Yin Yang 1 negatively regulates the differentiation-specific E1 promoter of human papillomavirus type 6

Human papillomavirus type 6 (HPV-6) is a low-risk HPV whose replication cyc le, like that of all HPVs, is differentiation dependent. We have previously shown that CCAAT displacement protein (CDP) binds the differentiation-indu ced HPV-6 E1 promoter and negatively regulates its activity in undifferenti ated cells (W. Ai, E. Toussaint. and A. Roman, J. Virol, 73:4220-1229, 1999 ). Using electrophoretic mobility shift assays (EMSAs), we now report that Yin Yang 1 (YY1), a multifunctional protein that can act as a transcription al activator or repressor and that can also inhibit NPV replication in vitr o, binds the HPV 6 E1 promoter. EMSAs, using subfragments of the promoter a s competitors, showed that the YY1 binding site is located at the 5' end of the E1 promoter. When a putative YY1 site was mutated, the ability of YY1 to bind was greatly decreased. The activity of the mutated E1 promoter, mon itored with the reporter gene luciferase, was threefold greater than that o f the wild-type promoter, suggesting that YY1 negatively regulates HPV-6 E1 promoter activity. Nuclear extracts from differentiated keratinocytes show ed decreased binding of YY1 to the wild-type promoter. Consistent with this , in differentiated keratinocytes, the activity of the transfected lucifera se gene transcribed from the mutated promoter was comparable to that of the wild-type promoter; both promoters were up-regulated in differentiated ker atinocytes compared to undifferentiated cells. These data suggest that YY1 functions in undifferentiated keratinocytes but not in differentiated kerat inocytes. Both the wild-type and mutated promoters could be negatively regu lated by overexpression of a plasmid encoding CDP. Thus, both YY1 and CDP a ppear to be negative regulators of the differentiation-induced HPV-6 E1 pro moter and thereby the HPV life cycle. In contrast, only binding of CDP was detected using the E1 promoter of the high-risk HPV-31.