1,25-Dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) suppresses c-myc expression during differentiation of HL-60 cells along the monocytic pathway by blocking transcriptional elongation at the first exon/intron border of the c-myc gene. In the present study, the physiological relevance of three putative regulatory protein binding sites found within a 280-base pair region in intron 1 of the c-myc gene was explored. HL-60 promyelocytic leukemia cells were transiently transfected with three different c-mye promoter constructs cloned upstream of a chloramphenicol acetyltransferase (CAT) reporter gene. With the wild-type c-myc promoter construct (pMPCAT), which contains MIE1, MIE2, and MIE3 binding sites, 1,25-(OH)(2)D-3 was able to decrease CAT activity by 45.4 +/- 7.9% (mean +/- S.E,, n = 8). The ability of 1,25-(OH)(2)D-3 to inhibit CAT activity was significantly decreased to 18.5 +/- 4.3% (59.3% reversal, p < 0.02) when examined with a MIE1 deletion construct (pMPCAT-MIE1). Moreover, 1,25-(OH)(2)D-3 was completely ineffective at suppressing CAT activity in cells transfected with pMPCAT-287, a construct without MIE1, MIE2, and MIE3 binding sites (-6.5 +/- 10.9%, p < 0.002), MIE1- and MIE2-binding proteins induced by 1,25-(OH)(2)D-3 had similar gel shift mobilities, while MIE3-binding proteins migrated differently. Furthermore, chelerythrine chloride, a selective protein kinase C (PKC) inhibitor, and a PKC beta antisense oligonucleotide completely blocked the binding of nuclear proteins induced by 1,25-(OH)(2)D-3 to MIE1, MIE2, and MIE3, A 1,25-(OH)(2)D-3-inducible MIE1-binding protein was identified to be HOXB4. HOXB4 levels were significantly increased in response to 1,25-(OH)(2)D-3. Taken together, these results indicate that HOXB4 is one of the nuclear phosphoproteins involved in c-mye transcription elongation block during HL-60 cell differentiation by 1,25-(OH)(2)D-3.