BATF regulates the expression of Nfil3, Wnt10a and miR155hg for efficient induction of antibody class switch recombination in mice

Abstract

BATF functions in T cells and B cells to control the host response to antigen and promote the production of class switched immunoglobulins. In this study, we demonstrate that BATF expression increases rapidly, and transiently, following B cell stimulation and use an inducible murine model of BATF deletion to show that this induction is necessary, and sufficient, for immunoglobulin (Ig) class switch recombination (CSR). We examine two genes (Nfil3 and miR155gh) that are positively regulated, and one gene (Wnt10a) that is negatively regulated by BATF during CSR. These genes play essential roles in CSR and each impacts the expression and/or function of the others. Our observations allow these targets of BATF regulation to be positioned in a network upstream of the activation of germline transcripts (GLT) from the IgH locus and of transcriptional activation of Aicda - the gene encoding the enzyme directing Ig gene rearrangements. This work extends the knowledge of the molecular control of CSR and, importantly, positions the induction and function of BATF as an early event in this process.

Keywords: B cells; BATF; class switch recombination; signaling pathways; transcription regulation.

Figure 1.. BATF is required for B cells to produce IgG.

Splenic B cells, isolated from Batf^KI/KI and Batf^ΔZ/ΔZ mice were labeled with CFSE and cultured with LPS and IL-4. CFSE dilution was analyzed by flow cytometry. The gating strategies to identify lymphocytes using (upper panel) and the percent of those cells that are proliferating (bar, middle panel) are shown. Results in the histogram (lower panel) are the mean±SEM of data collected from four independent CFSE experiments, each using cells from one mouse per genotype. SSC (side scatter); FSC (forward scatter) (B) B cells isolated from Batf^KI/KI and Batf^ΔZ/ΔZ spleens were cultured in the presence of absence of LPS and IL-4 and protein extracts immunoblotted to visualize the indicated proteins. Signals were normalized to HSP90 and expressed relative to unstimulated Batf^KI/KI B cells (set to 1.0). Shown is a representative blot from two independent experiments using protein from two mice per genotype. (C) Media from cultured Batf^KI/KI and Batf^ΔZ/ΔZ B cells were screened by ELISA for the indicated Ig. Results shown are the mean±SEM pooled from two independent experiments, each using cells from two mice per genotype. (***p<0.0001; ns=not significant; two-tailed, unpaired Student’s t-test)

Figure 2.. BATF has a biphasic expression pattern.

(A, B) RNA was prepared from WT splenic B cells at the indicated times following stimulation. RT-qPCR, performed in duplicate, was used to detect the indicated transcripts. Results were normalized to β-actin and expressed relative to time 0 (set to 1.0). Results shown are the mean±SEM of data obtained from four independent experiments, each using RNA from one mouse. (C) Protein was isolated from WT B cells at the indicated times following stimulation. Immunoblotting was used to detect the expression of proteins. Signals were normalized to HSP90 and expressed relative to time 0 (set to 1.0). Shown is a representative blot from four independent experiments, each using protein pooled from two mice.

Figure 3.. Early expression of BATF is necessary and sufficient for CSR.

(A) Schematic of TAM treatment to generate the timed deletion of Batf in the Δ0 and Δ12 IzT B cell cultures. (B and D) Control (C), Δ0 and Δ12 cells were sorted and RNA analyzed by RT-qPCR for the indicated transcripts, in duplicate. Results are normalized to β-actin (B) or Gapdh (D) and expressed relative to the control (set to 1.0). Results shown are the mean±SEM of data from four independent experiments, using RNA from one mouse per treatment. (C) Δ0 and Δ12 B cells were surface stained for IgM, IgG1, and IgE and profiled by flow cytometry. Gating strategy is in Suppl. Figure 2. Results are presented as the mean±SEM of data from six independent experiments, each using cells from one mouse per treatment. (**p<0.01; ***p<0.0001; ns=not significant; two-tailed, unpaired Student’s t-test)

Figure 4.. Analysis of Batf ^ΔZ/ΔZ B cell transcripts by RNA-Seq.

(A) Heatmap of the top 50 significantly altered genes in Batf^ΔZ/ΔZ B cells at 6hrs post-stimulation. (B) Shown are the top 5 GO:Biological Processes pathways into which genes identified by the RNA-Seq cluster. (C) RNA isolated from Batf^KI/KI and Batf^ΔZ/ΔZ splenic B cells cultured in the presence or absence of LPS and IL-4 was analyzed by RT-qPCR, in duplicate, to confirm mis-expression of three putative BATF targets genes. Results were normalized to β-actin and expressed relative to unstimulated Batf^KI/KI B cells (set to 1.0). Results shown are the mean±SEM of data collected from two independent experiments, each using RNA from two mice per genotype. (*p<0.05; **p<0.01; ***p<0.0001; ns=not significant; two-tailed, unpaired Student’s t-test)

Figure 5:. BATF re-expression restores gene expression and CSR in Batf^ΔZ/ΔZ B cells.

