A genomic regulatory element that directs assembly and function of immune-specific AP-1-IRF complexes

Abstract

Interferon regulatory factor 4 (IRF4) and IRF8 regulate B, T, macrophage, and dendritic cell differentiation. They are recruited to cis-regulatory Ets-IRF composite elements by PU.1 or Spi-B. How these IRFs target genes in most T cells is enigmatic given the absence of specific Ets partners. Chromatin immunoprecipitation sequencing in T helper 17 (T(H)17) cells reveals that IRF4 targets sequences enriched for activating protein 1 (AP-1)-IRF composite elements (AICEs) that are co-bound by BATF, an AP-1 factor required for T(H)17, B, and dendritic cell differentiation. IRF4 and BATF bind cooperatively to structurally divergent AICEs to promote gene activation and T(H)17 differentiation. The AICE motif directs assembly of IRF4 or IRF8 with BATF heterodimers and is also used in T(H)2, B, and dendritic cells. This genomic regulatory element and cognate factors appear to have evolved to integrate diverse immunomodulatory signals.

Fig. 1

IRF4 and BATF cistromes in T_H17 cells are enriched for composite AP-1–IRF motifs that direct cooperative binding. (A to C) ChIP-seq of in vitro differentiated T_H17 cells (42 hours) with antibodies directed against IRF4 or BATF. (A) MEME analysis of highly represented motifs within the IRF4 cistrome (2333 peaks) along with their statistical significance (E) values. (B) Union analysis of the IRF4 and BATF cistromes. Numbers indicate unique or coincident peaks, the latter with peak maxima within 100 bp of each other. (C) MEME analysis of highly represented motifs within the coincident peak sequences of IRF4 and BATF (1936 peaks). (D to G) EMSAs using nuclear extracts from 293T cells overexpressing IRF4, BATF, or JunB. The AP-1 (red) and IRF (blue) sites are indicated. The sequences were derived from IRF4 and BATF co-targeted regions within the CTLA-4 or Bcl11b loci. (D) Protein-DNA complexes were supershifted with antibodies indicated above. (E to G) EMSAs using wild-type (WT) or mutant AICE motif probes. CTLA-4 or Bcl11b DNA probes containing base substitution mutations in either the IRF site (IRF mut), the AP-1 site (AP-1 mut), or both (IRF/AP-1 mut) (E); inverted IRF sites (F); or inserted nucleotides between motifs (G) were used in indicated binding reactions (probes in tables S6 and S7). Red and blue arrows mark the positions of the BATF-JunB-DNA and IRF4-BATF-JunB-DNA complexes, respectively. Data are representative of at least two independent experiments.

Fig. 2

IRF4-BATF-JunB complexes assemble on presumptive regulatory sequences in key T_H17 genes. (A) (Left) ChIP-seq tracks for IRF4 (blue) or BATF (red) at the Il17a, Il21, Il23r, and Il12rb1 loci. Coincident peaks containing AICE motifs are highlighted (boxed regions). (Right) EMSAs using nuclear extracts from T_H17 cells and the indicated DNA probes and antibodies. (B) EMSAs using nuclear extracts from 293T cells overexpressing indicated proteins. Reactions were performed with increasing amounts (fivefold) of IRF4 protein. (C) ChIP analysis of IRF4 binding on indicated sequences in wild-type and Batf−/− T cells. (D) ChIP analysis of BATF binding on indicated sequences in Irf4+/− and Irf4−/− T cells. Purified CD4 cells were polarized for 42 hours under T_H17 differentiating conditions. The average ± SD of two independent experiments is shown for each target sequence after normalization to control T cells.

Fig. 3

IRF4 and BATF complexes activate gene expression and T_H17 differentiation. (A) T_H17-polarized cells were transiently transfected with luciferase reporter plasmids containing the wild-type Il12rb1 promoter or mutant derivatives and a control Renilla luciferase reporter plasmid. Data are from five experiments (average ± SD) (B and C) Naïve CD4 cells activated under nonpolarizing conditions (T_H0) or T_H17-polarizing conditions, respectively, were transduced with the indicated retroviruses. Four days after infection, cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4 hours and analyzed by flow cytometry for intracellular CTLA-4 or IL-17A after gating on transduced cells. (D) EMSAs using nuclear extracts from 293T cells overexpressing indicated proteins. Reactions used increasing amounts (fivefold) of IRF4. (E) Western blot analysis of wild-type and BATF mutant proteins used in (D). (F) Batf−/− T cells were activated under T_H17-polarizing conditions and transduced with retroviruses encoding wild-type BATF or indicated mutants. Six days after infection, cells were restimulated under T_H17 conditions and analyzed 5 days later for expression of IL17 and IFNγ by intracellular staining. (G) Network diagram of IRF4 and BATF co-targeted and regulated genes. Genes marked with * were differentially regulated in Batf−/− T cells based on genome-wide expression analysis but could not be confirmed via quantitative polymerase chain reaction (qPCR). RORγt and STAT3 inputs on these genes are indicated in blue (H). T_H0 cells were transduced with indicated retroviruses and analyzed as described in (B). Data are representative of at least two experiments and where indicated include average values ± SD. n.s., not significant.

Fig. 4

Specificity of AP-1–IRF complexes that cooperatively assemble on AICE motifs. (A to D) EMSAs using the Bcl11b probe and nuclear extracts from 293T cells overexpressing the indicated proteins: (A) c-Jun, BATF, IRF4; (B) FosL2, JunB, IRF4; (C) JunB, BATF, IRF8; or (D) JunB, BATF, IRF3, and IRF4. Complexes were supershifted with indicated antibodies. Red and blue arrows mark the ternary and quaternary protein-DNA complexes, respectively, as in Fig. 1. Data are representative of at least two independent experiments. (E to G) ChIPseq analysis of in vitro differentiated T_H0 and T_H2 cells (42 hours) and LPS-activated dendritic cells (6 hours) with antibodies directed against IRF4. Highly represented motifs and their respective E values within the IRF4 cistrome in T_H0 (241 peaks), in T_H2 (725 peaks), and in dendritic cells (10,364 peaks) are displayed.