Thy28, also known as ThyN1, is a highly conserved nuclear protein. We previously showed that in a chicken mature B cell line, Thy28 binds to the promoter of the gene encoding Pax5, a transcription factor essential for B cell development, and positively regulates its expression. Here, we generated a Thy28-deficient mouse line to analyze its potential role in B cell development in mice. Thy28-deficient mice showed normal development of B cells, and the expression of Pax5 was comparable between wild-type and Thy28-deficient primary B cells. Thus, species-specific mechanisms regulate Pax5 expression and B cell development.
Citation: Kitaura F, Yuno M, Fujita T, Wakana S, Ueda J, Yamagata K, et al. (2019) Normal B cell development and Pax5 expression in Thy28/ThyN1-deficient mice. PLoS ONE 14(7): e0220199. https://doi.org/10.1371/journal.pone.0220199
Editor: Sebastian D. Fugmann, Chang Gung University, TAIWAN
Received: March 7, 2019; Accepted: July 10, 2019; Published: July 22, 2019
Copyright: © 2019 Kitaura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by the Takeda Science Foundation (T.F.), Grant-in-Aid for Scientific Research (C) (#15K06895) (T.F.), and Grant-in-Aid for Scientific Research (B) (#15H04329) (T.F., H.F.), ‘Transcription Cycle’ (#15H01354) (H.F.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: T.F. and H.F. are founders and directors of Epigeneron, Inc. H.F. is an Academic Editor of PLOS ONE. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
B cell development is a complex process regulated by the concerted actions of many gene products. Pax5 is an essential transcription factor in the process of B cell development . Expression of the mouse Pax5 gene is regulated by many transcription factors and DNA-binding proteins. Examples of such regulators include PU.1, IRF4, IRF8, NF-κB, and EBF1 [2, 3]. We previously used a locus-specific chromatin immunoprecipitation (ChIP) approach to analyze the mechanisms regulating the expression of Pax5 in a chicken mature B cell line, DT40 . We found that Thy28, which is also known as ThyN1, binds to the promoter region of the Pax5 gene in a B cell-specific manner and positively regulates its expression .
Thy28 is an evolutionarily-conserved protein [7, 8] that is highly expressed in the bursa of Fabricius and in other lymphoid tissues in the chicken . It is also expressed in the liver, heart, and brain in chickens . In contrast to its relatively limited tissue distribution in the chicken, Thy28 is more broadly expressed in the mouse .
In the present study, we generated a mutant mouse strain lacking expression of Thy28 to examine its in vivo function in mice. The Thy28-deficient (Thy28-/-) mice were viable and showed normal development. Interestingly, B cell development in Thy28-/- mice was normal, suggesting that Thy28 is dispensable for B cell development in mice. Expression of Pax5 was comparable between wild-type and Thy28-/- primary B cells. These results suggest a species-specific role of Thy28 in B cell development and function.
Materials and methods
The targeting vector for the mouse Thy28 gene (PG00147_X_4_A07) was obtained from the European Conditional Mouse Mutagenesis Program (EUCOMM). The linearized plasmid was transfected into an embryonic stem (ES) cell line, EGR-G101, which was previously established from C57BL/6-Tg(CAG/Acr-EGFP)C3-N01-FJ002Osb mice, as described previously . After G418 selection, surviving colonies were subjected to screening by PCR. ES cells retaining the transgene in the Thy28 locus were injected into blastocysts derived from ICR mice (Japan SLC) to generate chimeras. The chimeric mice were crossed with C57BL/6 mice to generate heterozygous Thy28KI/+ mice (strain name: C57BL/6-Thyn1tm1a(EUCOMM)Osb/Osb) (RIKEN BioResource Center RBRC09564). The Thy28KI/+ mice were then crossed with CAG-FLPe mice  to generate Thy28flox/+ mice (strain name: B6.Cg-Thyn1tm1c(EUCOMM)Osb/Osb) (RIKEN BioResource Center RBRC09563), and the Thy28flox/+ mice were crossed with CAG-Cre mice  to generate Thy28+/- mice (strain name: B6.Cg-Thyn1tm1d(EUCOMM)Osb/Osb) (RIKEN BioResource Center RBRC09565). Finally, the Thy28+/- mice were crossed with each other to generate Thy28+/, Thy28+/-, and Thy28-/- mice.
All animal experiments were approved by the Institutional Animal Care and Use Committee at the Research Institute for Microbial Diseases, Osaka University.
