Objective       To investigate the transcriptional regulation of caudal-related homeodomain transcription factor 2 (CDX2) gene expression by Achaete scute-like 2 (Ascl2) in colon cancer epithelial cells.        Methods       Transcription factor binding sites of CDX2 promoter were predicted. Vector pGL3-Basic was used to construct 8 recombinant plasmids (pGL3-CDX2-C1 to pGL3-CDX2-C8) containing different truncated CDX2 proximal promoters. After transfection of recombinant plasmids into shRNA-Ascl2/LS174T and shRNA-Ctr/LS174T cells

the activity of those luciferase reporters was detected by luciferase reporter assay. We used chromatin immunoprecipitation (ChIP) assay with an anti-Ascl2 antibody to immunoprecipitate the possible Ascl2-binding DNA fragments within CDX2 proximal promoter. The immunoprecipitates were amplified through PCR using specific CDX2 primers.        Results       Predicted by transcription factor databases

the CDX2 proximal promoter contained the abundant E-box(s) and the probable cis-regulatory elements for Nkx-2

GATA-1

CdxA

Sox-5 and SRY. The inserts within those luciferase reporters were proved to be correct by enzyme digestion and sequencing. These luciferase reporters had higher luciferase activity in shRNA-Ascl2/LS174T cells than in shRNA-Ctr/LS174T cells (P0.01). However

further truncation of the CDX2 promoter resulted in an increased luciferase activity. The specific DNA fragments in CDX2 proximal promoter were detected in the immunoprecipitates by the anti-Ascl2 antibody. The semi-quantitation of specific DNA fragments within -841 to -646

-291 to -134 or -7 to +138 of CDX2 proximal promoter showed their significant reduction in shRNA-Ascl2/LS174T cells as compared to shRNA-Ctr/LS174T cells (P0.05).        Conclusion       Ascl2 transcriptionally suppresses CDX2 gene expression by direct binding to its proximal promoter in colon cancer epithelial cells.