P., and A.D. designed and performed with T2B2 SCID, whose defects may lie in mediators of T and BCR experiments, prepared figures, and analyzed data; D.M.B. codesigned experiments and provided essential tools and guidance; and J.M.P. designed and supervised the rearrangement, including RAG1, RAG2, and Artemis, or in research, performed experiments, and wrote the article with D.P. and K.S. adenosine deaminase or purine nucleoside phosphorylase, which Address correspondence are reprint requests to Dr. Jennifer M. Puck, Department of normally remove purine intermediates that are toxic to lymphocytes. Pediatrics, University of California San Francisco, Box 0519, 513 Parnassus Avenue, Up to 10% of humans with SCID have other rare, or as yet un- HSE 301A, San Francisco, CA 94143-0519. E-mail address: [email protected] identified, genetic defects. The online version of this article contains supplemental material. In contrast to humans, the most studied mouse models for SCID Abbreviations used in this article: ALP, all-lymphoid progenitor; BAC, bacterial have the T2B2 phenotype, including the scid mouse deficient in artificial chromosome; BM, bone marrow; BMT, bone marrow transplantation; CLP, common lymphoid progenitor; DN, double-negative; DP, double-positive; the DNA-dependent kinase DNA-PKc, and Rag1/Rag2-deficient eGFP, enhanced GFP; ES, embryonic stem; FISH, fluorescence in situ hybridization; mice, all of which fail to complete V(D)J rearrangement of Ab and HDAC, histone deacetylase; HSC, hematopoietic stem cell; IRES, internal ribosome entry site; MPP, multipotent progenitor; MSCV, murine stem cell virus; tg, trans- TCR genes during B and T cell development (12–17). These and genic; Zbtb, zinc finger BTB-POZ. the the athymic nude mouse lacking transcription factor Foxn1

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200623 2 Zbtb1 IS ESSENTIAL FOR LYMPHOCYTE DEVELOPMENT

(18, 19) have greatly facilitated basic studies of lymphoid cell BM transplantation maturation after the commitment to the T and B lineages has been Donor BM cell suspensions were harvested by flushing femurs and tibias made, but they have not permitted dissection of prethymic lym- with PBS and straining. HSCs were enriched by depletion with biotinylated phoid development. Moreover, lymphocyte phenotypes are dis- Abs (BD Biosciences) to lineage markers CD3, CD4, CD8, B220, Gr-1, and cordant between mice and humans with disruption of most human Ter-119, followed by addition of streptavidin microbeads and purification SCID genes, limiting the utility of mouse knockouts for illumi- using a MACS column (Miltenyi Biotec, Auburn, CA). Two million cells were injected by tail vein into nine gray irradiated recipients. Lineage nating mechanisms for human T lineage commitment or to serve engraftment was determined in blood and tissues after 4 mo. Experiments as faithful models for optimizing human SCID therapies. For were performed in duplicate. example, while humans with gc and JAK3 defects have T2B+ NK2 SCID, mice with targeted disruption of these genes have Bacterial artificial chromosome isolation, cytogenetics, and T cells, but lack B cells (T+B2NK2) (20). mapping 2 2 AT B+NK SCID phenotype was unexpectedly observed in Metaphases from Con A-stimulated splenocytes from heterozygous trans- homozygotes of one of five mouse lines transgenic for a Fas genic (tg/+) animals were prepared as described (23). After fluorescence in cDNA bearing a dominant death domain mutation (21). No off- situ hybridization (FISH) with the mFas plasmid labeled using cychrome (yellow) showed a signal on chromosome 12C-D, PCR-amplified segments spring of other transgenic founders lacked T cells, suggesting a from genes in this region were used to screen murine RPCI-23 bacterial recessive defect related to the insertion site rather than to the artificial chromosome (BAC) library filters (24). DNA from positive BAC expression of mutant Fas. This mouse presented a unique oppor- clones containing Sos2 (RP23-353H11), Esr2 (RP23-351E10), Hspa2 tunity to develop a phenotypic model similar to human T2B+NK2 (RP23-901C4), and Psen1 (RP23-291K4) was prepared and labeled by standard methods. Cychrome-labeled mFas DNA and BACs labeled with Downloaded from SCID, to dissect requirements for lineage differentiation of lym- either rhodamine (red) or FITC (green) were used together as FISH probes. phoid progenitors, and to identify a critical step in the decision of At least 20 cells with each combination were scored. CLPs and ALPs to become B versus T versus NK cells. Moreover, Additional mouse BAC clones containing Hspa2 (RP23-191N23 and the underlying defect turned out to be in Zbtb1, a transcription RP23-90K4) were obtained and aligned by PCR content in wild-type versus repressor of the zinc finger BTB domain family very recently homozygous transgenic (tg/tg) mice. Primer pairs from nearby flanking genes and intragenic segments were used to walk from centromeric and telomeric recognized to be involved in lymphoid cell development (22). undisturbed regions toward the insertion/deletion locus. PCR was performed

While the absence of peripheral T cells in our Zbtb1 knockout using RTG PCR beads (Amersham Pharmacia Biotech, Piscataway, NJ). http://www.jimmunol.org/ mouse confirms findings recently reported in the scanT mouse with a missense variant of Zbtb1 (22), our more detailed study of BAC transgenic mice for complementation hematopoietic stem cells, lymphoid progenitors, and lymphocyte BAC DNA for transgenic injections was isolated by a NucleoBond BAC subpopulations sheds light on Zbtb1 and its role in the develop- Maxi Kit (Clontech, Mountain View, CA) and verified by pulse field gel ment of T, B, and NK cells. electrophosesis after NotI digestion. BACs 90K4 and 191N23 were line- arized with BsiWI and AscI, respectively. BAC DNAwas injected into FVB/ N fertilized eggs, and three resultant animals for each BAC with germline Materials and Methods transmission were crossed to generate BAC-bearing tg/tg F2 mice for study. PCR with primers specific for genes located within the BACs confirmed

