N-Myc represses <i>RD3</i> gene transcription by direct binding to the <i>RD3</i> gene promoter.

<p>(<b>A</b>) BE(2)-C and Kelly cells were transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N-Myc siRNA-2 for 48 hours, followed by RNA extraction and real-time RT-PCR analyses of RD3 mRNA expression. (<b>B</b>) SHEP-21N cells were incubated with or without tetracycline for 48 hours, followed by RNA extraction and RT-PCR analysis of RD3 RNA expression. (<b>C</b>) Schematic representation of the <i>RD3</i> gene promoter. TSS represented transcription start site, and | represented Sp1-binding sites. (<b>D</b>) ChIP-Seq data from Dr. Michael Snyder's group at Yale University for the ENCODE/SYDH project generated from K562 cells. (<b>E</b>) ChIP assays were performed with a control or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the <i>RD3</i> gene core promoter region enriched in Sp1-binding sites in BE(2)-C cells. Fold enrichment was calculated by dividing PCR products from DNA samples immunoprecipitated with the anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was artificially set as 1.0. (<b>F</b>) BE(2)-C cells were transfected with control siRNA or N-Myc siRNA-1, followed by co-transfection with Cypridina TK control construct plus empty vector or <i>RD3</i> gene promoter pLightSwitch_Prom construct. Luciferase activities were measured with a LightSwitch Dual Assay System kit, and expressed as a percentage change relative to control siRNA transfected samples. Error bars represented standard error. * indicated <i>P</i><0.05.