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The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells.

Ise W, Kohyama M, Schraml BU, Zhang T, Schwer B, Basu U, Alt FW, Tang J, Oltz EM, Murphy TL, Murphy KM - Nat. Immunol. (2011)

Bottom Line: Restoring T(FH) cell activity to Batf(-/-) T cells in vivo required coexpression of Bcl-6 and c-Maf.In B cells, BATF directly controlled the expression of both activation-induced cytidine deaminase (AID) and of germline transcripts of the intervening heavy-chain region and constant heavy-chain region (I(H)-C(H)).Thus, BATF functions at multiple hierarchical levels in two cell types to globally regulate switched antibody responses in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT
The transcription factor BATF controls the differentiation of interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) by regulating expression of the transcription factor RORγt itself and RORγt target genes such as Il17. Here we report the mechanism by which BATF controls in vivo class-switch recombination (CSR). In T cells, BATF directly controlled expression of the transcription factors Bcl-6 and c-Maf, both of which are needed for development of follicular helper T cells (T(FH) cells). Restoring T(FH) cell activity to Batf(-/-) T cells in vivo required coexpression of Bcl-6 and c-Maf. In B cells, BATF directly controlled the expression of both activation-induced cytidine deaminase (AID) and of germline transcripts of the intervening heavy-chain region and constant heavy-chain region (I(H)-C(H)). Thus, BATF functions at multiple hierarchical levels in two cell types to globally regulate switched antibody responses in vivo.

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Batf directly regulates AID mRNA expression. (a) Relative expression levels of genes involved in class switch recombination determined by microarray analysis of Batf+/+ or Batf−/− B cells activated for 2 days with LPS. Expression is shown as a ratio of expression in Batf−/− B cells compared to Batf+/+ B cells. Data are from one microarray experiment. (b) Quantitative RT-PCR analysis of AID mRNA expression in Batf+/+ or Batf−/− B-cells stimulated for 2 or 4 days with LPS. Results are normalized to hprt expression and are presented as arbitrary unit (AU). *P<0.05 (unpaired student t-test). Data are from three independent experiments. (c) EMSA analysis was performed using B-cell nuclear extract from Batf+/+ B cells (WT) or Batf−/− B cells (KO) stimulated with LPS plus IL-4 for 24 hours and either a consensus AP-1 probe or +17 kb (2) AID probe (Supplementary Table 2) in the presence (+) or absence (−) of anti-BATF antibody. (d) B cells were stimulated as in c and ChIP performed with DNA precipitated using anti-BATF antibody and amplified using primers specific to −1.5 kb or +17 kb regions of AID locus. *P<0.05 (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (e) ChIP was performed as in d but DNA was precipitated using anti-acetylated histone H3. ***P<0.005 (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (f) FACS analyses (left) was performed on wild-type, Batf−/−or AID−/− B cells on the C57/BL6 background, activated with LPS and IL-4 and infected with the indicated retrovirus, and analyzed after 3 days. Data are representative data of two experiments. Frequency (right) of IgG1 positive B220+ B cells as determined in (f) from three biological replicates (mean and s.e.m.). *P<0.05, **P<0.01, and ***P<0.005, versus empty-RV control value (unpaired student t-test). NS, not significant.
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Figure 7: Batf directly regulates AID mRNA expression. (a) Relative expression levels of genes involved in class switch recombination determined by microarray analysis of Batf+/+ or Batf−/− B cells activated for 2 days with LPS. Expression is shown as a ratio of expression in Batf−/− B cells compared to Batf+/+ B cells. Data are from one microarray experiment. (b) Quantitative RT-PCR analysis of AID mRNA expression in Batf+/+ or Batf−/− B-cells stimulated for 2 or 4 days with LPS. Results are normalized to hprt expression and are presented as arbitrary unit (AU). *P<0.05 (unpaired student t-test). Data are from three independent experiments. (c) EMSA analysis was performed using B-cell nuclear extract from Batf+/+ B cells (WT) or Batf−/− B cells (KO) stimulated with LPS plus IL-4 for 24 hours and either a consensus AP-1 probe or +17 kb (2) AID probe (Supplementary Table 2) in the presence (+) or absence (−) of anti-BATF antibody. (d) B cells were stimulated as in c and ChIP performed with DNA precipitated using anti-BATF antibody and amplified using primers specific to −1.5 kb or +17 kb regions of AID locus. *P<0.05 (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (e) ChIP was performed as in d but DNA was precipitated using anti-acetylated histone H3. ***P<0.005 (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (f) FACS analyses (left) was performed on wild-type, Batf−/−or AID−/− B cells on the C57/BL6 background, activated with LPS and IL-4 and infected with the indicated retrovirus, and analyzed after 3 days. Data are representative data of two experiments. Frequency (right) of IgG1 positive B220+ B cells as determined in (f) from three biological replicates (mean and s.e.m.). *P<0.05, **P<0.01, and ***P<0.005, versus empty-RV control value (unpaired student t-test). NS, not significant.

