Transcription from human papillomavirus type 16 (HPV16) P₆₇₀, a promoter in the E7 open reading frame, is repressed in undifferentiated keratinocytes but becomes activated upon differentiation. We showed that the transient luciferase expression driven by P₆₇₀ was markedly enhanced in HeLa cells cotransfected with an expression plasmid for human Skn-1a (hSkn-1a), a transcription factor specific to differentiating keratinocytes. The hSkn-1a POU domain alone, which mediates sequence-specific DNA binding, was sufficient to activate the expression of luciferase. Electrophoretic mobility shift assay revealed the presence of two binding sites, sites 1 and 2, upstream of P₆₇₀, which were shared by hSkn-1a and YY1. Site 1 bound more strongly to hSkn-1a than site 2 did. YY1 complexing with a short DNA fragment having site 1 was displaced by hSkn-1a, indicating that hSkn-1a's affinity with site 1 was stronger than YY1's. Disrupting the binding sites by nucleotide substitutions raised the basal expression level of luciferase and decreased the enhancing effect of hSkn-1a. In HeLa cells transfected with circular HPV16 DNA along with the expression plasmid for hSkn-1a, the transcript from P₆₇₀ was detectable, which indicates that the results obtained with the reporter plasmids are likely to have mimicked the regulation of P₆₇₀ in authentic HPV16 DNA. The data strongly suggest that the transcription from P₆₇₀ is repressed primarily by YY1 binding to the two sites, and the displacement of YY1 by hSkn-1a releases P₆₇₀ from the repression.