THE KERATINOCYTE-SPECIFIC EPSTEIN-BARR-VIRUS ED-L2 PROMOTER IS REGULATED BY PHORBOL 12-MYRISTATE 13-ACETATE THROUGH 2 CIS-REGULATORY ELEMENTS CONTAINING E-BOX AND KRUPPEL-LIKE FACTOR MOTIFS

We previously employed 782 base pairs of the Epstein-Barr virus ED-LP early lytic cycle promoter in a transgenic mouse model to target cycli n D1 to the stratified squamous epithelium of the tongue and esophagus , This promoter is located 5' to the transcriptional start site of a s hort open reading frame BNLF-2A and is immediately 3' to the BNLF-1 (L MP-1 oncogene) open reading frame. We studied transcriptional regulati on of the ED-LB promoter by phorbol 12-myristate 13-acetate (PMA) as a means of understanding the tissue specificity of this promoter. The t ranscriptional activity of the ED-LS promoter was stimulated 40-fold b y PMA and could be blocked with the compound H7 through antagonism of protein kinase C. 5' deletion analysis of the 782-base pair promoter d emonstrated that the sequences necessary for PMA-stimulated trans-acti vation were located in two separate cis-regulatory regions of the prom oter: -187 to -164 and -144 to -114 base pairs from the transcription start site of BNLF-2A. Importantly, mutation of critical base pairs in each region was sufficient to abolish PMA-stimulated trans-activation in the native ED-L2 promoter. Region -187 to -164 contains a CACACCC (E-box) motif, and region -144 to -114 contains a CACACCC motif. Both of these motifs are necessary for trans-activation by PMA. These regio ns do not, however, demonstrate enhancer characteristics when tested i n a heterologous minimal promoter system. Variations of the CACACCC mo tif are found in other keratinocyte-specific promoters, as well as in the DNA binding motifs of the Kruppel-like family of transcription fac tors. Electrophoretic mobility shift assays with specific competitors and factor-specific antibody supershift assays demon strated that one complex binding the -187 to -164 region containing the CACCTG nucleoti des has characteristics of the helix loop-helix protein upstream stimu latory factor, whereas a factor binding the CACACCC motif may be a mem ber of the Kruppel-like family, These experiments show how ubiquitous and tissue-specific transcription factors induced by PMA regulate the ED-LP promoter in squamous epithelial cells.