Mad3 negatively regulates B cell differentiation in the spleen by inducing Id2 expression

Abstract

Immature B cells migrate to the spleen where they differentiate into mature cells. This final maturation step is crucial to enable B cells to become responsive to antigens and to participate in the immune response. Previously, we showed that Id2 acts as a negative regulator of the differentiation of immature B cells occurring in the spleen. Id2 expression has been found to depend on Myc-Max-Mad transcriptional complexes in mammary epithelial cells. Nearly all studies to date have shown that Mad proteins inhibit proliferation, presumably by antagonizing the function of Myc proteins. In the current study, we followed the Mad family members during peripheral B cell differentiation. We show that Mad3 actively regulates B cell differentiation. Our results demonstrate that high expression levels of Mad3 in immature B cells induce Id2 expression, which inhibits transcription of genes essential for B cell differentiation. During their differentiation to mature cells, B cells reduce their Mad3 expression, enabling the maturation process to occur.

Figure 1.

Mad3 expression is down-regulated as part of the transition from immature to mature B cells. (A–E) A and B, control immature (IgD⁻) and mature (IgD⁺) cells; C, sorted T1 B cell (CD21-CD23⁻ CD24⁺), T2 B cell (CD21⁺, CD23⁺ CD24⁺), and mature B cell (CD21⁺, CD23⁺, and CD24⁻) populations derived from control mice were purified. (D and E) B cells from CD74−/− or control mice. (A, C, and D) RT-PCR for Mad3 and HPRT was performed. (B and E) Quantitative RT-PCR results are expressed as fold change in Mad3 expression in stimulated cells compared with nonstimulated cells, which was defined as 1 (N = 3). (F) Western blot showing steady-state levels of Mad3 in total cell lysates of B cells from control and CD74−/− mice. Results presented are representative of at least three separate experiments.

Figure 2.

Binding activity to the Id2 promoter in immature and mature B cells. Control or CD74−/− B cells extracts were subjected to EMSA using labeled probes representing the Id2 or Oct-1 promoters in the presence or absence of unlabeled Id2 probes (34-fold excess [B and C] or 74-fold excess [A–C]). The probes used were CAAATG (A), CACATG (B), and CACGTG (C). Arrows indicate complexes of proteins and probes. Results presented are representative of at least three separate experiments.

Figure 3.

Mad3 binds to the Id2 promoter. (A) Cell extracts from CD74−/− or control B cells were incubated in with or without an anti-Mad3 Ab (Mad3) or an isotype control antibody (Con) and analyzed by EMSA with the oligonucleotide CAAATG. (B) Cell extracts from CD74−/− or control B cells were incubated with or without anti-Mad4 (Mad4), anti-Max (Max), or isotype control (Con) Abs and analyzed by EMSA with the CAAATG oligonucleotide probe. (C and D) ChIP analysis of Mad3 (C) or Mad 4 (D) binding to the Id2 promoter. Chromatin prepared from CD74−/− (C) or control (D) B cells was immunoprecipitated with control or anti-Mad3 (C) or anti-Mad4 (D) antibodies. Presence of the promoter sequence was then quantified by quantitative real-time PCR. Results presented are representative of at least three separate experiments.

Figure 4.

Mad3 activates Id2 expression. (A and B) 293 cells were transfected with Mad3 (A) or Mad4 (B), and an Id2 promoter/luciferase construct, or with an empty vector. After 24 h, the cells were lysed and luciferase expression from the Id2 promoter was determined. Results presented are representative of at least three separate experiments.

Figure 5.

Mad3 binds to HAT p300, which induces histone H3 acetylation. (A and B) ChIP analysis of histone H3 acetylation in the Id2 promoter. Chromatin prepared from CD74−/− or control B cells was immunoprecipitated with control or anti-acetylated H3 antibodies. Presence of the promoter sequence was then quantified by RT-PCR (A) and quantitative real-time PCR (B). Results presented are representative of two separate experiments. (C and D) Immunoprecipitations: CD74−/− B220+ B cells were lysed. (C) After anti-p300 immunoprecipitation, proteins were separated on SDS-PAGE and transferred onto nitrocellulose. Mad3 was detected by Western blot analysis. (D) After anti-Mad3 immunoprecipitation, proteins were separated on SDS-PAGE and transferred onto nitrocellulose. p300 was detected by Western blot analysis. The results presented are representative of at least three separate experiments.

Figure 6.

Knockdown of Mad3/Mad4 expression in immature and mature cells using antisense oligos. Purified B cells derived from CD74−/− or control mice were incubated in the presence of 10 nM Mad3 (A) or Mad4 (B) antisense (A) or sense (S) oligonucleotides for 24 h at 37°C. Id2 transcription levels were analyzed using RT-PCR. Results presented are representative of at least seven independent experiments. (C and D) Quantitative RT-PCR results are expressed as a fold change in Id2 expression after treatment with antisense Mad3 (C) or Mad4 (D). Results presented are representative of at least three independent experiments. (E and F) Western blot showing levels of Mad3 or Mad4 (E) or Id2 (F) in S- or A-treated B cells from control and CD74−/− mice.

Figure 7.

Mad3 negatively regulates B cell differentiation. (A–C) Total splenocytes derived from CD74−/− mice were incubated in the presence of 10 nM Mad3 (A) or Mad4 (B) antisense or sense oligonucleotides at 37°C. After 48 h, B cell subpopulations were analyzed for the mature B population by using anti-B220, anti-CD21, and anti-CD24 antibodies. (C) Graph summarizes the results of four independent experiments. (D) Total splenocytes derived from CD74−/− mice were incubated in the presence of 10 nM Mad3 or Mad4 antisense or sense oligonucleotides at 37°C. After 48 h, B cell subpopulations were analyzed for the mature B population by using anti-AA4.1 antibody. Graph summarizes the results of four independent experiments.