Genome-wide mapping of Ikaros binding sites in pre-B cells. (A) Ikaros ChIP-seq data for Cd79b, Igll1/Vbreb1, Ccnd2, and Lig4 with ChIP- polymerase chain reaction (PCR) validation. Endogenous Ikaros (top) and HA-Ikaros (bottom) tracks for B3 cells are shown alongside corresponding input samples. Red horizontal bars indicate the position of primers used for ChIP-PCR validation. Prom, promoter; enh, enhancer; control: Chr16: 16 853 772 near Igll1. (B) Genes bound by Ikaros in B3 cells (gray) and primary pre-B cells (blue) overlap significantly (P < 10−16, hypergeometric test, gene body plus 2 kb upstream of the transcription start site). (C) The percentage of Ikaros-regulated genes that is bound by Ikaros at 2, 6, and 48 h. (D) Odds ratios of Ikaros binding at differentially expressed genes after 2, 6, and 48 h. (E) Most Ikaros binding sites map in and around genes. (F) Enrichment of Ikaros binding at genomic features of Ikaros-responsive genes (gene = TSS to TTS ± 2 kb), TSS (±1 kb), TTS (+2 kb), and enhancers. Enhancers were defined by the presence in pro-B cells of the enhancer mark H3K4me1 or –me2 and the binding of Ikaros in addition to at least one of the transcription factors E2A, EBF1, or FOXO1 within a 1-kb window and the absence of the promoter mark H3K4me3 within 3 kb. These criteria were met by 771 putative enhancers surrounding 499 genes (3.06% of 16291 genes). (G) Experimental co-occurrence of Ikaros binding with other hematopoietic transcription factors (TFs).