Mice BAC content. Animals were bred to determine fertility; immunopheno- by guest on September 28, 2021 Transgenic mouse line 26A with six tandem copies of mFas D231V has typing was carried out as above. been described previously (21). The SCID phenotype in homozygotes was achieved by heterozygous crosses. Wild-type mouse strains in this study Retrovirus rescue of the SCID phenotype were from The Jackson Laboratory (Bar Harbor, ME). Mice were fed A 2.3-kb EcoRI fragment containing Zbtb1 coding exon 2 was inserted into autoclaved chow and antibiotics and were housed in sterile isolator cages, the 6.5-kb EcoRI-linearized murine stem cell virus (MSCV)-internal undergoing procedures according to approved protocols at the National ribosome entry site (IRES)-GFP vector (provided by Art Nienhuis, St. Human Genome Institute at the National Institutes of Health (Bethesda, Jude Children’s Research Hospital, Memphis, TN). Full sequencing as- MD) and the University of California San Francisco (San Francisco, CA). sured forward orientation and integrity. MSCV-Zbtb1-IRES-GFP was Hspa2 knockout mice were provided by Dr. Mitchell Eddy (National In- transfected into AmphoPack 293 cells (Clontech) using a CellPhect kit stitute on Environmental Health Sciences, Research Triangle Park, NC). (GE Healthcare/Amersham Biosciences, Piscataway, NJ), and supernatant was used for transduction of GP+E-86 cells (25). GP+E-86 clones Lymphocyte subset enumeration expressing GFP were selected by FACS and expanded. Supernatants were Cell suspensions were prepared from blood, thymus, spleen, and BM and titered on NIH 3T3 cells and analyzed by slot blot to determine the clone stained according to standard protocols with mAbs (BD Biosciences, San with the highest GFP expression. Jose, CA; BioLegend/eBioscience, San Diego, CA). Flow cytometry was BM was harvested from tg/tg mice and their wild-type littermates 48 h after performed on an LSRII FACS machine (BD Biosciences) and analyzed with injection with 5-fluorouracil (Fluka/Sigma-Aldrich, St. Louis, MO) (26). BM FlowJo software (Tree Star, Ashland, OR). Complete and differential white cells were cultured and transduced with MSCV-Zbtb1-IRES-GFP as previ- 3 6 blood counts were performed by the Clinical Pathology Department at the ously described (27). Tg/tg mice irradiated with 5 Gy received 2 10 cells National Institutes of Health. in 200 ml by tail vein. Four months after BM transplantation (BMT), they were euthanized and immune phenotype was determined. DNA from Con A- Histopathology stimulated T cells and peripheral blood was extracted using a Puregene kit (Qiagen, Valencia, CA). PCR was performed using vector primers MSCV- Tissues collected in fixative according to standard protocols were sectioned and GFP 4128F (59-TGCCCAGAAGGTACCCCATTGT-39) and MSCV-IRES- stained with H&E at American Histolabs (Gaithersburg, MD) and analyzed 4475R (59-ACGCCGTAGGTCAGGATGGTCAC-39). Amplification con- with the assistance of Dr. Michael Eckhaus (Veterinary Resource Program, ditions were 94˚C for 2 min, followed by 94˚C for 30 s, 65˚C for 30 s, and National Institutes of Health). Testes from mice were fixed in Bouin’s solu- 72˚C for 45 s for 35 cycles, then 72˚C for 5 min. tion. Peroxidase immunostaining with polyclonal rabbit anti-Hspa2 antiserum 2A (provided by Dr. Mitchell Eddy) was performed by Histoserv (German- Production of Zbtb1 gene-targeted mice town, MD). Zbtb1 coding exon 2 was replaced by enhanced GFP (eGFP) using a pPNT- T cell functional tests pgk-Neo vector. Homologous regions flanking Zbtb1 exon 2 were made from C57BL/6 genomic DNA by PCR using SuperMix High Fidelity Splenocytes were collected and culturedinConAfor48hasdescribed(21). (Invitrogen, Carlsbad, CA). The coding region of eGFP from pIRES2- BrdU incorporation after 48 h incubation was analyzed using a BD Bio- eGFP (Clontech) was cloned into pBluescript SK+. For assembly, the 4.5- sciences LSRII flow cytometer. Cytotoxicity was measured using the Prom- kb 59 homologous segment released by XhoI and BspHI digestion was ega Cytotox 96 kit (Promega, Madison, WI) following the manufacturer’s ligated into the pBluescript-eGFP digested with NcoI and XhoI. The 2.4- instructions. kb 39 homologous segment was ligated into pPNT using KpnI and EcoRI The Journal of Immunology 3 sites. Finally, the 59 arm and eGFP fragment were released from pBlue- deficiency when the transgene was bred to homozygosoity. Het- script with XhoI and SalI, gel purified, and ligated into the XhoI site of the erozygotes from all lines and homozygotes from lines other than pPNT construct, followed by electroporation into GeneHog competent 26A had mild hepatic infiltration with T cells, but did not have cells (Invitrogen). Sequence-verified targeting construct DNA was linear- ized and injected at 1 mg/ml into C57BL/6 embryonic stem (ES) cells. the phenotype of Fas-deficient MRLlpr mice or humans with Injected ES clones under Neo selection were analyzed for homologous autoimmune lymphoproliferative syndrome/autoimmunity, excess recombination by Southern blotting. Only one of three clones gave rise to CD42CD82 DN T cells, and defective apoptosis (29). Instead, the germ line-transmitting chimeras following injection of ES cells into C57BL/ transgenic animals were healthy and indistinguishable from wild- 6 blastocysts. Heterozygotes bred from this clone generated seven wild- type, nine heterozygote, and eight homozygote offspring of both sexes, type (+/+) mice. However, homozygotes of line 26A (tg/tg) had indicating that Zbtb1 is not necessary for embryonic development. undetectable T and markedly reduced B and NK cells in peripheral blood, although B cells were present in spleens (Fig. 1A, 1B). Real-time PCR for Zbtb1 expression Peripheral blood myeloid cells were slightly decreased in tg/tg Expression levels of the two Zbtb1 splice variants were determined by mice whereas erythrocytes and platelets were normal (Supple- quantitative real-time PCR. RNA was isolated from tissues and FACS- mental Fig. 1A). Spleens in tg/tg mice were 50% of normal sorted subpopulations of BM, thymus, and spleen from 6- to 8-wk-old C57BL/6 mice. B cell subsets were sorted using markers described by weight, with only 3% T cells but with an increased proportion of Hardy and Hayakawa (28). RNA was prepared using the Qiagen RNeasy B cells to 60% (Fig. 1B) and normal absolute B cell numbers. Tg/tg kit (Qiagen, Chatsworth, CA). All samples were treated with DNase I. mice showed poor proliferation to Con A and absent T cell cyto- RNA quality was assessed by A260/280 ratio (ND-100 spectrophotometer; toxicity (Fig. 1C, 1D). They also had impaired isotype switching of NanoDrop Technologies, Wilmington, DE) and gel electrophoresis. A the B cells, indicated by increased serum concentrations of IgM and high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA) was used to prepare cDNA with 1 mg RNA from tissues or 200 ng from decreased IgG and IgA (Fig. 1E). The phenotype was invariant with Downloaded from flow-sorted subpopulations. Absolute copy numbers of the two splice ageupto8mo. variant transcripts, Zbtb1713 and Zbtb1644, as well as those of housekeeping In contrast to their unaffected +/+ littermates, tg/tg mice had only genes Pol2Ra and HPRT were assessed for normalization (primer a vestigial thymus (Fig. 1F). Typical of most human SCID cases, sequences are available on request). Amplified cDNA fragments were cloned into TOPO vector pCR2.1. thymic epithelial and stromal cells were present, but cortical areas Plasmids were prepared, sequence verified, and serially diluted to generate were undeveloped and lymphoid cells were rare to absent. Mes- 3 7 standards from 4 to 400 10 copies/well. Real-time PCR was performed enteric lymph nodes in tg/tg animals, when located, were small; http://www.jimmunol.org/ in TaqMan Universal PCR Master Mix using 100 nM each primer and 200 axillary and inguinal lymph nodes were not found. The infertility nM FAM- and TAMRA-labeled oligonucleotide probe (primer and probe of male tg/tg mice was due to arrested spermatocyte development sequences are available on request) in an ABI Prism 7900HT system (Applied Biosystems). Cycle conditions were 10 min at 95˚C, followed by (Supplemental Fig. 1B). 