For genotyping, genomic DNA was extracted and subjected to PCR with KOD FX (Toyobo). PCR conditions were as follows. Thy28KI/+ mice: heating at 94°C for 2 min, followed by 35 cycles of 98°C for 10 s, 68°C for 10 min, and 68°C for 2 min. Thy28flox/+ mice: heating at 94°C for 2 min, followed by 37 cycles of 94°C for 20 s, 64°C for 20 sec, 72°C for 30 sec, and 72°C for 10 min. Tny28+/- mice: heating at 94°C for 2 min; followed by 35 cycles of 98°C for 10 s, 62°C for 30 sec, 68°C for 6 min, and 68°C for 2 min. Primers used for genotyping PCR are shown in Table 1.
Nuclear extracts (NE) were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Aliquots of NE (10 μg) were subjected to immunoblot analysis with an anti-Thy28 Ab (kindly gifted by Dr. Compton) , as described previously .
Cell staining and flow cytometry
Cells were stained for 30 min at 4°C with fluorochrome-conjugated antibodies (Abs). Abs used for surface staining were fluorescein isothiocyanate (FITC)-conjugated mouse CD19 (130-102-494, Miltenyi), phycoerythrin (PE)-Cy7-conjugated mouse CD3 (552774, BD Bioscience), allophycocyanin (APC)-conjugated mouse IgD (405713, BioLegend), APC-Cy7-conjugated mouse MHC class II (107628, BioLegend), BV510-conjugated mouse CD19 (562956, BD Pharmingen), BV421-conjugated CD5 (562739, BD Pharmingen), and PE-conjugated CD21/35 (552957, BD Pharmingen).
For detection of Pax5 protein, splenocytes from 7-week-old mice were stained with FITC-labeled anti-CD19 in autoMACS Running Buffer—MACS Separation Buffer (130-091-221, Miltenyi), followed by staining with a PE-conjugated anti-Pax5 Ab (12–9918, eBioscience/Thermo Fisher Scientific) according to the manufacture's protocol. Flow cytometric analysis was performed on a FACSCalibur (BD Biosciences) and data was analyzed with FlowJo software (TreeStar).
Results and discussion
Generation of Thy28-/- mice
To examine the potential role of Thy28 in B cell development in mice, we generated mutant mice in which the Thy28 gene was inactivated by deletion of its exons 3–7 (Thy28-/- mice) (Figs 1 and 2, Table 1). The linearized targeting vector for the mouse Thy28 gene was transfected into an ES cell line, EGR-G101 . After G418 selection, surviving colonies were subjected to screening by PCR. ES cells retaining the transgene in the Thy28 locus were injected into blastocysts derived from ICR mice to generate chimeras. The chimeric mice were crossed with C57BL/6 mice to generate heterozygous Thy28KI/+ mice. The Thy28KI/+ mice were crossed with CAG-FLPe mice  to generate Thy28flox/+ mice, and the Thy28flox/+ mice were crossed with CAG-Cre mice  to generate Thy28+/- mice. Finally, the Thy28+/- mice were crossed each other to generate Thy28+/, Thy28+/-, and Thy28-/- mice. The Thy28-/- mice were viable and born in the expected Mendelian ratios (Table 2), suggesting that the Thy28 gene is dispensable for normal development. As expected, the expression of Thy28 protein was lost in Thy28-/- mice, and reduced in heterozygous Thy28+/- mice (Fig 3). These results indicated that our targeting strategy effectively knocked out the Thy28 gene in these mice.
Schematic diagrams of the Thy28 locus (A), the targeting vector (B), the targeted allele (C), the floxed allele (D), and the deleted allele (E).
Detection of the 5' integration site (A) and the 3' integration site (B) in the ES cell clones. (C) Detection of 5' and 3' integration in an individual mouse. (D) Confirmation of deletion of selection markers by FLPe-mediated FRT recombination. (E) Confirmation of deletion of exons 3–7 of the Thy28 gene by Cre-mediated loxP recombination.
Expression of Thy28 in murine spleocytes was detected by immunoblot analysis with an anti-Thy28 Ab. Coomassie Brilliant Blue (CBB) staining is shown as a protein loading control.
Normal development of B cells in Thy28-/- mice
To examine the potential role of Thy28 in the development of mouse B cells and other lymphocytes, we analyzed the B cell population in Thy28-/- mice. As shown in Figs 4 and 5, no abnormalities were detected in cellularity in the B cell population. B cell numbers in Thy28-/- mice were normal, as determined by the percentages of CD19+ cells in the spleen and lymph node (LN) (Figs 4 and 5). The percentages of total B cells, B1B cells, B2B cells, follicular B cells, marginal zone B (MZB) cells, and pre-B cells in spleens from Thy28-/- mice were normal (Fig 6). These data suggest that Thy28 is dispensable for B cell development in mice.
(A) Percentages of B cells and T cells in the spleen. (B) Expression of IgD on splenic B cells. (C) Percentages of CD19+ B cells in the inguinal lymph nodes (LNs). (D) Expression of IgD on B cells in the inguinal LNs.
