The keratinocyte-specific Epstein-Barr virus ED-L2 promoter is regulated by phorbol 12-myristate 13-acetate through two cis-regulatory elements containing E-box and Krüppel-like factor motifs.
We previously employed 782 base pairs of the Epstein-Barr virus ED-L2 early lytic cycle promoter in a transgenic mouse model to target cyclin D1 to the stratified squamous epithelium of the tongue and esophagus. This promoter is located 5' to the transcriptional start site of a short open reading frame BNLF-2A and is immediately 3' to the BNLF-1 (LMP-1 oncogene) open reading frame. We studied transcriptional regulation of the ED-L2 promoter by phorbol 12-myristate 13-acetate (PMA) as a means of understanding the tissue specificity of this promoter. The transcriptional activity of the ED-L2 promoter was stimulated 40-fold by PMA and could be blocked with the compound H7 through antagonism of protein kinase C. 5' deletion analysis of the 782-base pair promoter demonstrated that the sequences necessary for PMA-stimulated trans-activation were located in two separate cis-regulatory regions of the promoter: -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 CACCTG (E-box) motif, and region -144 to -114 contains a CACACCC motif. Both of these motifs are necessary for trans-activation by PMA. These regions do not, however, demonstrate enhancer characteristics when tested in a heterologous minimal promoter system. Variations of the CACACCC motif are found in other keratinocyte-specific promoters, as well as in the DNA binding motifs of the Krüppel-like family of transcription factors. Electrophoretic mobility shift assays with specific competitors and factor-specific antibody supershift assays demonstrated that one complex binding the -187 to -164 region containing the CACCTG nucleotides has characteristics of the helix-loop-helix protein upstream stimulatory factor, whereas a factor binding the CACACCC motif may be a member of the Krüppel-like family. These experiments show how ubiquitous and tissue-specific transcription factors induced by PMA regulate the ED-L2 promoter in squamous epithelial cells.
Jenkins
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TD
Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
Nakagawa
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H
Rustgi
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AK
D019070:Cell Lineage; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D009687:Nuclear Proteins; D014157:Transcription Factors; D013755:Tetradecanoylphorbol Acetate