The newly identified ZICEs correlate with gene downregulation. (A) Motif analysis of IRF4 binding sequences obtained from downregulation target genes. IRF4 direct targets in B1-8^hi splenic B cells were selected using IRF4 ChIP-seq in B1-8i splenic B cells (Gene Expression Omnibus accession number GSE46607) as described in supplemental Figure 1B. IRF4 binding targets belonging to cluster 3 (supplemental Table 1) were further classified into those with and without PU.1 binding (PU.1 coincident and noncoincident, respectively). In total, 47, 28, 113, and 47 regions were extracted for these categories, respectively. These sequences were analyzed with the MEME algorithm to identify overrepresented motifs within 100 bp in either direction for PU.1 coincident or 200 bp in either direction for PU.1 noncoincident of the peak maxima. Results are represented for the enriched motifs. (B) The ZICEs correlate with efficient downregulation of IRF4 direct target genes. Left: IRF4 downregulated target genes were classified into 2 subgroups depending on the presence of the ZICEs within the IRF4 bound regions (supplemental Table 3). ZICE (−), 52 genes with 84 IRF4 binding peaks that lacked the ZICEs and included IcosL and Pik3ap1; ZICE (+), 26 genes with 62 IRF4 binding peaks that contained the ZICEs and included Ebf1, Haao, and Setd2. Among these 62 peaks, 29 peaks were detected in PU.1 ChIP-seq as well, whereas 33 peaks were not detected. The y-axis shows the percentage of downregulated genes. Right: The amounts of transcripts of each ZICE (−) or ZICE (+) IRF4 direct target genes in CD138 positive (CD138^posi) cells were divided by that of transcripts at 0 hours (from Figure 1B). Data are shown with box-and-whisker plot and the P value. (C) Gene ontology (GO) analysis of subgroups of IRF4 downregulation target genes. 52 ZICE (−) genes or 26 ZICE (+) genes were analyzed for their enrichment in GO focusing on biological process using the David v6.8 algorithm. The x-axis shows the P value of pathway-specific enrichment.