95˚C for 30 s and 60˚C for 1 min for 40 cycles. All samples were analyzed in duplicate. Copy numbers were determined by SDS 2.1 detection soft- BMT to define the cellular lesion in tg/tg SCID mice ware (Applied Biosystems), and data were normalized to obtain test gene To determine whether the SCID phenotype of tg/tg mice was HSC copies per 1000 copies of HPRT and Polr2a. autonomous or attributable to a dysfunctional thymic environment, B cell proliferation assays lineage-depleted BM from +/+ mice was injected into irradiated tg/ by guest on September 28, 2021 Splenocytes were collected from Zbtb1 knockout, wild-type, and TCRa tg mice as well as into irradiated control heterozygous littermates. knockout mice, and T cells were depleted using the Pan T cell isolation kit After 4 mo, reconstitution in tg/tg mice was equivalent to that in (Miltenyi Biotec). B cells (1 3 105) were added to each well of a 96-well controls (Supplemental Fig. 1D, Supplemental Table IA). Not only round-bottom plate and incubated with LPS (0.1 or 10 ng/ml), anti-IgM did CD3, CD4, and CD8 T cells appear in numbers equivalent to (0.1 or 10 ng/ml), or anti-CD40 (0.1 or 10 ng/ml) in RPMI 1640 with 10% FCS, penicillin and streptomycin, nonessential amino acids, and 5 mM those of control mice, but also T cell proliferation to Con A and BrdU (Invitrogen). BrdU incorporation after 48 h incubation was analyzed cytotoxicity against P815 target cells was normalized. Addition- using a BD Biosciences LSRII flow cytometer. ally, serum IgM, IgG, and IgA levels became normal, indicating Serum Ig assays recovery of isotype switching (data not shown). When the reciprocal BMT was performed using lineage-depleted Serum collected in BD Microtainer separator tubes (Becton Dickenson, FVB/N tg/+ or tg/tg SCID mouse cells to reconstitute irradiated Franklin Lakes, NJ) had levels of IgM, IgG, and IgA assayed using an 3 ELISA mouse Ig quantitation kit (Bethyl Laboratories, Montgomery, TX). C57BL/6 FVB/N F1 mice, the recipients developed fewer T and Data were analyzed using an unpaired t test with a Welch correction (Prism B cells than did recipients of tg/+ heterozygous BMT. Even 5; GraphPad Software, La Jolla, CA). though hemoglobin analysis indicated 100% donor engraftment, Western blotting and immunoprecipitation lineage chimerism using PCR with primers specific for C57BL/6 and FVB/N strains showed failure to engraft T and B cells (Sup- Whole-cell, nuclear, and cytoplasmic 293T cell extracts were prepared 24 plemental Fig. 1E, Supplemental Table IB). Proliferation and and 48 h following transfection of cloned human or murine cDNA encoding cytotoxic function remained poor, and IgM levels rose even Zbtb1713 and Zbtb1644 isoforms with N- or C-terminal, FLAG, or myc epitope tags. Aliquots were subjected to electrophoresis, transfer, and through IgA and IgG levels were normal, with the latter attributed immunoblotting using reagents and methods from eBioscience. Anti- to radio-resistant recipient mouse plasma cells (data not shown). FLAG (Sigma-Aldrich) or anti-myc (Santa Cruz Biotechnology, Santa Thus, the SCID defect was intrinsic to HSCs, did not affect my- Cruz, CA) mouse mAbs followed by HRP-anti-mouse IgG (Santa Cruz eloid progenitors, and was limited to lymphoid lineages, mainly Biotechnology) were applied and detected by the SuperSignal substrate detection system (Pierce Protein Research Products/Invitrogen). affecting the generation of T and NK cells. For immunoprecipitations, cell lysates from transfected 293T cells were mixed with primary Ab and rocked overnight with Sepharose beads conju- Mapping and identification of the genetic locus for gated to protein A. After washing the beads, protein was released by boiling in immunodeficiency gel-loading buffer. Electrophoresis and detection were carried out as above. Labeled plasmid containing the 6-kb mFas transgene was used as a FISH probe to map the integration site on metaphase chromo- Results some spreads from heterozygous tg/+ animals; a single hybrid- SCID and infertility in a transgenic mouse ization signal was found on mouse chromosome 12qC-D. BAC Of five lines of FVB/N mice transgenic for mutant Fas D231V (21), clones containing known mouse genes were used as probes in one line, 26A, exhibited male infertility and profound immuno- three-color hybridizations to narrow the region (Fig. 2A). The 4 Zbtb1 IS ESSENTIAL FOR LYMPHOCYTE DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Characterization of homozygous transgenic mice. (A) Peripheral blood and (B) splenic lymphocyte subsets from wild-type (+/+), hetero- zygous (tg/+), and homozygous transgenic (tg/tg) mice. (C) Proliferation of splenic T cells after Con A stimulation, measured by BrdU incorporation. (D) Killing of P815 target cells. (E) Serum Ig levels. (F) Thymus histology of wild type (+/+) compared with homozygous transgenic (tg/tg) mice, using H&E staining, original magnification 340. transgene signal (yellow) was found between BACs containing Fig. 1C). Lack of Hspa2 thus explained the male infertility in tg/tg Sos2 (green) and Psen1 (red) (Fig. 2Ai). Additional BACs within mice. this interval were further used to refine the transgene location. A Boundaries of the tg insertion/deletion were established by PCR BAC containing Esr2 (red) was centromeric to the transgene, mapping with primer pairs selected from the mouse genome se- whereas one with Hspa2 (green) was replaced by the plasmid quence (Fig. 2B). Positive PCR amplification in +/+ and tg/+ mice signal (yellow) on one copy of chromosome 12 (Fig. 2Aii), sug- with failure in tg/tg mice indicated deleted sequences (Fig. 2B, gesting that partial or full deletion of Hspa2, perhaps with sur- hatched area, top). The tg allele was deleted for hypothetical genes rounding genes, had occurred when the transgene was integrated AK133227, AK136613, and a portion of Plekhg3 on the telomeric into mouse chromosome 12. We thus hypothesized that one or side of Hspa2 and two genes in the Zbtb family, Zbtb1 and Zbtb25, more genes disrupted by the transgene integration accounted for on the centromeric side. the SCID phenotype of tg/tg mice. Hspa2, encoding a 70-kDa heat shock chaperone protein, had Implication of Zbtb1 in immunodeficiency been knocked out in mice and found to be essential for male With Hspa2 ruled out as the immunodeficiency gene, we used fertility (30, 31). However, a role for Hspa2 in the immune a complementation approach with transgenesis of BAC clones system had not been studied. Hspa2 knockouts, provided by Dr. 191N23 and 90K4, extending to the telomeric and centromeric Mitchell Eddy, had normal T and NK cells and normal Ab levels sides of Hspa2, respectively (Fig. 2B), to evaluate the ability of (data not shown), ruling out Hspa2 as a candidate gene for the tg/ the candidate genes to rescue immunity and fertility. After injec- tg immunodeficiency. However, Hspa2 protein was absent in tg/ tions into blastocysts from the original tg/tg mice, three lines of tg mice as expected, shown by lack of staining with polyclonal mice with each BAC were produced and crossed to mFas tg/+ anti-Hspa2 Ab, also provided by Dr. Mitchell Eddy (Supplemental mice to generate tg/tg BAC-bearing animals. Tg/tg mice carry- The Journal of Immunology 5

