# Tumor suppressors BTG1 and BTG2 regulate early mouse B-cell development

E. Tijchon, L. van Emst, L. Yuniati, D. van Ingen Schenau, J. Havinga, Jean-Pierre Rouault, P. Hoogerbrugge, F. van Leeuwen, B. Scheijen

► To cite this version:

E. Tijchon, L. van Emst, L. Yuniati, D. van Ingen Schenau, J. Havinga, et al., Tumor suppressors BTG1 and BTG2 regulate early mouse B-cell development. Haematologica, 2016, 101 (7), pp.e272-e276. 10.3324/haematol.2015.139675. hal-02998446

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Distributed under a Creative Commons Attribution 4.0 International License
Tumor suppressors BTG1 and BTG2 regulate early
mouse B-cell development

Transcription factors that regulate B-cell differentiation are frequently targeted by genetic aberrations in B-cell malignancies. The B-cell translocation gene 1 (BTG1), which encodes a transcription coregulator,¹,² is recurrently affected by gene deletions in B-cell precursor acute lymphoblastic leukemia (BCP-ALL),³,⁵ and targeted by missense mutations in diffuse large B-cell lymphomas (DLBCL).⁶,⁷ Similarly, the closely related BTG2 gene is subject to mutations in DLBCL, Burkitt lymphoma and follicular lymphoma.⁶,⁹ To establish the role of BTG1 and BTG2 in B lymphopoiesis, we studied the composition of the hematopoietic compartment in mice deficient for

either *Btg1* or *Btg2*, as well as in *Btg1*;*Btg2*-double knock-
out animals. We found that *Btg1* and *Btg2* are both
required at multiple stages of early B-cell development,
but also display specific functions in progenitor B-cells.
Our data demonstrate that transcriptional cofactors
BTG1 and BTG2 act as important regulators of B-cell dif-
ferentiation, providing an important link to their role as
tumor suppressors in B-cell malignancies.

To determine the function of *Btg1* and *Btg2* during nor-
mal hematopoiesis we performed flow cytometric analy-
sis of the bone marrow (BM) compartment and spleen.
The fraction of B220⁺ progenitor B-cells was significantly
reduced in *Btg1*- and *Btg2*-deficient mice, ranging from a
10% decrease in the *Btg2*⁻/⁻ mice, 20% in *Btg1*⁻/⁻ mice, to
40% in the *Btg1*⁻/⁻;*Btg2*⁻/⁻ double knockout animals relative
to control mice (Figure 1A). Similarly, B-cell counts in the

<figure><img src="image_2.png" /></figure>

**Figure 1.** Decreased B-cell numbers in mice deficient for *Btg1* and *Btg2*. (A-B) Flow cytometric analysis was performed on mononuclear cells isolated from bone marrow (BM) and spleen of wild-type (WT) control (n=16), *Btg1*⁻/⁻ (n=7), *Btg2*⁻/⁻ (n=8) and *Btg1*⁻/⁻;*Btg2*⁻/⁻ mice (n=11). The fractions of B220⁺ BM cells (A) and B220⁺IgM⁺ splenocytes (B) are indicated. Each dot represents an individual sample and the horizontal line indicates the mean. (C) Competitive repopulation assay of wild-type and *Btg1*⁻/⁻;*Btg2*⁻/⁻ donor cells (CD45.2) derived of BM mixed 1:1 with CD45.1 wild-type cells which were transplanted into 9Gy (Gray) irradiated C57BL6/J mice (CD45.1). Flow cytometric analysis was performed on blood and spleen. The fraction of B220⁺, CD3⁺ and Mac-1⁺ of WT:WT (CD45.1:CD45.1) (n=4) and WT:*Btg1*⁻/⁻;*Btg2*⁻/⁻ (CD45.1:CD45.2) (n=6) cells are indicated. (D) Schematic representation of the different B-cell developmental stages according to Hardy nomenclature. Cell surface markers that distinguish specific Hardy fractions are indicated. (E) The percentage for the different Hardy B-cell fractions within BM is indicated as determined in WT control (n=16), *Btg1*⁻/⁻ (n=7), *Btg2*⁻/⁻ (n=8), and *Btg1*⁻/⁻;*Btg2*⁻/⁻ (n=11) mice. Each bar represents the mean of each group of mice ± the standard error of the mean (SEM). \*P<0.05, \*\*P<0.01, \*\*\*P<0.001.
spleen were most significantly reduced in *Btg1*⁻/⁻; *Btg2*²⁻/⁻ mice, with about 30% fewer B220⁺IgM⁺ B-cells (Figure 1B). In contrast, the fraction of myeloid progenitor cells in BM was not altered in the *Btg1*⁻ and *Btg2*⁻-deficient animals (*Online Supplementary Figure S1*). In addition, we did not observe any specific defects in T-cell development in mice lacking *Btg1* and *Btg2* expression (*Online Supplementary Figure S2*).

