Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Normal B cell development and Pax5 expression in Thy28/ThyN1-deficient mice

  • Fusako Kitaura,

    Roles Investigation

    Affiliation Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka, Japan

  • Miyuki Yuno,

    Roles Investigation

    Affiliation Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka, Japan

  • Toshitsugu Fujita,

    Roles Funding acquisition, Investigation, Writing – review & editing

    Affiliations Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka, Japan, Department of Biochemistry and Genome Biology, Hirosaki University Graduate School of Medicine, Zaifu-cho, Hirosaki, Aomori, Japan

  • Shigeharu Wakana,

    Roles Investigation

    Affiliations Technology and Development Team for Mouse Phenotype Analysis, RIKEN BioResource Research Center, Koyadai, Tsukuba, Ibaraki, Japan, Department of Gerontology, Institute of Biomedical Research and Innovation, Minatojima Minamiachi, Chuo-ku, Kobe, Japan

  • Jun Ueda,

    Roles Investigation

    Affiliations Center for Genetic Analysis of Biological Responses, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Japan, Center for Advanced Research and Education (CARE), Asahikawa Medical University, Asahikawa, Japan

  • Kazuo Yamagata,

    Roles Investigation

    Affiliations Center for Genetic Analysis of Biological Responses, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Japan, Faculty of Biology-Oriented Science and Technology, Kindai University, Nishimitani, Kinokawa City, Wakayama, Japan

  • Hodaka Fujii

    Roles Conceptualization, Funding acquisition, Investigation, Supervision, Writing – original draft, Writing – review & editing

    hodaka@hirosaki-u.ac.jp

    Affiliations Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka, Japan, Department of Biochemistry and Genome Biology, Hirosaki University Graduate School of Medicine, Zaifu-cho, Hirosaki, Aomori, Japan

Normal B cell development and Pax5 expression in Thy28/ThyN1-deficient mice

  • Fusako Kitaura, 
  • Miyuki Yuno, 
  • Toshitsugu Fujita, 
  • Shigeharu Wakana, 
  • Jun Ueda, 
  • Kazuo Yamagata, 
  • Hodaka Fujii
PLOS
x

Abstract

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.

Introduction

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 [1]. 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 [4][5]. 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 [6].

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 [7]. It is also expressed in the liver, heart, and brain in chickens [7]. In contrast to its relatively limited tissue distribution in the chicken, Thy28 is more broadly expressed in the mouse [8].

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

Mice

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 [9]. 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 [10] 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 [11] 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.

Genotyping

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.

Immunoblot analysis

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) [7], as described previously [12].

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).

Statistics

Prism 8 software (GraphPad) was used for statistical analyses. One-way analysis of variance (ANOVA) or Student t-tests were used to calculate p-values.

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 [9]. 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 [10] to generate Thy28flox/+ mice, and the Thy28flox/+ mice were crossed with CAG-Cre mice [11] 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.

thumbnail
Fig 1. Generation of the Thy28-deficient 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).

https://doi.org/10.1371/journal.pone.0220199.g001

thumbnail
Fig 2. Genotyping of the Thy28-deficient mice.

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.

https://doi.org/10.1371/journal.pone.0220199.g002

thumbnail
Fig 3. Expression of Thy28 in Thy28-mutant mice.

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.

https://doi.org/10.1371/journal.pone.0220199.g003

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.

thumbnail
Fig 4. B cell profiles.

(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.

https://doi.org/10.1371/journal.pone.0220199.g004

thumbnail
Fig 5. Numbers of B cells in spleens and 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.

https://doi.org/10.1371/journal.pone.0220199.g005

thumbnail
Fig 6. Cellularity of B cells in the spleen.

(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.

https://doi.org/10.1371/journal.pone.0220199.g006

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 [6]. 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.

thumbnail
Fig 7. Expression of Pax5 in splenic B cells.

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.

https://doi.org/10.1371/journal.pone.0220199.g007

Conclusions

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.

Supporting information

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.

https://doi.org/10.1371/journal.pone.0220199.s001

(PDF)

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.

https://doi.org/10.1371/journal.pone.0220199.s002

(PDF)

Acknowledgments

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.

References

  1. 1. Medvedovic J, Ebert A, Tagoh H, Busslinger M. Pax5: a master regulator of B cell development and leukemogenesis. Adv Immunol. 2011;111:179–206. pmid:21970955
  2. 2. Decker T, di Magliano MP, McManus S, Sun Q, Bonifer C, Tagoh H, et al. Stepwise activation of enhancer and promoter regions of the B cell commitment gene Pax5 in early lymphopoiesis. Immunity. 2006;30:508–20.
  3. 3. O'Riordan M, Grosschedl R. Coordinate regulation of B cell differentiation by the transcription factors EBF and E2A. Immunity. 1999;11:21–31. pmid:10435576
  4. 4. Buerstedde JM, Takeda S. Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell. 1991;67:179–88. pmid:1913816
  5. 5. Fujita T, Fujii H. Biochemical analysis of genome functions using locus-specific chromatin immunoprecipitation technologies. Gene Regul Syst Bio. 2016;10(Suppl. 1):1–9. pmid:26819551
  6. 6. Fujita T, Kitaura F, Fujii H. A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells. PLoS One. 2015;10:e0116579. pmid:25607658
  7. 7. Compton MM, Thomson JM, Icard AH. The analysis of cThy28 expression in avian lymphocytes. Apoptosis. 2001;6:299–314. pmid:11445672
  8. 8. Miyaji H, Yoshimoto T, Asakura H, Komachi A, Kamiya S, Takasaki M, et al. Molecular cloning and characterization of the mouse thymocyte protein gene. Gene. 2002;297:189–96. pmid:12384300
  9. 9. Fujihara Y, Kaseda K, Inoue N, Ikawa M, Okabe M. Production of mouse pups from germline transmission-failed knockout chimeras. Transgenic Res. 2013;22:195–200. pmid:22826106
  10. 10. Schaft J, Ashery-Padan R, van der Hoeven F, Gruss P, Stewart A. Efficient FLP recombination in mouse ES cells and oocytes. Genesis. 2001;31:6–10. pmid:11668672
  11. 11. Matsumura H, Hasuwa H, Inoue N, Ikawa M, Okabe M. Lineage-specific cell disruption in living mice by Cre-mediated expression of diphtheria toxin A chain. Biochem Biophys Res Commun. 2004;321:275–9. pmid:15358172
  12. 12. Fujita T, Fujii H. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem Biophys Res Commun. 2013;439:132–6. pmid:23942116