Stimulated B cells isolated from spleens of Batf^KI/KI and Batf^ΔZ/ΔZ mice were transduced with either MSCV or MSCV-BATF and Thy1.1 + cells isolated by FACS. (A) Protein extracts were immunoblotted, in duplicate, to detect the indicated proteins. Signals were normalized to HSP90 and expressed relative to MSCV-transduced, stimulated Batf^KI/KI B cells (set to 1.0). Shown is a representative blot from three individual experiments in which protein from two Batf^KI/KI or Batf^ΔZ/ΔΖ mice were blotted in parallel for each treatment. (B, D and E) RNA and microRNA from each group was analyzed by RT-qPCR to detect the indicated transcripts, in duplicate. Aicda, Nfil3 and Wnt10a transcripts are normalized to β-actin (B and E), miR155 to RNU6 (E) and GLT transcripts to Gapdh (D) and are expressed relative to levels in unstimulated Batf^KI/KI B cells (B and E) (set to 1.0, but not shown) or MSCV-transduced Batf^KI/KI B cells (set to 1.0) (D). Results shown are the mean±SEM of data collected from three independent experiments, each using RNA or microRNA from two mice per genotype and treatment. (C) Transduced cells from each group were profiled for Thy1.1 and surface IgG1 and IgE expression by flow cytometry. See Suppl. Figure 7 for gating strategy. Results shown are the mean±SEM of data collected from three independent experiments, each using cells from two mice per genotype and treatment. (F) RNA and microRNA were prepared from control (C) and TAM-treated Δ0 and Δ12 GFP+ IzT B cells (see Figure 3). RT-qPCR was used to detect the indicated RNAs, in duplicate. Results are normalized to β-actin (mRNA) or RNU6 (miRNA) and expressed relative to levels in Izt control cells (set to 1.0). Results are presented as the mean±SEM of data from four independent experiments, using RNA or microRNA isolated from one mouse per treatment. (*p<0.05; **p<0.01; ***p<0.0001; ns=not significant; two-tailed, unpaired Student’s t-test)

Figure 6:. BATF binding accompanies regulation of Nfil3, miR155hg and Wnt10a expression.

Chromatin was prepared from B cells isolated from spleens of WT and Batf^ΔZ/ΔZ mice and stimulated with LPS and IL-4. ChIP was performed to detect binding of BATF (A) or acetylation of H3K27 (B) at the indicated AP-1 or AICE motifs identified within Nfil3, mir155gh and Wnt10a (see Suppl. Figure 5A). BATF binding to AP-1(E) of Myb was used as a positive control while non-BATF bound AP-1 (N) of Myb was used as a normalization control for background [29]. (B) DNA associated with H3K27 acetylation is expressed as a % of total input. Results shown are the mean±SEM of data obtained from two independent ChIP experiments, each performed with DNA isolated from two mice per genotype and time point. (**p<0.01; ***p<0.0001; ns=not significant; two-tailed, unpaired Student’s t-test)

Figure 7:. NFIL3, miR155 and WNT10A impact Aicda expression and CSR.

Stimulated B cells isolated from spleens of Batf^KI/KI and Batf^ΔZ/ΔZ mice were transduced with the indicated retroviruses. Transduced, Thy1.1 cells isolated by FACS. (A, D and E) RNA from each group was analyzed by RT-qPCR to detect the indicated transcripts, in duplicate. Results are normalized to β-actin (A) or Gapdh (D and E) and are expressed relative to levels in MSCV-transduced, Batf^KI/KI B cells (set to 1.0). Results are presented as the mean±SEM of data collected from three independent experiments, each using RNA from two mice per genotype. (B and C) Transduced cells from each group were profiled for Thy1.1 and surface IgG1 and IgE expression by flow cytometry. Gating strategy is in Suppl. Figure 7. Results shown are the mean±SEM of data obtained from six independent experiments, each using cells from one mouse per treatment. (*p<0.05; **p<0.01; ***p<0.0001; ns=not significant; two-tailed, unpaired Student’s t-test)

Figure 8:. Hierarchy of BATF, NFIL3, miR155 and WNT10A in regulating CSR.

(A) Stimulated B cells from Batf^KI/KI and Batf^ΔZ/ΔZ spleens were transduced with the indicated retroviruses and Thy1.1 cells isolated by FACS. RNA from each group was analyzed by RT-qPCR to detect the indicated transcripts, in duplicate. Results are normalized to β-actin and expressed relative to the level in MSCV-transduced Batf^KI/KI B cells (set to 1.0). Results shown are the mean±SEM of data collected from three independent experiments, each with RNA from two mice per genotype. (*p<0.05; **p<0.01; ***p<0.0001; ns=not significant; two-tailed, unpaired Student’s t-test) (B) Working model depicting the primary site of action of the molecules examined in this study (upper) and the hierarchy of their actions in inducing IgH GLT, AID expression and CSR in B cells (lower).