Fig. 4
From: IRF8-dependent molecular complexes control the Th9 transcriptional program
IRF8 needs cooperative factors to induce Th9 polarisation. a Transactivation of the Il9 (left) and Il21 (right) promoters by IRF8. Il9 or Il21 promoter reporter plasmids were transfected into NIH-3T3 cells with an IRF8-encoding vector. b Determination of IRF8-binding motifs by analysis of IRF8 ChIP-Seq peaks from WT Th9 cells. Left, letter size indicates frequency of nucleotide. Right, significance of motif occurrence. c Immunoprecipitation assay on Th9 cells differentiated for 24 h using anti-IRF8 antibody (above) or anti-PU.1 antibody (below), followed by immunoblot analysis with indicated specific antibodies. Ig is used as a control. d, e PLA in WT naive CD4+ T cells and in Th9 cells after 24 h of differentiation. DAPI staining (blue) indicates the nucleus. Green dots represent proximity between IRF8/PU.1, IRF4/PU.1 or IRF8/IRF4 (d) or between BATF and IRF8 or PU.1 or IRF4 (e) Magnifications are ×63, scale bar represents 5 µm. f Top panel represent a screenshot from the ECR (evolutionary conserved regions) Browser web site of the mouse Il9 (left) and Il21 (right) genes. Exons are in blue, introns in pink, UTRs in yellow and CNS are in red. Bottom panel is a representation of binding peaks of ChIP-Seq data of BATF, IRF4, PU.1 and IRF8 in WT Th9 cells differentiated for 24 h. The red square shows the overlap between BATF, IRF4, PU.1 and IRF8 peaks in Il9 or Il21 CNS1. g Co-location of IRF8, PU.1, IRF4 and BATF-binding peaks in Th9 cells were determined by multiple overlap analysis of the respective ChIP-Seq data. IRF8 can bind DNA alone or in combination with one, two or three factors (IRF4, BATF and PU.1). h The average density of IRF4, PU.1 and BATF in Th9 cells is shown for regions centred on the summits of the IRF8 peaks. i Transactivation of the Il9 promoters by IRF8, PU.1, IRF4 and BATF. Il9 promoter reporter plasmids were transfected into NIH-3T3 cells with vectors encoding IRF8, PU.1, IRF4 and BATF alone or in combination. ns, not significant; *P < 0.05, **P < 0.01; ***P < 0.001 (Kruskal–Wallis test (a) or two-way ANOVA (i)). Data are from three independent experiments (mean and s.e.m.)
