Deletion hotspots in AMACR promoter CGI are the cis-acting elements.
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A: Promoter assays showed that AMACR599 with the CGI in it had promoter activity comparable to that of AMACR1818, suggesting that the 599 bp region is critical for the gene regulation. Thus, we selected AMACR599 for further investigation. The promoter activity was normalized as relative light units. B: The location of the deletion hotspots in AMACR promoter. Various sequence variants were compared with the wild-type promoter. A previously identified CCAAT box is illustrated. C: Compared with the wild-type AMACR599, deletion of CCAAT enhancer element at CG5, or in combination with other deletion hotspots at CG3, 10 and 12-16, significantly reduced the promoter activity (58∼67%, p<0.0001, one-way analysis of variance, followed by Tukey's HSD post hoc test). No significant differences among the CCAAT deletion groups (p = 0.014 to 1) were observed. When the CCAAT enhancer was maintained intact, the single deletion at CG12-16 or in combination with deletion hotspots at CG3 and 10 resulted in an increase in the promoter activity by 83−105% (p<0.0001) but no significant difference among the deletion groups (p = 0.060 to 1). Further, when the CCAAT enhancer was maintained and CG12-16 was intact, deletion of either CG3 or CG10 did not change the promoter activity significantly (p = 0.26 and 0.69, respectively). In contrast, double-deletion at CG3 and 10 decreased the promoter activity by 69% (p<0.001).