Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors

Abstract

Interleukin-21 (IL-21) is a pleiotropic cytokine that induces expression of transcription factor BLIMP1 (encoded by Prdm1), which regulates plasma cell differentiation and T cell homeostasis. We identified an IL-21 response element downstream of Prdm1 that binds the transcription factors STAT3 and IRF4, which are required for optimal Prdm1 expression. Genome-wide ChIP-Seq mapping of STAT3- and IRF4-binding sites showed that most regions with IL-21-induced STAT3 binding also bound IRF4 in vivo and furthermore revealed that the noncanonical TTCnnnTAA GAS motif critical in Prdm1 was broadly used for STAT3 binding. Comparing genome-wide expression array data to binding sites revealed that most IL-21-regulated genes were associated with combined STAT3-IRF4 sites rather than pure STAT3 sites. Correspondingly, ChIP-Seq analysis of Irf4(-/-) T cells showed greatly diminished STAT3 binding after IL-21 treatment, and Irf4(-/-) mice showed impaired IL-21-induced Tfh cell differentiation in vivo. These results reveal broad cooperative gene regulation by STAT3 and IRF4.

Figure 1. Characterization of an IL-21 Response Element 3′ of the Prdm1 Gene

(A) IL-21 induces Prdm1 gene expression. Prdm1 mRNA (means ± SEM of ≥3 independent experiments, total combined samples ≥4) was determined by quantitative RT-PCR 6 hr after treatment with 50 ng/ml IL-21. (B) Cells were activated with 1 µg/ml anti-CD40 and/or 3 µg/ml anti-IgM-Fab for 3 days, rested 24 hr, and treated with 100 ng/ml IL-21. Prdm1 mRNA (means ± SEM of two independent experiments, total combined samples = 4) was determined 24 hr later by quantitative RT-PCR. (C) Cells were activated with 1 µg/ml anti-CD40 for 3 days, rested for 24 hr, and treated with 100 ng/ml IL-21 for 24 hr and immunoblotted with antibodies to BLIMP1 or actin. Representative results of three independent experiments. (D) Schematic of the Prdm1 gene (defined by NM_007548 version 2; version 3 of May 2008 starts 61 bp downstream and lacks the 79 bp second exon) and locations of luciferase reporter constructs. pGL4-pro, minimal promoter (−972 to +183) reporter; K, KpnI; X, XhoI; S, SmaI; open triangle is the luciferase insertion site in exon 1 for R1, R8, R9, and R10. (E) Luciferase assays in NFS201 cells were performed 18 hr after treatment with 50 ng/ml IL-21. (F) Shown is luciferase activity 6 hr after treatment with 50 ng/ml IL-21 for NFS201 (means ± SEM of ≥4 independent experiments, total combined samples ≥6) and with 100 ng/ml IL-21 for splenic B cells (means ± SEM of ≥2 independent experiments, total combined samples ≥4). (G and H) More detailed mapping of the Prdm1 IL-21 response element in preactivated splenic B cells. Shown is luciferase activity 6 hr after treatment with 100 ng/ml IL-21 (means ± SEM of ≥3 independent experiments, total combined samples ≥6).

Figure 2. STAT3 and IRF4 Bind to the Prdm1 IL-21 Response Element

(A) DNA sequence of the 212 bp (26,237 to 26,448 region relative to the TSS) R37 construct containing the IL-21 response element. The STAT3-binding motif is underlined, IRF4 and IRF8-binding motifs are boxed, and probes #1, #2, and #3 are indicated. (B) EMSAs with nuclear extracts from NFS201 cells treated for 7 hr with 50 ng/ml IL-21. The solid and open arrows indicate IL-21-induced complexes, with the solid arrow corresponding to the STAT3-DNA complex. The open and closed circles indicate the supershifted STAT3 and IRF4 complexes, respectively. The WT probe is probe #1 in (A). (C) EMSAs were performed with probes #2 and #3 (bold and italic sequences in A) with NFS201 nuclear extracts. The solid arrowhead indicates an IL-21-induced complex, the closed circle indicates supershifted IRF4-DNA complexes, and the open arrowhead indicates a supershifted IRF8-DNA complex. (D) ChIP was performed with crosslinked splenic B cell lysates prepared 30 min after treatment with 100 ng/ml IL-21 (means ± SEM). Input was determined by quantitative PCR. Shown are representative results from two (B and C) or three (D) independent experiments.

