Activation of protein kinase C induces nuclear translocation of RFX1 and down-regulates c-myc via an intron 1 X box in undifferentiated leukemia HL-60 cells

Treatment of human promyelocytic leukemia cells (HL-60) with phorbol la-myr istate 13-acetate (PMA) is known to decrease c-myc mRNA by blocking transcr iption elongation at sites near the first exon/intron border. Treatment of HL-60 cells with either PMA or bryostatin 1, which acutely activates protei n kinase C (PKC), decreased the levels of myc mRNA and Myc protein. The inh ibition of Myc synthesis accounted for the drop in Myc protein, because PMA treatment had no effect on Myc turnover. Treatment with PMA or bryostatin 1 increased nuclear protein binding to MIE1, a c-myc intron 1 element that defines an RFX1-binding X box. RFX1 antiserum supershifted MIE1-protein com plexes. Increased MIE1 binding was independent of protein synthesis and abo lished by a selective PKC inhibitor, which also prevented the effect of PMA on myc mRNA and protein levels and Myc synthesis. PMA treatment increased RFX1 in the nuclear fraction and decreased it in the cytosol without affect ing total RFX1. Transfection of HL-60 cells with myc reporter gene construc ts showed that the RFX1-binding X box was required for the downregulation o f reporter gene expression by PMA. These findings suggest that nuclear tran slocation and binding of RFX1 to the X box cause the down-regulation of myc expression, which follows acute PKC activation in undifferentiated HL-60 c ells.