Dissection of ASE enhancer activity using extrachromosomal reporter assays. (A and B) Diagrams show relative positioning of the ASE with respect to mouse Rag1 and Rag2 promoters (A) and conserved binding sites for transcription factors within the ASE core region (B). (C) ChIP assays of transcription factor binding to the ASE core region and the inactive MageA2 promoter in sorted DP thymocytes. The data represent mean ± SEM of three independent experiments, with enrichment expressed relative to control IgG ChIP. (D) Activity of wild-type and mutant ASEs tested in luciferase reporters containing the Rag1 promoter (top) or Rag2 promoter (bottom). Test ASE fragments of 1.2 kb were cloned downstream of Rag1 or Rag2 promoter–driven luciferase gene, and plasmids were assayed for luciferase activity following transient transfection into VL3-3M2 cells. The data represent mean ± SEM of four independent experiments, with values for mutant ASEs normalized to wild-type in each experiment and the average value for the wild-type ASE set as 1. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-way ANOVA with Holm-Sidak’s multiple comparisons test (C) or one-way ANOVA with Holm-Sidak’s multiple comparisons test (D).