FIGURE 2. Mapping and identification of the ge- netic locus for immunodeficiency. (A) FISH localiza- tion of tg insertion (cychrome, yellow): (i) telomeric to a BAC encoding Sos2 (FITC, green) and centromeric to Psen1 (rhodamine, red); and (ii) telomeric to Esr2 (rhodamine, red) and colocalizing with and replacing Hspa2 (FITC, green). Nuclei counterstained with DAPI (blue), original magnification 3100. (B) Map of mouse chromosome 12C3 showing order and direction of transcription of genes Hspa2 (open box) and Zbtb1 Downloaded from (black) and transcripts Zbtb25, AK133227, AK136613, and Plakhg3 (gray); hatched rectangle above shows transgene insertion/deletion region; black lines below show BAC genomic clones (ends indicated by black dots) used for rescue transgenesis. (C) Immune and fertility phenotypes of wild-type, original mFas tg/tg, http://www.jimmunol.org/ and tg/tg BAC animals containing the indicated BAC sequences 90K4 partial, 90K4 complete, and 191N23 (means 6 SD; six mice per group). by guest on September 28, 2021

ing BAC clone 191N23 were fertile, as expected with restoration Targeted disruption of Zbtb1 of Hspa2, but retained the immunodeficient phenotype with absent To prove that Zbtb1 was necessary and sufficient to cause the T cells and T cell function, elevated IgM, and low IgG and IgA observed immunodeficiency of tg/tg mice, we replaced exon 2 of (Fig. 2C, right column). Only mice with the full-length 90K4 BAC Zbtb1 with a GFP-pgk-Neo cassette to produce a gene-targeted were fertile with intact immunity, as shown in Fig. 2C (90K4 mouse (Fig. 3A). The first exon of the Zbtb1 gene is noncoding, complete). In one mouse line, spontaneous breakage of BAC 90K4 with the ATG start of translation at nucleotide 19 in exon 2; resulted in restoration of the coding portion of Zbtb25 (Fig. 2C, therefore, disruption of exon 2 would obliterate Zbtb1 gene ex- 90K4 partial). Crosses of the BAC transgenics with the mFas tg/tg pression. After transfection of C57BL6/J ES cells and injection mice revealed that triple transgenics homozygous for the original into blastocysts, we obtained three founder mice, one of which tg/tg insertion/deletion, and carrying the 191N23 BAC (restoring passed the targeted disruption to offspring. Zbtb1 knockout F2 Hspa2, AK133227, AK136613, and Plekhg3) plus the partial 90K4 mice were generated and were fertile (but unfortunately failed to BAC (possibly restoring Zbtb25, but not Zbtb1), were fertile, but express GFP). Whereas Zbtb1 heterozygous (+/2) and wild type were affected with SCID. These experiments suggested that loss (+/+) littermates had normal T, NK, and B cell numbers in the of Zbtb1, or of both Zbtb1 and Zbtb25, was necessary and suffi- periphery, Zbtb1 knockout (2/2) mice showed nearly absent 2 + 2 cient to produce T B NK SCID in the mouse. T cells, reduced NK cells, and normal B cells (Fig. 3B). Addi- To determine whether absent Zbtb1 alone was the cause of SCID, tionally, splenic T cells, which were very few in number, showed we used a retroviral vector containing the single large coding exon a complete lack of proliferative and cytotoxic function (Supple- of Zbtb1 to transduce BM stem cells from tg/tg or +/+ control donor mental Fig. 2A). The thymus and lymph nodes were vestigial in mice and injected these transduced cells to rescue lethally irradiated Zbtb1 knockout mice, and the spleen was half the weight of tg/tg mice. At 4 mo after BMT, T cells were found in peripheral spleens of littermate controls (data not shown). Nonlymphoid cells blood and spleen, although normal numbers were not completely in the peripheral blood were unaffected (Supplemental Fig. 2B). restored (Supplemental Table IC). However, serum Ig levels were The phenotype of the Zbtb1 knockout mice therefore replicated comparable to wild-type mice, and T cell proliferation and cyto- that of the original transgenic mice, as well as the recently re- toxicity were normal (Supplemental Fig. 1F), indicating that res- ported scanT mice with a missense mutation in Zbtb1 (22). De- toration of Zbtb1 restored immunity, curing the SCID phenotype. tailed characterization of knockout mice enabled us to define the 6 Zbtb1 IS ESSENTIAL FOR LYMPHOCYTE DEVELOPMENT Downloaded from

