A distal element in the HPV-11 upstream regulatory region contributes to promoter repression in basal keratinocytes in squamous epithelium

In benign squamous lesions and in organotypic epithelial cultures, the huma n papillomavirus (HPV) E6 and 57 genes are transcriptionally up-regulated i n differentiated, spinous keratinocytes. We previously identified sequence elements in the enhancer-promoter regions of HPV types 18 and 11 important for this promoter regulation by using the bacterial LacZ reporter gene in s tratified raft cultures of primary human keratinocytes (PHKs) or in submerg ed, proliferating cultures acutely transduced with recombinant retroviruses . Notably, mutations in the promoter-proximal Sp1, Oct1, and AP1 sites each significantly reduce reporter activity in differentiated cells, indicating that the bound factors are transcription transactivators. In the present s tudy, we performed further mutagenesis on distal motifs in the HPV-11 regul atory region in PHKs in submerged and raft cultures. Mutations in an AP2-li ke site,three individual NF-1 sites, or five NF-1 sites collectively reduce d promoter activity slightly in differentiated cells. A mutation in a putat ive glucocorticoid response element had no discernable effect in the presen ce or the absence of dexamethasone However, mutations in a C/EBP binding si te, especially the distal site, strikingly up-regulated reporter gene expre ssion, particularly in basal and lower spinous cells, implicating bound pro tein as a transcription repressor. Collectively, these results demonstrate that the overall differentiation-dependent papillomaviral gene expression o bserved in vivo and in vitro involves promoter repression in the lower stra ta and activation in the upper, differentiated strata. (C) 1999 Academic Pr ess.