CCAAT displacement protein binds to and negatively regulates human papillomavirus type 6 E6, E7, and E1 promoters

Expression of human papillomavirus genes increases as the target cell, the keratinocyte, differentiates. CCAAT displacement protein (CDP) is a cellula r protein which has been shown in other cell types to negatively regulate g ene expression in undifferentiated cells but not in differentiated cells. W e have previously shown that a 66-bp purine-thymidine-rich sequence (the 66 -mer) binds CDF and negatively regulates the human papillomavirus type 6 (H PV-6) E6 promoter (S, Pattison, D. G. Skalnik and A, Roman, J, Virol, 71:20 13-2022, 1997), Cotransfection experiments with a plasmid expressing lucife rase from the HPV-6 E6, E7, or E1 regulatory region and a plasmid carrying the CDP gene indicate that CDP represses transcription from all three HPV-6 promoters. Using electrophoretic mobility shift assays (EMSAs), we have sh own that CDP binds HPV-6 both upstream and downstream of the E6, E7, and El transcription initiation start sites. Furthermore, when keratinocytes were induced to differentiate, all three promoter activities Increased. Consist ent with this, immunoblotting and EMSAs revealed that endogenous nucleus CD P and, correspondingly, DNA binding activity decreased when keratinocytes w ere induced to differentiate. The elevated promoter activities were abrogat ed by exogenously transfected CDP, Our data demonstrate that CDP fulfills t he requirement of a differentiation-dependent negative regulator that could tie the HPV life cycle to keratinocyte differentiation.