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The promoter of the ornithine decarboxylase (ODC) gene contains two E-boxes, which are critical sites for transcriptional activation by the binding of c-Myc-Max heterodimers. We have identified heterogeneous nuclear ribonuclear protein U (hnRNP U) as a component of the complex formed on the E-box-containing promoter region of the ODC gene by using DNA-affinity chromatography, immunoprecipitation and chromatin immunoprecipitation assays. The N-terminal domain of hnRNP U was responsible for the association with c-Myc-Max complex. Down-regulation of hnRNP U with RNA interference blocked the induction of the ODC gene and cell growth by serum stimulation, suggesting that hnRNP U is a coactivator of the c-Myc-Max complex and essential for cell proliferation. Electrophoretic mobility-shift assays revealed that the segment between the two E-boxes in the promoter is the primary binding site of hnRNP U. The putative binding sequence was narrowed-down to a 13-nucleotide segment by comparing the sequence between the E-boxes with the binding sites of hnRNP U, which were recently identified in the promoter of Bmal1, a core component of the circadian molecular oscillator. These findings increase our knowledge of how the c-Myc-Max complex exerts its transcriptional regulatory role and suggest that hnRNP U may be a coactivator of this transcriptional activator complex.

hnRNP U interacts with the c-Myc-Max complex on the E-box promoter region inducing the ornithine decarboxylase gene