FIGURE 3. Targeted disruption of Zbtb1 and characterization of knockout mice. (A) Gene-targeting construct, pPNT-Zbtb1 59-eGFP-Neo- Zbtb1-39 (15.338 kb, above), and homologous B6 genomic target locus (below). (B) PBL phenotype of knockout transmitting founder offspring: http://www.jimmunol.org/ wild-type, Zbtb1 heterozygous (+/2), and Zbtb1 knockout (2/2) mice. T cells detected as CD3+; B cells, CD19+; NK cells, NK1.1+. phenotype resulting from complete absence of the Zbtb1 protein versus that resulting from published missense mutation C74R, therefore further delineating the role of Zbtb1 in hematopoietic stem cell differentiation and lymphoid development. FIGURE 4. Analysis of thymocytes and BM subsets in knockout mice. 2 2 2 Investigation of embryonic thymi in Zbtb1-deficient mice (A) Day 15.5 and day 17.5 fetal TCRb CD4 CD8 DN thymocytes in wild- by guest on September 28, 2021 type (+/+), heterozygous (tg/+), and homozygous transgenic (tg/tg) mice. Because of the absence of mature T cells in the periphery and 2 Percentages are means 6 SD of 10 fetuses/group. DN1, CD25 CD44+; a hypocellular, vestigial thymus in adult knockout mice lacking DN2, CD25+CD44+;DN3,CD25+CD442.(B) DN subsets from adult wild- Zbtb1, embryonic thymi were analyzed to determine at which stage type and Zbtb1 knockout mice; DN1–DN3, upper plots; subdivided DN1 T cell development was arrested. Thymocytes were identified accord- (lower plots) with DN1a, CD117+CD242; DN1b, CD117+CD24lo;DN1c, ing to their differentiation status, with the earliest arrivals from the CD117+CD24+; DN1d, CD1172CD24+; and DN1e, CD1172CD242 (34). 2 2 2 2 BM being DN1 cells, CD4 CD8 TCRb CD25 CD44+;successive (C) DN1 cell subsets per thymus (n = 2/genotype). (D) BM cell subsets from maturation to DN2 CD25+CD44+ and then DN3 CD25+CD442 cells wild-type and Zbtb1 knockout mice, long-term HSCs (LT HSC; LSK, 2 2 + 2 was monitored by cell surface marker content (32), with progression CD34 ,CD48 ), short-term HSCs (ST HSC; LSK, CD34 ,CD48 ), MPPs + + + + beyond the DN3 stage indicated by rearrangement and expression of (LSK, CD34 ,CD48), and CLPs (LSK, CD127 , CD135 ). Data represent 6 , , the TCRb genes (33). When day 15.5 and day 17.5 mouse embryos means SD (10 mice/group). *p 0.05, **p 0.001. from the original tg insertion/deletion were genotyped by FISH and examined for thymocyte maturation, tg/tg mice had reduced but detectable DN1, but very few DN2 and DN3 cells (Fig. 4A) and long-term HSCs (LSK, CD34-, CD48-), short-term HSCs (LSK, + + essentially no DN4, DP, or single-positive T cells (data not shown). CD34 , CD48-), multipotent progenitors (MPPs) (LSK, CD34 , + + + Further analysis of the DN1 population as described by Porritt et al. CD48 ), and CLPs (LSK, CD127 , CD135 ) (35, 36) were de- (34) (Fig. 4B, 4C) showed low, but detectable, numbers of DN1a termined from the Zbtb1 knockout versus wild-type mice . Zbtb1 (c-Kit/CD117+,HSA/CD242) and DN1b (c-Kit/CD117+, HSA/ knockout mice had equivalent numbers of long-term HSCs but CD24lo) cells, which are thought to represent the major pathway more cells of the other early subsets, short-term HSCs, MPPs, and for expansion and differentiation from BM progenitors to DN2 CLPs (Fig. 4C). This could reflect feedback mechanisms in an thymocytes. DN1c cells (c-Kit/CD117+, HSA/CD24+), which are attempt by the BM to make up for the reduced number of mature thought to retain B lineage developmental potential, were also T cells in the periphery. Although the BM was able to produce detectable, but there were essentially no CD1172 DN1d or DN1e lymphoid progenitors in the absence of Zbtb1, these progenitors cells, which have poor proliferative potential and do not mature did not appear in the thymus. into T cells. This pattern indicates that T cell progenitors lacking Our observations contrast with the report on scanT mice in Zbtb1, although present in the thymus in small numbers, were which numbers and percentages of all hematopoietic subsets were unable to progress through thymic differentiation. equal to those of wild-type mice (22). This could be because the 2/2 Zbtb1 protein, although mutated in the scanT mice, could still Stem cell subpopulations in Zbtb1 mice retain residual function and influence HSC numbers and differ- To determine prethymic effects of knocking out Zbtb1, absolute entiation, whereas in our knockout mice, the complete absence of numbers of cells comprising the different BM stem cell subsets, Zbtb1 protein affects the maintenance of stem cell numbers. The Journal of Immunology 7