Numbers of (A) splenocytes, (B) splenic CD19+ B cells, (C) cells in inguinal LNs, and (D) CD19+ B cells in inguinal LNs are shown. The age range of the mice was 8.1–38.7 weeks. One-way ANOVA was used to calculate p-values.
(A) B cells, (B) B1B cells, (C) B2B cells, (D) follicular B cells, (E) marginal zone B (MZB) cells, and (F) pre-B cells. Gating steps are as follows: B cells: MHC class II+ CD19+; B1B cells: MHC class II+ CD19+ CD5+; B2B cells: MHC class II+ CD19+ CD5-; follicular B cells: MHC class II+ CD19+ CD5- CD21/35+; MZB cells: MHC class II+ CD19+ CD5- CD21/35high; and pre-B cells: MHC class II+ CD19+ CD5- CD21/35low. Student's t-tests were used to calculate p-values.
Normal expression of Pax5 in Thy28-/- B cells
Finally, we examined the effect of the loss of Thy28 on the expression of Pax5. Expression of Pax5 in CD19+ splenic B cells was comparable between Thy28+/, Thy28+/-, and Thy28-/- mice (Fig 7). Expression of Pax5 in mature B cells in inguinal LNs was also comparable between Thy28+/, Thy28+/-, and Thy28-/- mice (S1 Fig). These data show that Thy28 is dispensable for Pax5 expression in mature B cells in the mouse. We previously showed that Thy28 binds to the promoter region of the Pax5 gene in a B cell-specific manner in a chicken mature B cell line, DT40, and down-regulation of Thy28 resulted in a decrease in the expression of the Pax5 gene . These results in a chicken B cell line were in clear contrast with the present results in mice. We also knocked down Thy28 in the human B cell lines Nalm-6 and Raji. As shown in S2 Fig, down-regulation of Thy28 in these cell lines did not affect the expression of Pax5. These results demonstrate that Thy28 is dispensable for Pax5 expression in B cells from at least two mammals, mice and humans, and suggest a species-specific mechanism for the regulation of Pax5 expression.
Splenocytes from 7-week-old mice were stained with a FITC-conjugated anti-CD19 Ab and a PE-conjugated anti-Pax5 Ab. The expression level of Pax5 in CD19+ B cells is shown. The mean fluorescence intensity (MFI) of Pax5 staining is shown. Black: unstained control; red: Pax5 staining. Percentages of Pax5+ cells in CD19+ splenic B cells from Thy28+/, Thy28+/-, and Thy28-/- mice were 95.6%, 94.7%, and 95.6%, respectively.
We generated Thy28-deficient mice to investigate the potential role of Thy28/ThyN1 in B cell development. Thy28-deficient mice were viable and showed a Mendelian birth ratio. Thy28-deficient mice had normal B cell numbers as well as normal percentages of subclasses of B cell lineages. Finally, the expression of Pax5 was normal in B cells from Thy28-deficient mice. These results indicate that Thy28/ThyN1 is dispensable for the regulation of Pax5 expression and the development of B cells in the mouse and suggest a species-specific role of Thy28/ThyN1 in Pax5 expression and B cell development.
S1 Fig. Expression of Pax5 in mature B cells in inguinal lymph nodes.
Splenocytes from 9-week-old mice were stained with a FITC-conjugated anti-CD19 Ab, an APC-conjugated IgD Ab, and a PE-conjugated anti-Pax5 Ab. The expression of Pax5 in CD19high and IgD+ B cells is shown. The mean fluorescence intensity (MFI) of Pax5 staining is shown. Percentages of Pax5+ cells in CD19high and IgD+ B cells from Thy28+/, Thy28+/-, and Thy28-/- mice were 99.6%, 99.7%, and 99.9%, respectively.
S2 Fig. Expression of Pax5 in human B cell lines.
(A, B) shRNA-mediated knock-down of Thy28 in a human pre-B cell line, Nalm-6. Expression of Pax5 protein (A) and Pax5 mRNA (B) was analyzed in Nalm-6 cells stably expressing an shRNA against GFP or human Thy28. The expression of Pax5 mRNA was quantified by real-time RT-PCR and normalized to the expression of GAPDH mRNA (mean +/- SEM, n = 4). (C, D) Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated knock-out of Thy28 in a human Burkitt′s lymphoma cell line, Raji. (C) Nucleotide insertions or deletions generated by CRISPR/Cas9 in the human Thy28 gene. The ATG codons in blue and the TGA codon in red indicate start codons and an inserted stop codon, respectively. The CRISPR/Cas9 target sequence is underlined. (D) Expression of Pax5 was analyzed in Thy28 mutant (KO) Raji cells.
We thank Mitsuko Mori for injection of ES cells into blastocysts, and EUCOMM for providing the targeting construct (PG00147_X_4_A07). We also thank M. Compton for providing the anti-Thy28 Ab.
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