Figure 3. STAT3 and IRF4 Mediate IL-21-Induced Prdm1 Expression in B Cells

(A) Sequences for STAT3 and IRF4-binding motif mutants in the R37 reporter. (B) Luciferase assays were performed with WT or mutated R37 constructs shown in (A). Shown is luciferase activity 6 hr after treatment with 100 ng/ml IL-21 (means ± SEM of ≥3 independent experiments, total combined samples ≥ 6). (C) Prdm1 mRNA expression (means ± SEM of two independent experiments, total combined samples = 4) was determined in preactivated splenic B cells by quantitative RT-PCR 24 and 48 hr after treatment with 100 ng/ml IL-21. (D) Shown is luciferase activity of the R37 construct 6 hr after treatment with 100 ng/ml IL-21 (means ± SEM of two independent experiments, total combined samples = 4). (E) IL-21 receptor expression in preactivated and rested Stat3-deficient B cells (dashed line). The solid line refers to WT mice. The isotype antibody control is shown in gray. (F) Prdm1 and Pim1 mRNA expression (means ± SEM of ≥3 independent experiments, total combined samples ≥4) was determined by quantitative RT-PCR 24 hr after treatment with 100 ng/ml IL-21. (G) IL-21 receptor expression of Irf4^−/− B cells (dashed line) and WT control cells (solid line) was determined. The isotype antibody control is shown in gray. (H) Splenic B cells were treated with 100 ng/ml IL-21 (means ± SEM of two independent experiments, total combined samples = 4) and Irf4 expression was determined. (I) Prdm1 mRNA expression of IL-21 stimulated cells expressing PU.1 (Sfpi1f/f-Cre^−/−) or lacking PU.1 (Sfpi1f/f-Cre^−/+) was determined (means ± SEM of two independent experiments, total combined samples ≥ 4). (J) IL-21 receptor expression of PU.1-deficient B cells (dashed line) and WT control (solid line). The isotype antibody control is shown in gray. In (C) and (D), *p < 0.05 (compared with IL-21-treated WT control mice). In (H), *p < 0.05 for IL-21-treated versus Ctrl.

Figure 4. STAT3 and IRF4 Control IL-21-Induced Prdm1 Expression in CD4⁺ T Cells

(A) T cells were preactivated with anti-CD3 plus soluble anti-CD28, and IL-2 for 3 days and rested for 24 hr in fresh RPMI medium, and Prdm1 mRNA in CD4⁺ T cells was determined by quantitative RT-PCR 9, 24, and 48 hr after treatment with IL-2, IL-4, IL-5, IL-6, IL-10, IL-21, or IFN-γ (see Experimental Procedures for details). Shown are means ± SEM of ≥5 independent experiments (total combined samples ≥10). (B) Prdm1 mRNA expression was determined in preactivated CD8⁺ T cells by quantitative RT-PCR at the indicated times after treatment with 100 ng/ml IL-21. (C) Preactivated splenic CD4⁺ and CD8⁺ T cells were treated with 100 ng/ml IL-21 for 24 hr and BLIMP1 or Actin protein levels were determined by immunnoblotting. (D) Luciferase assays were performed with WT or mutated forms of R37 for STAT3 and/or IRF4-binding motifs 6 hr after treatment of CD4⁺ T cells with 100 ng/ml IL-21. (E) ChIP of STAT3 and IRF4 was performed with splenic CD4⁺ T cell lysates prepared 20 min after treatment with 100 ng/ml IL-21. Input was determined by quantitative PCR. Results are representative of three independent experiments. (F) Prdm1 mRNA expression was measured in splenic CD4⁺ T cells from Stat3^f/f-Cre^−/− or Stat3^f/f-Cre^−/+ mice; cells were not treated or were stimulated with IL-21. The asterisk indicates no induction as compared to IL-21-treated Stat3^f/f-Cre^−/− cells. (G) IL-21R expression was measured in CD4⁺ T cells from Stat3-deficient (dashed line) or WT control (solid line) mice. The isotype antibody control is shown in gray. (H and I) Prdm1 mRNA expression was measured in WT (control), Irf4^−/−, and Irf8^−/− CD4⁺ T cells (H) or CD8⁺ T cells (I) by quantitative RT-PCR 24 hr after treatment with 0 or 100 ng/ml IL-21mRNA levels (means ± SEM of two independent experiments, total combined samples = 4). The asterisk indicates decreased induction as compared to Ctrl. (J) IL-21R expression was measured in CD4⁺ T cells from Irf4^−/− (dashed line) or WT (solid line) mice. The isotype antibody control is shown in gray. (K) IL-21 significantly induces Irf4 expression in splenic CD4⁺ T cells. Cells were treated with 100 ng/ml IL-21 (means ± SEM of two independent experiments, total combined samples = 4). (L) CD4⁺ T cells were preactivated and then not stimulated or stimulated with IL-21, IL-6, or IL-10, and subsequently, lysates were immunoblotted with antibodies to phospho-STAT3, STAT3, or Actin. A representative result from three independent experiments is shown. (M) CD4⁺ T cells from Irf4^+/+ or Irf4^−/− mice were preactivated and then not stimulated or stimulated with IL-21, IL-6, or IL-10 for 1 or 6 hr. Socs3 mRNA expression (means ± SEM of two independent experiments, total combined samples = 4) was determined by quantitative RT-PCR. *p < 0.05 (comparing similar samples to cells from Irf4^+/+ and Irf4^−/− mice).