Mentions: Although AID expression was reported to be reduced in Batf−/− B cells5, the basis for this reduction was not determined. Further, the ability of AID to restore switching defects in Batf−/− B cells was not demonstrated, so that defects beyond reduced AID expression could contribute to defective class switching in Batf−/− B cells. Therefore, to identify mechanisms contributing to defective CSR, we compared global gene expression of wild-type and Batf−/− B cells activated in vitro with LPS (Fig. 7a, Supplementary Fig. 12). Aicda mRNA was reduced by approximately 10-fold in Batf−/− B cells relative to wild-type B cells by microarray and q-PRC (Fig. 7a, b). Among 15 additional proteins known to be involved in class switching, AID was the only one to be significantly reduced in Batf−/− B cells (Fig. 7a). And in contrast to T cells, Batf−/− B cells did not show any known transcription factors that were reduced significantly relative to wild-type cells (Supplementary Fig. 12). For example, expression of Bach2 and IRF4, which are both required for isotype switching26, 27, were not reduced in Batf−/− B cells, suggesting that Batf might directly control AID expression rather than controlling expression of subordinate transcription factors as it does in TFH cells.


The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells.

Ise W, Kohyama M, Schraml BU, Zhang T, Schwer B, Basu U, Alt FW, Tang J, Oltz EM, Murphy TL, Murphy KM - Nat. Immunol. (2011)

