C-MYC ACTIVATES RCC1 GENE-EXPRESSION THROUGH E-BOX ELEMENTS

Proto-oncogene c-myc is implicated in proliferation of Although c-Myc protein has been function as a transcription factor recognizing an E-b ox (CACGTG) element, few c-myc-regulated genes have been identified an d the specific role of c-myc is still unclear, RCC1 is necessary for m ammalian cells to proliferate, Four CACGTG elements exist within 1.3 k b downstream of the major transcription start site for the human RCC1 gene in HeLa cells, Stimulation of HeLa cells with serum increased c-m yc expression and RCC1 expression, Therefore the relationship between the expression of RCC1 and c-myc was investigated, Rat 3Y1 cells overe xpressing c-myc contained about tw ice as much RCC1 mRNA as control ce lls, When a chimeric protein comprised of c-myc and the estrogen bindi ng domain of estrogen receptor was activated by addition of 4-hydroxyt amoxifen (OHT), expression of RCC1 mRNA increased twofold, To examine whether c-MSc functions through the CACGTG elements, a DNA fragment of RCC1 intron 4, exon 5 and part of intron 5 was joined to firefly luci ferase cDNA to construct a reporter plasmid, In transient expression e xperiments using HeLa cells, co-transfection with c-myc stimulated the luciferase activity up to 2.5-fold in a dose-dependent manner, When t he CACGTG elements in the reporter plasmid were destroyed, stimulation by c-myc was not observed, The four CACGTG elements did not contribut e equally to the stimulation by c-myc. Gel retardation experiments sug gest that c-Myc with Max binds to the CACGTG elements in the context o f the RCC1 gene sequence in vitro. These results indicate that c-Myc c an regulate expression of RCC1 through the E-box elements.