Figure 5. STAT3- and IRF4-Binding Sites Colocalize

(A) Venn diagram of IRF4-binding sites before and after IL-21 stimulation. Shown are STAT3 sites after stimulation; the rare STAT3 sites before IL-21 treatment were omitted. (B) High-resolution mapping of STAT3 and IRF4 ChIP-Seq tags at the Prdm1 IL-21 response element. The number of tags/50 bp is plotted. The 212 bp IL-21 response element is drawn to scale and exactly overlaps the STAT3 and two IRF4-binding sites. The maxima of the curves coincide with the essential motifs shown in Figures 3 and 4; the sequences at these maxima are high-lighted in blue and yellow, with DNA-binding motifs for STAT3 and IRF4 boxed and underlined, respectively. The shoulder of the largest IRF4 peak suggests a secondary binding site (gray region). (C) The mouse Prdm1 gene includes three alternative transcript variants a, b, and c (AceView at NCBI). The STAT3- and IRF4-binding sites before and after IL-21 treatment are aligned (scale bar is 10 kb and 20 tags/200 bp window vertically), with binding sites corresponding to peaks 1–13 (Table S11). Although only one STAT3-IRF4 site (site 12, see asterisk, shown in detail in B) was found to regulate IL-21-mediated Prdm1 expression, several other major STAT3 peaks were induced by IL-21, and all overlapped IRF4 peaks. In intron 5 of variant a, site 9 strongly binds STAT3 but weakly binds IRF4, whereas site 10, which is 1.59 kb 3′ of site 9, strongly binds IRF4 but weakly binds STAT3. Additionally, IRF4 associated to the two promoter areas at sites 1 to 5.

Figure 6. IRF4 Promotes STAT3 Binding In Vivo

(A) CD4⁺ T cells from Irf4^−/− and littermate control mice were preactivated and rested as in Figure 5A. mRNA for Socs3, Bcl3, and Tha1 determined by quantitative RT-PCR 1 and 6 hr after treatment with 100 ng/ml IL-21. Shown are means ± SEM of two independent experiments (total combined samples = 4). *p < 0.05 (compared with WT control mice). (B) High-resolution mapping of STAT3 and IRF4-ChIP-Seq tags in the Socs3, Bcl3, and Tha1 genes. (C) The distance in base pairs between the precise position of IRF4 binding, before (blue) or after (red) IL-21 treatment, and the precise position of STAT3 binding after IL-21 is plotted for all composite IRF4- and STAT3-binding sites with >30 tags. (D) CD4⁺ T cells from Irf4^+/+ and Irf4^−/− mice were preactivated and stimulated with 100 ng/ml IL-21 as indicated, followed by immunoblotting with antibodies to phospho-STAT3, STAT3, or Actin. The representative result of two independent experiments is shown. (E) High-resolution mapping of the STAT3 ChIP-Seq tags in the Prdm1, Socs3, Bcl3, and Tha1 genes in CD4⁺ T cells from Irf4^−/− and littermate control mice.

Figure 7. Distance between the Maxima of STAT3 Binding and GAS or GAS-like Motifs

(A) The presence of TTCnnnGAA GAS motifs and single-nucleotide variants in the 4397 STAT3-binding sites were analyzed genome-wide. The position of each motif relative to the maximum of STAT3-binding tags (within 250 bp on each side) was recorded and the distribution over all sites was plotted. (B) Shown are the number of sites with the motif within 100 bp of maximal STAT3 binding, the number of observed and expected motifs, assuming a locally flat distribution, and p value of the observed overrepresentation of each motif in the center (Pearson χ² test).