BM and spleen B cell subsets in Zbtb1 knockout mice impaired at the pre-B cell stage in the absence of Zbtb1, suggesting Because the absence of Zbtb1 resulted in lack of T cells in the that Zbtb1 is important during early B cell development, the number periphery and spleen, but did not prevent B cell development, B cell of mature B cells in the periphery was comparable to that in wild- type controls. This could be due to redundancy of the function of subsets (28) in both BM and spleen were analyzed to determine Zbtb1 in peripheral B cells and/or compensation for its functions by whether the absence of Zbtb1 affected their proportions. Wild-type other members of the same protein family or through different and TCRa knockout mice were used as controls to evaluate the signaling pathways. absence of T cell help as a contributor to any alterations in B cell It is difficult to distinguish a B cell-intrinsic defect from altered subsets. No differences in pro-B cell frequencies in the BM were proportions of B cells at different stages of development. However, observed across the three mouse phenotypes. However, Zbtb1 in vitro proliferation of B cells from Zbtb1 knockout mice, wild- knockout mice had fewer pre-B cells compared with TCRa type littermates, and TCRa knockout mice, in response to anti- knockout and wild-type mice (Fig. 5A, Supplemental Fig. 2Ci). IgM, LPS, and anti-CD40, showed that B cells from the Zbtb1 When the B cell subsets in the spleens were analyzed, Zbtb1 knockout mice proliferated at least as much or more than those knockout mice were found to exhibit higher proportions of marginal from wild-type or TCRa knockout mice (Fig. 5C). The brisk zone B cells and higher proportions of follicular B cells than did proliferation of B cells from the Zbtb1 knockout mice could be either wild-type or TCRa knockout mice (Fig. 5B, Supplemental accounted for by the increased proportion of marginal zone cells Fig. 2Cii). These findings were consistent with the phenotype of the in their spleens (Fig. 5B), since marginal zone cells proliferate scanT mice (22). Thus, although B cell development appeared to be more than follicular B cells (37–39). Downloaded from http://www.jimmunol.org/

FIGURE 5. Analysis of B cells of knockout mice. (A) Relative pro- portions of BM B cell subsets (wild- type, Zbtb12/2, and TCRa2/2 mice) as defined by the Hardy and Hay- by guest on September 28, 2021 akawa gating scheme: I, pro-B (B220+CD43+); II, pre-B (B220+ CD432); III, pre-B late (B220+ CD432IgD2IgM2); IV, immature B (B220+CD432IgD2IgM+); V, ma- ture recirculating B (B220+CD432 IgD+IgM+). (B) Relative proportions of splenic B cell subsets in wild type, Zbtb12/2,andTCRa2/2 mice: I, im- mature B (B220+IgDhiIgMloCD232 CD21/352); II, marginal zone (B220+ IgDhiIgMloCD23loCD21/CD35hi); III, follicular (B220+IgDloIgMhiCD432 CD52CD23hiCD21/CD35mid). Data represent means 6 SD (four mice per genotype). *p , 0.1, **p , 0.05. (C) Wild-type, Zbtb12/2,andTCRa2/2 splenic B cell proliferation after stim- ulation with LPS (0.1 and 10 ng/ml), anti-IgM (0.1 and 10 ng/ml), or anti- CD40(0.1and10ng/ml), measured by BrdU incorporation at 48 h. 8 Zbtb1 IS ESSENTIAL FOR LYMPHOCYTE DEVELOPMENT

Tissue expression of Zbtb1 Analysis of the expression of the two isoforms of Zbtb1 mRNA Zbtb1 in BM revealed maximum expression in long-term HSCs (LSK, Copy number of mRNA in lymphoid tissues of wild-type 2 2 + 2 mice was determined by quantitative real-time PCR and normal- CD34 , CD48 ), less in short-term HSCs (LSK, CD34 , CD48 ), + + ized to the housekeeping gene HPRT (Fig. 6). Maximum tissue and still less in multipotent progenitors (LSK, CD34 , CD48 ) expression was found in thymus and spleen, followed by lymph (35, 36) (Fig. 6C). nodes and PBMCs. Unfractionated and lineage-depleted BM had B lymphoid subsets of the BM showed a striking increase in + + + expression, but at lower levels (Fig. 6A). Zbtb1 expression from pro-B (B220 , CD43 ) to pre-B (B220 , CD432) cell stages, with levels then declining upon further dif- Two isoforms were found, Zbtb1713 and Zbtb1644, that share an + 2 2 + initial noncoding exon 1 and the 59 end of exon 2. Zbtb1713 is not ferentiation into immature B cells (B220 , CD43 , IgD , IgM ) + 2 + + further spliced and encodes a BTB/POZ domain followed by eight and low levels in mature B cells (B220 , CD43 , IgD , IgM ) zinc finger domains in a single open reading frame (40). The (Fig. 6D). The increased expression of Zbtb1 mRNA in pre-B cells shorter Zbtb1644, formed by a splice from within exon 2 to an suggests that Zbtb1 plays an important role at this stage of B cell alternative exon 3, lacks the last three C-terminal zinc finger development and could possibly explain the above-described de- domains found in Zbtb1713 and was less abundant in all cell types creased number of pre-B cells observed in BM of Zbtb1-deficient studied (Fig. 6B, 6C). Although DN1–DN4 thymocytes expressed compared with wild-type mice. In splenic B cells, overall Zbtb1 both Zbtb1 isoforms, maximum expression was observed in the mRNA levels were 10-fold lower, with predominant expression in + + + lo hi 2 2 hi CD4 CD8 DP population, in which Zbtb1644 was 20% as abun- follicular (B220 , IgD , IgM , CD43 , CD5 , CD23 , CD21/ + + med + hi lo lo/2 dant as the Zbtb1713. CD4 and CD8 single-positive cells had 35 ) and marginal zone (B220 , IgD , IgM CD23 , CD21/ lower expression of both isoforms (Fig. 6B). 35hi) fractions (Fig. 6E). Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Zbtb1 mRNA expres- sion measured by quantitative PCR in lymphoid populations in wild-type mice. (A) Spleen, PBMCs, thymus, lymph node, BM, and BM depleted of mature lineages (Lin 2ve BM). (B) Thymocyte subsets, as described in Fig. 4A. (C) BM progenitor pop- ulations, as described in Fig. 4B. (D and E) BM and splenic B lineage subsets, as described in Fig. 5; y-axis shows copy numbers of Zbtb1 per 1000 copies of housekeeping gene HPRT. Data represent means 6 SD (five mice per group). The Journal of Immunology 9

Expression and nuclear localization of Zbtb1 protein Expression vectors with human and mouse cDNAs encoding the 713- and 644-aa isoforms of Zbtb1 were constructed with epitope tags at either N- or C-terminal positions. Human Zbtb1713, with an N-terminal FLAG sequence following the initial methionine codon (N-FLAG-hZBTB1713) was expressed in 293T cells. To determine the cellular localization of the protein, Western blotting was carried out with the nuclear and cytoplasmic protein fractions and blots were probed with anti-FLAG Ab to detect ZBTB1, as well as anti-histone deacetylase (HDAC)1 and anti–a1a-tubulin Abs to document purity of nuclear and cytoplasmic extracts, re- spectively (Fig. 7A). The tagged protein was readily detectable in nuclear extracts (Fig. 7Ai). Consistent with its structural similarity to transcriptional repressors and its previously described nuclear location (41), we found the ZBTB1 signal with the anti-FLAG Ab confined to the nuclear fraction, and not in the cytoplasmic frac- tion (Fig. 7Aii).

