Sox4 induces the expression of Rag1 and Rag2 and controls gene recombination at the Igh loci. (A) Real-time RT-PCR analysis of Sox4, Rag1, and Rag2 expression in cDNAs prepared from primary (left) or immortalized (middle) Sox4^fl/+SE-Cre and Sox4^fl/flSE-Cre pro-B cells or from HA- or HA-Sox4-expressing and immortalized pro-B cells that had the endogenous floxed Sox4 deletion (right). Fraction B cells that were sorted out 9 days after the introduction of SE-Cre were used in the analysis (left and middle). In each pair, the higher level was set as 1 (error bars, SEM). (B) Genome browser view of Sox4 binding in the Rag1 locus identified by ChIP-Seq. Rag1 transcript in relation to Sox4 binding is shown. The arrow points to the Sox4-binding site. chr, chromosome. (C) qPCR detection of the Sox4 binding to Rag1 locus. Sox4 binding to the first intron of Rag1 was detected by qPCR amplification of bioChIP DNA from BAP or BAP-Sox4 cells, and the results were normalized to those from corresponding input DNA samples (normalized results from BAP cells were set as 1). (D) Semiquantitative PCR analysis of D_H-J_H3, V_HQ52-DJ_H3, V_H7183-DJ_H3, and V_HJ558-DJ_H3 gene rearrangements in fivefold serial dilutions of DNA prepared from Sox4^fl/+SE-Cre and Sox4^fl/flSE-Cre pro-B cells. α-Actin served as a normalization control. (E) Semiquantitative RT-PCR analysis of rearranged D_H-JC_μ, V_HQ52-DJC_μ, V_H7183-DJC_μ, and V_HJ558-DJC_μ transcripts in fivefold serial dilutions of cDNA prepared from Sox4-deficient pro-B cells (left) and HA-Sox4-complemented pro-B cells (right). * denotes nonspecific amplification. β-Actin served as a normalization control. Data are representative of 1 (B-C) or 3 (A,D-E) independent experiments.