Figure 4. Function of activated and repressed Ikaros target genes in pro-B cells.

(a) Pie diagram indicating the different functional classes of activated and repressed Ikaros target genes in pro-B cells. Regulated Ikaros target genes coding for cell surface protein, signal transducer and transcription factors are shown below and ranked according to their fold expression changes observed between in vitro cultured Ikzf1^Δ/– and Ikzf1^Δ/+ pro-B cells (Fig. 3f; Supplementary Table 1). The color code refers to >10-fold (green), 5-10-fold (blue), 4-5-fold (red) and 3-4-fold (black). Underlined target genes were jointly activated or repressed by Ikaros and Pax5 (ref. ²⁰). The number sign (#) indicates genes with low expression levels in ex vivo sorted pro-B cells, i.e. < 5 RPKM in Ikzf1^Δ/+ pro-B cells for activated target genes and < 5 RPKM in Ikzf1^Δ/– pro-B cells for repressed target gene. Asterisks indicate genes, which were not regulated (< 1.4-fold) in short-term cultured Ikzf1^Δ/– Rag2^–/– and Ikzf1^Δ/+ Rag2^–/– pro-B cells. (b) Validation of regulated Ikaros target genes at the protein level. In vitro cultured and ex vivo c-Kit^hi pro-B cells (CD19⁺CD2^–IgM^–IgD^–) of the Cd79a-Cre Ikzf1^fl/– (Δ/–; black line) and Cd79a-Cre Ikzf1^fl/+ (Δ/+; grey) genotypes were analyzed by flow cytometry for the expression of cell surface proteins encoded by activated (Icam1, Bst1) and repressed (Cd22) target genes as well as by the indirectly repressed gene Cd72. (c) Aiolos expression in Ikzf1 mutant pro-B cells. Aiolos expression was analyzed by intracellular staining of in vitro cultured and ex vivo c-Kit^hi pro-B cells (CD19⁺CD2^–IgM^–IgD^–) from the Cd79a-Cre Ikzf1^fl/– (Δ/–; black line) and Cd79a-Cre Ikzf1^fl/+ (Δ/+; grey) mice. Small pre-B cells (c-Kit^–CD19⁺B220⁺CD2⁺IgM^–IgD^–; dashed line) of control mice are shown for comparison.