Dimerization of Zbtb1 isoforms sharing identical BTB/POZ Downloaded from domains

We studied dimerization using Zbtb1713 tagged with FLAG and Zbtb1644 tagged with myc at either end to minimize the chance of steric hindrance (42). After transfection into 293T cells, N- and C- FLAG-Zbtb1713 were detected at 83 kDa using mouse anti-FLAG and HRP-anti-mouse IgG (Fig. 7B, upper panel); similarly, http://www.jimmunol.org/ somewhat smaller N- and C-myc-Zbtb1644 were detected using mouse anti-myc (not shown). Upon reciprocal immunoprecipita- tion, the N- and C-FLAG-Zbtb1713 proteins were coprecipitated with either the N- or C-myc-Zbtb1644, and vice versa (Fig. 7B, FIGURE 7. Zbtb1 protein detection by Western blotting. (Ai) Expres- middle and lower panels). sion of tagged N-FLAG-hZBTB1 in nuclear extracts of transfected Thus, the epitope tags did not affect protein expression or in- 293T cells. Upper panel, Anti-FLAG mAb (clone M2) detects the FLAG- teraction, and the short and long isoforms, with identical 59 BTB tagged Zbtb1. Increasing proportions of nuclear extract from 293T cells dimerization domains, were shown to undergo dimerization. Thus, transfected with the pTargeT-N-FLAG-hZBTB1 were mixed with extract the short isoform, although lacking the three C-terminal zinc fingers from untreated cells, keeping the amount of protein loaded constant. The by guest on September 28, 2021 positive signal increased accordingly. Lower panel, Anti-HDAC1 loading and expressed at a considerably lower level than the predomi- control. (Aii) Nuclear localization of FLAG-tagged Zbtb1. Panel 1, Anti- nant 713-aa isoform, may bind to the long form under physi- FLAG in fractionated cytoplasmic versus nuclear protein extracts of ologic conditions, potentially modifying the activity of Zbtb1 transfected 293T cells. Panels 2 and 3, HDAC1 and a1a-tubulin, nuclear DNA-binding complexes. and cytoplasmic controls, respectively. (B) Western blot of 293T cell ZBTB1 sequencing in human SCID patients lysates 24 (left) and 48 h (right) after transfection. Upper panel, Probed with anti-FLAG Ab. Lanes 1 and 5, Control insert pEF-BOS-EGFP (green The phenotoype of mouse Zbtb1 deficiency suggested that dele- fluorescence to ensure efficient transfection); lanes 2 and 6, N-FLAG- 2 + terious mutations in human ZBTB1 could cause T B immune mZBTB1713; lanes 3 and 7, C-FLAG-mZBTB1713; lanes 4 and 8, plasmid deficiency in humans. We examined the sequence of ZBTB1 in with no insert. Anti-FLAG signal is at the expected 83 kDa. Middle and 20 cases of human SCID with T2B+ phenotype, but without de- lower panels, Heterodimerization after cotransfection, demonstrated by monstrable mutations in previously reported SCID disease genes. immunoprecipitation (IP) and reciprocal detection of FLAG-Zbtb1713 However, none was found to have variants in ZBTB1 that were and myc-Zbtb1644. Lanes: 1, pEF-BOS-EGFP; 2, N-FLAG-Zbtb1713; 3, C-FLAG- mZbtb1 ; 4, N-myc- Zbtb1 ; 5, C-myc-Zbtb1 ; 6, predicted to be deleterious. 713 644 644 N-FLAG-Zbtb1713 plus N-myc-Zbtb1644; 7, N-FLAG- Zbtb1713 plys C- myc- mZbtb1644; 8, C-FLAG-Zbtb1713 plus N-myc-Zbtb1644; 9, C-FLAG- C Discussion Zbtb1713 plus C-myc-Zbtb1644.() Schematic representation of an es- Our results strengthen the evidence that Zbtb1 is essential for T sential role of Zbtb1 during T lineage commitment. cell development, as recently noted in scanT mice generated by chemical mutagenesis by Siggs et al. (22). The steps in T lineage a maximal level at the DP stage and then declining, and the total differentiation from HSCs are not fully understood, and identifi- lack of maturing thymocytes in mice without Zbtb1 implies that cation of the role of Zbtb1 in prethymic lymphoid pathways and the role of this factor in thymocyte differentiation is irreplaceable migration to the thymus will further elucidate this process. and cannot be compensated for by other members of the same Analysis of fetal thymi of our mice lacking Zbtb1 showed de- protein family or by alternate signaling pathways. The much re- creased numbers of the earliest DN1 thymocytes and failure to duced NK cell number in all mice lacking Zbtb1 indicates that it is progress beyond the DN1 stage. A lymphocyte-autonomous defect also important for NK cell development or expansion. rather than defective thymic microenvironment was demonstrated The B cell phenotype of our knockout mice is similar to that of by reciprocal transplants showing that knockout thymus supported scanT mice (22); neither the scanT C74R missense mutation nor generation of wild-type BM into T cells, whereas the converse the complete absence of Zbtb1 in our transgenic mouse affected transplant of knockout BM into wild-type mice did not. Zbtb1 the number of B cells. Mature B cells were found in the periphery mRNA was expressed in wild-type thymocytes, increasing to of Zbtb1-deficient animals. Their in vitro proliferation in response 10 Zbtb1 IS ESSENTIAL FOR LYMPHOCYTE DEVELOPMENT to various stimuli was comparable to wild-type B cells, arguing main is responsible for its nuclear localization and exhibits the against a B cell-intrinsic defect and suggesting that the observed strongest repressive activity. This suggests that some residual inability to class switch could be due to absence of T cell help. activity may therefore still be present in the scanT mice due to There are 49 known mammalian Zbtb gene family members their unaffected ZNF domains. (43). BTB/POZ domains at the N termini of these proteins mediate An explanation for the increased number of HSCs in Zbtb1 either homo- or heterodimerization with related proteins (44). All knockouts could be that there is an attempt to compensate for have multiple C2H2 zinc finger domains, which in canonical form increased apoptosis or otherwise decreased fitness that impairs have two Cys and two His residues in a C-2-C-12-H-3-H se- lymphocyte development; our preliminary mixed chimerism quence. The 12 central residues, when stabilized by a Zn atom, studies have suggested the latter, as did similar experiments with form a projection that can interact with the major groove of scanT mice (22). The increased number of HSCs and progenitors a dsDNA helix, recognizing around five specific bp of a G-rich in the BM, but decreased thymocyte number in Zbtb1-deficient sequence (45, 46). Proteins belonging to the Zbtb family, in- mice, could also reflect a defect in migration and homing to the cluding BCL6/ZNF51, Th-POK/ZFP67, PLZP, and PLZF/Zbtb16, thymus, a process facilitated by chemokine receptors CCR7 and to name a few, are transcription repressors implicated as switches CCR9 (55–57). Indeed, our preliminary experiments (not shown) at branch points between alternative developmental pathways. suggest a decrease in expression of CCR9 mRNA in Zbtb1 knock- Their underexpression causes failure to branch out from a default out HSCs. pathway and alters homeostasis of hematopoietic cell compart- Activation of the cAMP signaling pathway has been shown to ments, whereas overexpression can also produce uncontrolled modulate T cell development, and stimulation of cAMP-dependent