To assess whether the defect in B lymphopoiesis was not related to defective support from the BM microenvironment in the *Btg1* and *Btg2* knockout animals, competitive repopulation assays were performed. BM cells derived from either wild-type (CD45.2) or *Btg1*⁻;*Btg2*²⁻ mice (CD45.2) were mixed with competitor wild-type cells (CD45.1) and transplanted into lethally irradiated C57BL6/J mice (CD45.1). FACS analysis on peripheral blood and spleen demonstrated that contribution to the B-cell lineage was significantly reduced using *Btg1*⁻;*Btg2*²⁻ donor cells when compared to wild-type donor cells (Figure 1C), arguing that the observed defect in B-cell development is cell autonomous.

To establish at what stage of early B-cell development *Btg1*⁻ and *Btg2*⁻-deficiency affect B-cell differentiation, we investigated the relative frequencies of the different B-cell

subsets within the BM compartment. Analysis of the common lymphoid progenitor (CLP) fraction, defined as lineage negative (Lin⁻) Sca-1⁻c-Kit⁺Flt3⁺Il-7Rα⁺ cells, revealed no significant differences in *Btg1*⁻, *Btg2*²⁻ and *Btg1*⁻;*Btg2*²⁻ mice compared to wild-type controls (*Online Supplementary Figure S3*). Next, we employed the cell surface markers B220, CD43, CD24, IgD and IgM, to distinguish different B-cell precursor populations, generally known as Hardy fractions (Figure 1D). When we examined the single knockout animals, the *Btg2*²⁻ mice displayed decreased B-cell fractions at both the early and later stages of B-cell differentiation, while *Btg1*-deficiency resulted only in reduced B-cell fractions beyond the pre-B cell stage, including the immature B-cells (Hardy fractions E and F) (Figure 1E). In the *Btg1*⁻;*Btg2*²⁻ mice, we observed a similar phenotype as the *Btg2*²⁻ mice at the early stages of B-lineage differentiation, with a moderate decrease in the pool of pro-B cells (Hardy BC) compared to control mice. However, from the pre-B cell stage onwards, the *Btg1*⁻;*Btg2*²⁻ mice displayed a more pronounced phenotype in comparison to the *Btg1*⁻ and *Btg2*²⁻ single knockout animals, showing a 2-fold reduction in the B220⁺CD43⁻ cell fraction relative to control mice (Hardy D, E and F) (*Online Supplementary Figure S4*).

<figure><img src="image_3.png" /><figcaption>**Figure 2.** *Btg1* stimulates B-cell progenitor outgrowth in response to IL-7. (A) Bone marrow (BM) mononuclear cells (1 x 10⁵ cells) were isolated from wild-type (WT) control (n=18), *Btg1*⁻ (n=7), *Btg2*²⁻ (n=8) and *Btg1*⁻;*Btg2*²⁻ mice (n=11) and added in methylcellulose containing 10 ng/mL IL-7. (B) BM mononuclear cells were isolated from WT control (n=10), *Btg1*⁻ (n=7), *Btg2*²⁻ (n=4) and *Btg1*⁻;*Btg2*²⁻ mice (n=4) and added after CD19 isolation (2 x 10⁵ CD19⁺ cells) in methylcellulose containing 10ng/ml IL-7. (A, B) Mean colony counts (and SEM) were determined (>30 cells/colony) after seven days of culture. Data are representative of at least two independent experiments. **P<0.01, ***P<0.001. (C) BM cells from WT, *Btg1*⁻, *Btg2*²⁻, and *Btg1*⁻;*Btg2*²⁻ mice (all n=2) were stained with B220 and IL-7Rα/CD127 antibodies and the percentage of IL-7Rα-positive cells within the B220⁺ fraction was determined. The black line indicates IL-7Rα expression in the WT cells, while the blue peak shows the IL-7Rα expression in the *Btg1*⁻, *Btg2*²⁻ and *Btg1*⁻;*Btg2*²⁻ double knockout cells. (D) Apoptosis assay of CD19⁺ BM cells isolated from WT (n=4) and *Btg1*⁻ (n=7) cultured for 4 days in B-cell specific medium, by measuring Annexin V using flow cytometry. The percentage of Annexin V-positive cells in CD19⁺ BM cells is indicated. (E) Proliferation assay of CD19⁺ BM cells isolated from WT (n=3) and *Btg1*⁻ (n=3) cultured for 4 days in B-cell specific medium, measuring Ki-67 by flow cytometry. The cell cycle distribution according to Ki-67 staining in CD19⁺ BM cells is indicated. Each bar represents the mean of each group of mice ± the standard error of the mean (SEM). **P<0.01, ***P<0.001.</figcaption></figure>
We conclude from these results that *Btg1* and *Btg2* are both required for mouse B-cell development, while deficiency for each of the genes shows a different outcome on the relative abundance of the specific Hardy fractions.

