Human papillomavirus type 16 P670 promoter is negatively regulated by CCAAT displacement protein.
HPV16 late gene transcription from P670 is suppressed in undifferentiated keratinocytes. To identify DNA sites involved in the negative regulation, we examined the effect of a series of substitutions in the P670 promoter region (nucleotide (nt) 106-855) on the transcription, using an expression plasmid having the promoter fragment placed to drive the firefly-luciferase gene. Twenty-base pair-long segments covering the entire promoter region were replaced with a sequence lacking any so far known factor-binding motifs to produce 38 mutants. These plasmids were introduced by transfecton into undifferentiated or partially differentiated human HaCaT and HeLa cells, and transient expression of the reporter was examined with the cell extracts. The reporter expression from the wild-type promoter region was lower, half to one-third, in the undifferentiated cells than in the partially differentiated cells, which expressed hSkn-1a, a keratinocyte specific transcription factor that activates P670, and CCAAT displacement protein (CDP), a transcriptional repressor involved in cell differentiation. Two mutants with substitutions including the putative CDP-binding sites, one from nt 562 to 567 and the other from nt 673 to 678, induced markedly enhanced reporter expression particularly in the partially differentiated cells. Electrophoretic mobility shift analysis demonstrated that bacterially produced GST-CDP bound to the two sites in a sequence-specific manner. The data strongly suggest that CDP acts as a major suppressor for P670 transcription by binding to the promoter region in the undifferentiated cells and even in the partially differentiated cells that express the activator hSkn-1a.
Sato
Kaori
K
Center for Pathogen Genomics, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Takeuchi
Takamasa
T
Kukimoto
Iwao
I
Mori
Seiichiro
S
Yasugi
Toshiharu
T
Yano
Tetsu
T
Taketani
Yuji
Y
Kanda
Tadahito
T
D019677:Artificial Gene Fusion; D001665:Binding Sites; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D052162:Human papillomavirus 16 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D008954:Models, Biological; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D050810:Octamer Transcription Factors / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection
C080632:CUX1 protein, human; D004279:DNA, Viral; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D050810:Octamer Transcription Factors; C484169:POU2F3 protein, human; D012097:Repressor Proteins; D014157:Transcription Factors; D049409:Luciferases, Firefly