Batf directly regulates AID mRNA expression. (a) Relative expression levels of genes involved in class switch recombination determined by microarray analysis of Batf+/+ or Batf−/− B cells activated for 2 days with LPS. Expression is shown as a ratio of expression in Batf−/− B cells compared to Batf+/+ B cells. Data are from one microarray experiment. (b) Quantitative RT-PCR analysis of AID mRNA expression in Batf+/+ or Batf−/− B-cells stimulated for 2 or 4 days with LPS. Results are normalized to hprt expression and are presented as arbitrary unit (AU). *P<0.05 (unpaired student t-test). Data are from three independent experiments. (c) EMSA analysis was performed using B-cell nuclear extract from Batf+/+ B cells (WT) or Batf−/− B cells (KO) stimulated with LPS plus IL-4 for 24 hours and either a consensus AP-1 probe or +17 kb (2) AID probe (Supplementary Table 2) in the presence (+) or absence (−) of anti-BATF antibody. (d) B cells were stimulated as in c and ChIP performed with DNA precipitated using anti-BATF antibody and amplified using primers specific to −1.5 kb or +17 kb regions of AID locus. *P<0.05 (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (e) ChIP was performed as in d but DNA was precipitated using anti-acetylated histone H3. ***P<0.005 (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (f) FACS analyses (left) was performed on wild-type, Batf−/−or AID−/− B cells on the C57/BL6 background, activated with LPS and IL-4 and infected with the indicated retrovirus, and analyzed after 3 days. Data are representative data of two experiments. Frequency (right) of IgG1 positive B220+ B cells as determined in (f) from three biological replicates (mean and s.e.m.). *P<0.05, **P<0.01, and ***P<0.005, versus empty-RV control value (unpaired student t-test). NS, not significant.
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Figure 7: Batf directly regulates AID mRNA expression. (a) Relative expression levels of genes involved in class switch recombination determined by microarray analysis of Batf+/+ or Batf−/− B cells activated for 2 days with LPS. Expression is shown as a ratio of expression in Batf−/− B cells compared to Batf+/+ B cells. Data are from one microarray experiment. (b) Quantitative RT-PCR analysis of AID mRNA expression in Batf+/+ or Batf−/− B-cells stimulated for 2 or 4 days with LPS. Results are normalized to hprt expression and are presented as arbitrary unit (AU). *P<0.05 (unpaired student t-test). Data are from three independent experiments. (c) EMSA analysis was performed using B-cell nuclear extract from Batf+/+ B cells (WT) or Batf−/− B cells (KO) stimulated with LPS plus IL-4 for 24 hours and either a consensus AP-1 probe or +17 kb (2) AID probe (Supplementary Table 2) in the presence (+) or absence (−) of anti-BATF antibody. (d) B cells were stimulated as in c and ChIP performed with DNA precipitated using anti-BATF antibody and amplified using primers specific to −1.5 kb or +17 kb regions of AID locus. *P<0.05 (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (e) ChIP was performed as in d but DNA was precipitated using anti-acetylated histone H3. ***P<0.005 (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (f) FACS analyses (left) was performed on wild-type, Batf−/−or AID−/− B cells on the C57/BL6 background, activated with LPS and IL-4 and infected with the indicated retrovirus, and analyzed after 3 days. Data are representative data of two experiments. Frequency (right) of IgG1 positive B220+ B cells as determined in (f) from three biological replicates (mean and s.e.m.). *P<0.05, **P<0.01, and ***P<0.005, versus empty-RV control value (unpaired student t-test). NS, not significant.
Mentions: Although AID expression was reported to be reduced in Batf−/− B cells5, the basis for this reduction was not determined. Further, the ability of AID to restore switching defects in Batf−/− B cells was not demonstrated, so that defects beyond reduced AID expression could contribute to defective class switching in Batf−/− B cells. Therefore, to identify mechanisms contributing to defective CSR, we compared global gene expression of wild-type and Batf−/− B cells activated in vitro with LPS (Fig. 7a, Supplementary Fig. 12). Aicda mRNA was reduced by approximately 10-fold in Batf−/− B cells relative to wild-type B cells by microarray and q-PRC (Fig. 7a, b). Among 15 additional proteins known to be involved in class switching, AID was the only one to be significantly reduced in Batf−/− B cells (Fig. 7a). And in contrast to T cells, Batf−/− B cells did not show any known transcription factors that were reduced significantly relative to wild-type cells (Supplementary Fig. 12). For example, expression of Bach2 and IRF4, which are both required for isotype switching26, 27, were not reduced in Batf−/− B cells, suggesting that Batf might directly control AID expression rather than controlling expression of subordinate transcription factors as it does in TFH cells.

Bottom Line: Restoring T(FH) cell activity to Batf(-/-) T cells in vivo required coexpression of Bcl-6 and c-Maf.In B cells, BATF directly controlled the expression of both activation-induced cytidine deaminase (AID) and of germline transcripts of the intervening heavy-chain region and constant heavy-chain region (I(H)-C(H)).Thus, BATF functions at multiple hierarchical levels in two cell types to globally regulate switched antibody responses in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT
The transcription factor BATF controls the differentiation of interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) by regulating expression of the transcription factor RORγt itself and RORγt target genes such as Il17. Here we report the mechanism by which BATF controls in vivo class-switch recombination (CSR). In T cells, BATF directly controlled expression of the transcription factors Bcl-6 and c-Maf, both of which are needed for development of follicular helper T cells (T(FH) cells). Restoring T(FH) cell activity to Batf(-/-) T cells in vivo required coexpression of Bcl-6 and c-Maf. In B cells, BATF directly controlled the expression of both activation-induced cytidine deaminase (AID) and of germline transcripts of the intervening heavy-chain region and constant heavy-chain region (I(H)-C(H)). Thus, BATF functions at multiple hierarchical levels in two cell types to globally regulate switched antibody responses in vivo.

Show MeSH
Related in: MedlinePlus