To more specifically assess the function of *Btg1* and *Btg2* in early B lymphopoiesis, we investigated whether interleukin-7 (IL-7) dependent outgrowth of B-lymphoid progenitor cells was affected in the absence of *Btg1* and *Btg2* expression. BM cells derived from *Btg1*<sup>−/−</sup>, *Btg2*<sup>−/−</sup> single knockout and *Btg1*<sup>−/−</sup>*;Btg2*<sup>−/−</sup> double knockout mice were plated in methylcellulose and cultured for one week in the presence of cytokine IL-7. The number of colonies in methylcellulose was about 4-fold lower in cultures derived from *Btg1*<sup>−/−</sup> and *Btg1*<sup>−/−</sup>*;Btg2*<sup>−/−</sup> BM cells relative to

those derived from control BM cells (Figure 2A). Moreover, colony size was also reduced in these two knockout genotypes (data not shown). In contrast, *Btg2*-deficiency had no significant impact on the outgrowth of lymphoid progenitor cells in the IL-7-dependent methylcellulose colony formation assay. Given the fact that the number of B220<sup>+</sup> B-cell progenitors was decreased in the BM of the different *Btg1* and *Btg2* knockout lines, we also analyzed the colony outgrowth of similar amounts of purified CD19<sup>+</sup> progenitor B-cells under the same conditions. Again, colony formation was significantly impaired using CD19<sup>+</sup> cells derived from *Btg1*<sup>−/−</sup> and *Btg1*<sup>−/−</sup>*;Btg2*<sup>−/−</sup> mice, but not with *Btg2*<sup>−/−</sup> progenitor B-cells, which indicates that the loss of *Btg1* function is

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Aberrant T-lineage gene expression in progenitor B-cells deficient for *Btg1* and *Btg2*. (A) Relative expression levels of B-cell transcription factors E2A (Tcf3), Foxo1, Ebf1 and Pax5 was determined using cDNA generated from B220<sup>+</sup> bone marrow (BM) cells of wild type control (WT), *Btg1*<sup>−/−</sup>, *Btg2*<sup>−/−</sup> and *Btg1*<sup>−/−</sup>*;Btg2*<sup>−/−</sup> mice by qRT-PCR and normalized to the expression of the housekeeping gene TBP. (B) RNA was isolated from B220<sup>+</sup>CD43<sup>+</sup> and B220<sup>+</sup>CD43<sup>−</sup> cells derived from wild type control and *Btg1*<sup>−/−</sup>*;Btg2*<sup>−/−</sup> mice (n=4) and analyzed by microarray using the Illumina BeadArray platform. The data obtained were RMA normalized and relative gene expression differences of >1.6-fold was determined. KEGG pathway analysis was performed using the online Gene Set Enrichment Analysis (GSEA) tool and relevant pathways with highest *P*-value that are differentially expressed between WT and *Btg1*<sup>−/−</sup>*;Btg2*<sup>−/−</sup> cells are indicated. (C) Relative expression levels of Cd4, Izkzf2, Tcf7, Gata3 and Notch1 were determined on cDNA generated from B220<sup>+</sup> BM cells of the different genotypes by real-time PCR and normalized to TBP expression. (A and C) Data represent the mean and SEM of three independent experiments containing cDNA derived from 2 different biological samples. \*P<0.05, \*\*P<0.01, \*\*\*P<0.001.</figcaption></figure>
largely responsible for the defective outgrowth of B-lym-
phoid progenitors (Figure 2B).

