Figure 5
Figure 5. Genome-wide identification and characterization of Sox4-binding elements. (A) Schematic outline of the experimental model, in which fetal liver pro-B cells were immortalized, transduced with BirA ligase, Sox4-depleted, and transduced with BAP-Sox4 or BAP. (B) Immunoblot analysis of Sox4 in BAP-Sox4- or BAP-transduced pro-B cells, which were immortalized, transduced with BirA, and depleted of endogenous Sox4. BAP-Sox4 had bound to streptavidin and yielded an additional slow-migrating band. α-Tubulin served as a loading control. (C) Activation of Sox4-luciferase reporter gene expression by biotinylated BAP-Sox4 (in the presence of BirA ligase) in 293T cells. (D) Enrichment of Sox4-binding elements in bioChIP. The multimerized Sox4 canonical motif was introduced into BAP or BAP-Sox4 pro-B cells and examined for changes in bioChIP DNA by quantitative PCR (qPCR). The amount of bioChIP DNA detected in BAP cells was set as 1. #1 and #2 denote results from 2 different sets of the primers. (E) Pie diagram of the genome-wide distribution of Sox4-binding regions in pro-B cells. (F) Enrichment of the Sox4 signal over the transcription start sites (TSS) of gene promoters. (G) Identification of Sox4-binding motifs by CisFinder. Sox4 canonical motif from the UniProbe database and the Sox4 motif identified in this study (left). GA-binding protein α chain (GABPA) canonical motif from the UniProbe database and the Sox4 motif identified in this study (right). (H) Histograms of the distances from the Sox4 (left) and GABPA (right) binding motifs to their peak summits. (I) Sox4 transactivation function on canonical Sox4 (8 copies) and GABPA (4 copies) motifs, cloned in either the forward or the reverse orientation, in luciferase reporter assays of 293T cells. (J) Association between Sox4-binding signals and the concomitant gene expression. A total of 16 984 genes present in the gain-of-function microarray and the RefSeq database were ranked by log2 fold change (FC) in ascending order, and genes with low (1-1000), intermediate (7992-8991), and high (15 985-16 984) levels of change were analyzed for Sox4-binding signals surrounding the 10-kb region of the transcriptional start site. Data are representative of 2 (B-C,I), 3 (D), or 1 (E-H,J) experiment; error bars (C-D,I), SEM. RPKM, reads per kilobase per million tags.

Genome-wide identification and characterization of Sox4-binding elements. (A) Schematic outline of the experimental model, in which fetal liver pro-B cells were immortalized, transduced with BirA ligase, Sox4-depleted, and transduced with BAP-Sox4 or BAP. (B) Immunoblot analysis of Sox4 in BAP-Sox4- or BAP-transduced pro-B cells, which were immortalized, transduced with BirA, and depleted of endogenous Sox4. BAP-Sox4 had bound to streptavidin and yielded an additional slow-migrating band. α-Tubulin served as a loading control. (C) Activation of Sox4-luciferase reporter gene expression by biotinylated BAP-Sox4 (in the presence of BirA ligase) in 293T cells. (D) Enrichment of Sox4-binding elements in bioChIP. The multimerized Sox4 canonical motif was introduced into BAP or BAP-Sox4 pro-B cells and examined for changes in bioChIP DNA by quantitative PCR (qPCR). The amount of bioChIP DNA detected in BAP cells was set as 1. #1 and #2 denote results from 2 different sets of the primers. (E) Pie diagram of the genome-wide distribution of Sox4-binding regions in pro-B cells. (F) Enrichment of the Sox4 signal over the transcription start sites (TSS) of gene promoters. (G) Identification of Sox4-binding motifs by CisFinder. Sox4 canonical motif from the UniProbe database and the Sox4 motif identified in this study (left). GA-binding protein α chain (GABPA) canonical motif from the UniProbe database and the Sox4 motif identified in this study (right). (H) Histograms of the distances from the Sox4 (left) and GABPA (right) binding motifs to their peak summits. (I) Sox4 transactivation function on canonical Sox4 (8 copies) and GABPA (4 copies) motifs, cloned in either the forward or the reverse orientation, in luciferase reporter assays of 293T cells. (J) Association between Sox4-binding signals and the concomitant gene expression. A total of 16 984 genes present in the gain-of-function microarray and the RefSeq database were ranked by log2 fold change (FC) in ascending order, and genes with low (1-1000), intermediate (7992-8991), and high (15 985-16 984) levels of change were analyzed for Sox4-binding signals surrounding the 10-kb region of the transcriptional start site. Data are representative of 2 (B-C,I), 3 (D), or 1 (E-H,J) experiment; error bars (C-D,I), SEM. RPKM, reads per kilobase per million tags.

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