(often malignant) expansion of cells (6, 7, 44, 47, 48). Addition- signaling in the thymus prevents differentiation of T cell precursors Downloaded from ally, proteins with a similar structure have also been shown to and results in increased apoptosis (58). It has been reported that mediate transcriptional repression and interact with components of one function of Zbtb1 is a dose-dependent repression of a cAMP histone deactylase corepressor complexes (40, 48–51). The Zbtb25 response element, preventing it from binding to a CREB (40). gene located immediately adjacent to Zbtb1 is also implicated Moreover, a human multiple malformation syndrome with hy- in T cell development and negatively regulates NF of activated poplasia of the thymus was recently associated with CREB1

T cells, NF-AT (52). mutation (59). Thus, the absence of Zbtb1-mediated repression http://www.jimmunol.org/ Zbtb1 is a small gene now known to encode a transcriptional of c-AMP signaling pathways could be one of the reasons why repressor that may act through control of chromatin remodeling, T cells are unable to develop in the thymus of both complete which in turn leads to changes in gene expression (40). It is one of knockout and scanT mice. a few genes thought to be transcribed by spRNAP-IV, a single The mechanism of action of Zbtb1 during early lymphoid de- polypeptide nuclear polymerase expressed from an alternate termination could also be to repress the differentiation of B cell transcript of the mitochondrial RNA polymerase gene POLRMT progenitors from the ALPs in BM (2), favoring the differentia- (53, 54). We have shown that both predicted isoforms of the tion of ALPs into T cell precursors instead (Fig. 7C). Further protein are expressed, a 713-aa protein encoded by an unspliced experiments, such as with RNA expression arrays and chromatin open reading frame in exon 2 and an alternate 644-aa isoform immunoprecipitation, will elucidate the molecular targets of by guest on September 28, 2021 produced by a splice from within the distal coding region of exon Zbtb1. 2 to exon 3. Both isoforms share the N-terminal BTB/POZ di- A precise understanding of the development of lymphocytes merization domain and proximal five zinc finger domains (44), but from HSCs is critical for treating humans with immunodeficien- the last three zinc finger domains are truncated from the alter- cies, blood diseases and cancers by transplantation, gene therapy, nately spliced isoform. The longer Zbtb1713 is expressed at a and discovery of new drugs. Zbtb1 is similar to transcription factors higher level than Zbtb1644 in all tissues investigated. The two with proven roles in lymphocyte development and has now been isoforms not only form homodimers, as expected for members of shown repeatedly to be essential and specific for mouse thymocyte the Zbtb family (44), but also can dimerize with each other. Al- development (6, 7, 22). Our ability to elucidate and manipulate though the physiologic targets of Zbtb1 transcription repression Zbtb1 functions in mice and ultimately in humans will facilitate are unknown, the reduced complement of zinc finger domains in understanding and treating human immune disorders. SCID and the shorter isoform might confer reduced repressive activity upon related primary immunodeficiencies in humans are rare, but life- a heterodimeric Zbtb1713/Zbtb1644 complex, potentially modulat- threatening, and in many cases with genetic causes still unknown. ing or fine-tuning its effect. Further functional studies with the two Although to date we have not found mutations of human ZBTB1 in isoforms of the protein may also shed light on the regulation of patients with typical T2B+ SCID phenotypes, characteristics of repressive activity of other proteins with similar structures. humans with ZBTB1 defects may be distinct, and examining a Detailed analysis of our Zbtb1 knockout mice has revealed wider range of human phenotypes, including those with multiple a contrast between the scanT mice and the Zbtb1 knockout mice malformations associated with thymic hypoplasia, with deficient in the numbers of the BM progenitor subsets. Although the cortical T cells such as the CREB1 defect reported by Kitazawa numbers and percentages of BM stem cell subsets in the scanT et al. (59) may be fruitful. In any case, our new Zbtb1-deficient mice were reported to be comparable to wild-type mice (22), our model will be useful to study the role of this transcription fac- Zbtb1 knockout mice had significantly increased numbers of tor and its associations and targets in the development of T short-term HSCs, MPPs, and CLPs. This difference could be due lymphocytes. to residual function of the protein bearing a C74R mutation in the BTB domain of Zbtb1 of scanT mice. Complete absence Acknowledgments of Zbtb1 may result in a loss of repression of branching into Robert Nussbaum, Fabio Candotti, Mark Killian, Nigel Killeen, and Pam differentiation pathways, causing an increase in numbers of the Schwartzberg gave expert advice in mouse construction and flow cytometry, stem cell subsets in default pathways in our knockout mice. Art Nienhaus provided the MSCV-IRES-GFP vector, Mitchell Eddy pro- Analysis of the transcriptional activity of truncated Zbtb1 pro- vided Hspa2 mice and Ab, Michael Eckhaus helped with histology and teins by Liu et al. (40), each containing one of the two major Amy Chen, Beverly Hay, and Marla Vacek provided skillful experimental functional domains (BTB and ZNF), revealed that the ZNF do- assistance. The Journal of Immunology 11

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