To assess whether differences in colony formation
could be attributed to reduced expression levels of the IL-
7 receptor alpha (IL-7Rα/CD127), CD127 expression was
measured on B220⁺ BM cells by flow cytometry. We
observed that IL-7Rα expression was not decreased in
*Btg1*⁻⁺ progenitor B-cells, but was even upregulated in
*Btg1*⁻⁺;*Btg2*⁻⁺ cells (Figure 2C), arguing that the low num-
ber of colonies due to Btg1-deficiency was not related to
reduced IL-7Rα expression levels. Next, we examined
whether *Btg1*-deficient CD19⁺ cells displayed intrinsic
differences in the level of apoptosis and proliferation. We
observed a moderate but significant increase in the frac-
tion of Annexin V-positive cells derived from *Btg1*⁻⁺ mice
(Figure 2D; *Online Supplementary Figure S5*), while the cell
cycle distribution, as determined by Ki-67 staining, was
not affected (Figure 2E). Thus, Btg1 appears to be
required for optimal IL-7 dependent B-lymphoid colony
formation, in part by promoting cell survival in early pro-
genitor B-cells.

To elucidate how *Btg1* and *Btg2* regulate B-cell develop-
ment *in vivo*, we examined the expression of key tran-
scription factors involved in early B-cell differentiation,
including *E2a* (*Tcf3*), *Foxo1*, *Ebf1*, and *Pax5*.¹⁰,¹¹
Quantitative reverse transcription polymerase reaction
(qRT-PCR) on RNA isolated from B220⁺ sorted BM cells
of *Btg1*⁻⁺, *Btg2*⁻⁺ and *Btg1*⁻⁺;*Btg2*⁻⁺ mice demonstrated that
the expression of these master regulators of B-cell differ-
entiation was not decreased (Figure 3A). On the contrary,
mRNA expression levels of these transcription factors
were even somewhat elevated in the absence of Btg1 and
Btg2. Hence, the partial block in B-cell differentiation
observed in the *Btg1*⁻⁺;*Btg2*⁻⁺ animals cannot be explained
by diminished expression levels of these B-lineage tran-
scription factors.

Next, we performed microarray based gene expression
profiling using early (B220⁺CD43⁺) and late progenitor
B-cells (B220⁺CD43⁻) isolated from control and *Btg1*⁻⁺;
*Btg2*⁻⁺ mice and performed Gene Set Enrichment
Analyses (GSEA) using the Molecular Signatures
Database (MSigDB) to define KEGG pathways that were
specifically affected (Figure 3B). Interestingly, several sig-
naling pathways were identified, including the presence of
a T-cell receptor signaling signature in early B-cells of
*Btg1*⁻⁺;*Btg2*⁻⁺ mice showing upregulation of *Cd3d*
(Cd3delta) and *Cd3g* (Cd3gamma) (*Online Supplementary
Table S1*). Therefore, we aimed to assess whether
*Btg1*;*Btg2*-deficient progenitor B-cells displayed aberrant
expression of other T-lineage specific genes, including
*Cd4*, *Ikzf2*, *Tcf7*, *Gata3* and *Notch1*. Indeed, qRT-PCR
revealed that *Btg1*⁻⁺;*Btg2*⁻⁺ B220⁺ BM cells showed signif-
icantly increased expression levels of these T-lineage
marker genes (Figure 3C), while we also observed higher
expression of the CD3, CD4 and CD8 cell surface mark-
ers on *Btg1*⁻⁺;*Btg2*⁻⁺ committed B-cells (*Online
Supplementary Figure S6*). These findings suggest that *Btg1*
and *Btg2* are implicated in regulating B-lineage commit-
ment by suppressing the expression of lineage-inappro-
priate genes.

In summary, using genetic mouse models, we demon-
strate that both *Btg1* and *Btg2* promote mouse B-cell dif-
ferentiation at multiple stages of development, and most-
ly display overlapping functions during B lymphopoiesis,
which corresponds with an additive phenotype in the
*Btg1*⁻⁺;*Btg2*⁻⁺ mice. On the other hand, *Btg1* fulfills a more
prominent role in promoting the proliferative expansion
of lymphoid progenitors by inhibiting apoptosis. This is
in agreement with the finding that *Btg1* ablation in the

mouse brain decreases the pool of proliferating stem and
progenitor cells, which is accompanied by increased
apoptosis.¹² Importantly, *Btg1* and *Btg2* are both required
to repress T-lineage gene expression in progenitor B-cells.
These findings are partially overlapping with the pheno-
type observed in *Ebf1* and *Pax5* knockout mice, where it
has been shown that EBF1 and PAX5 are critical regula-
tors of B-lineage specification and commitment by
repressing alternative non-B-cell fates.¹³⁻¹⁵ As the expres-
sion of Ebf1 and Pax5 itself is not inhibited in
*Btg1*⁻⁺;*Btg2*⁻⁺ progenitor B-cells, the BTG1 and BTG2 tran-
scriptional coregulators may affect B-lineage commit-
ment by modifying the function of these B-cell specific
transcription factors or by epigenetic regulation of their
target genes. Notably, *PAX5* and *EBF1*, similar to *BTG1*,
are also subject to gene deletions in BCP-ALL, and com-
binations of these deletions often co-occur in the same
leukemia.⁴ Together our findings suggest that the block in
differentiation observed in BCP-ALL may result from
gene alterations affecting multiple master regulators of B-
cell differentiation.

