Deletion hotspots in <i>AMACR</i> promoter CGI are the <i>cis</i>-acting elements.

<p>A: Promoter assays showed that <i>AMACR599</i> with the CGI in it had promoter activity comparable to that of <i>AMACR1818</i>, suggesting that the 599 bp region is critical for the gene regulation. Thus, we selected <i>AMACR599</i> for further investigation. The promoter activity was normalized as relative light units. B: The location of the deletion hotspots in <i>AMACR</i> promoter. Various sequence variants were compared with the wild-type promoter. A previously identified CCAAT box is illustrated. C: Compared with the wild-type <i>AMACR599</i>, 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%, <i>p</i><0.0001, one-way analysis of variance, followed by Tukey's HSD <i>post hoc</i> test). No significant differences among the CCAAT deletion groups (<i>p</i> = 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% (<i>p</i><0.0001) but no significant difference among the deletion groups (<i>p</i> = 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 (<i>p</i> = 0.26 and 0.69, respectively). In contrast, double-deletion at CG3 and 10 decreased the promoter activity by 69% (<i>p</i><0.001).</p>