Esther Tijchon,¹ Liesbeth van Ernst,¹ Laurensia Yuniati,¹
Dorette van Ingen Schenau,¹ Jørn Havinga,¹ Jean-Pierre
Rouault,² Peter M. Hoogerbrugge,³ Frank N. van Leeuwen,¹
and Blanca Scheijen¹

*ET and LvE contributed equally to this work as joined first authors. FNvL and BS contributed equally to this work as joined senior authors.*

¹Laboratory of Pediatric Oncology, Radboud University Medical Centre, Nijmegen, the Netherlands; ²Institut de Génomique Fonctionnelle de Lyon, Ecole Normale Supérieure de Lyon, Université Lyon 1, France; and ³Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands

Acknowledgments: the authors would like to thank Marieke von Lindern for providing the *Btg1* knockout mice, and Felice Tironé for the *Btg2* knockout animals. The authors thank the staff of the Central Animal Facility (CDL) and Rob Woestenenk for their technical assistance.

Funding: this research was supported by a grant from the Stichting Kinderen Kankervrij (KiKa-77).

The online version of this letter has a Supplementary Appendix.

Correspondence: blanca.scheijen@radboudumc.nl
doi:10.3324/haematol.2015.139675

Key words: hematopoiesis, lymphocytes, pediatric acute lymphoblastic leukemia, aggressive non-Hodgkin lymphoma.

Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.

References

1. Lin WJ, Gary JD, Yang MC, Clarke S, Herschman HR. The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine N-methyltransferase. J Biol Chem. 1996;271(25):15034-15044.

2. Winkler GS. The mammalian anti-proliferative BTG/Tob protein family. J Cell Physiol. 2010;222(1):66-72.

3. Kuiper RP, Schoenmakers EF, van Reijmersdal SV, et al. High-resolution genomic profiling of childhood ALL reveals novel recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell cycle progression. Leukemia. 2007;21(6):1258-1266.

4. Mullighan CG, Goorha S, Radtke I, et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature. 2007; 446(7137):758-764.

5. Waanders E, Scheijen B, van der Meer LT, et al. The origin and nature of tightly clustered BTG1 deletions in precursor B-cell acute lymphoblastic leukemia support a model of multiclonal evolution. PLoS
Genet. 2012;8(2):e1002583.

6. Lohr JG, Stojanov P, Lawrence MS, et al. Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc Natl Acad Sci USA. 2012; 109(10):3879-3884.

7. Morin RD, Mendez-Lago M, Mungall AJ, et al. Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature. 2011; 476(7360):298-303.

8. Love C, Sun Z, Jima D, et al. The genetic landscape of mutations in Burkitt lymphoma. Nat Genet. 2012;44(12):1321-1325.

9. Pasqualucci L, Khiabanian H, Fangazio M, et al. Genetics of follicular lymphoma transformation. Cell Rep. 2014;6(1):130-140.

10. Mandel EM, Grosschedl R. Transcription control of early B cell differentiation. Curr Opin Immunol. 2010;22(2):161-167.

11. Ramirez J, Lukin K, Hagman J. From hematopoietic progenitors to B

cells: mechanisms of lineage restriction and commitment. Curr Opin Immunol. 2010;22(2):177-184.

12. Farioli-Vecchioli S, Micheli L, Saraulli D, et al. Btg1 is Required to Maintain the Pool of Stem and Progenitor Cells of the Dentate Gyrus and Subventricular Zone. Front Neurosci. 2012;6:124.

13. Cobaleda C, Jochum W, Busslinger M. Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors. Nature. 2007;449(7161):473-477.

14. Lukin K, Fields S, Guerrettaz L, et al. A dose-dependent role for EBF1 in repressing non-B-cell-specific genes. Eur J Immunol. 2011; 41(6):1787-1793.

15. Nechanitzky R, Akbas D, Scherer S, et al. Transcription factor EBF1 is essential for the maintenance of B cell identity and prevention of alternative fates in committed cells. Nat Immunol. 2013;14(8):867-875.