Numerous nuclear proteins bind the long control region of human papillomavirus type 16: a subset of 6 of 23 DNase I-protected segments coincides with the location of the cell-type-specific enhancer.
The long control region of the human papillomavirus type 16 genome is 856 base pairs (bp) long. It contains a cell-type-specific enhancer, a glucocorticoid response element, and sequences mediating the response to the viral gene products of open reading frame E2; all three regulate the promoter P97. We mapped binding sites of trans-acting proteins relevant for the cell-type-specific enhancer and other cis-acting elements by DNase I footprint experiments with nuclear extracts from HeLa cells. Throughout the human papillomavirus type 16 long control region 23 footprints protect 557 of 900 bp. Nine footprints fall into a 400-bp segment that was previously identified to contain the cell-type-specific enhancer. Variations of the protein concentration in the footprint reaction do not affect six of these nine footprints. At high protein concentrations, three footprints fuse to a 106-bp protected region, suggesting that this segment specifically binds several proteins of lower affinity or abundance. Unexpectedly, extracts from human MCF7 and mouse 3T3 cells, in which the enhancer is inactive, give footprints identical to those obtained with HeLa extracts. Seven footprints contain the sequence 5'-TTGGC-3'. Footprint competition experiments suggest that factor NFI binds to these seven motifs. Competition with cloned oligonucleotides in transfections suggests that these elements contribute to the enhancer function. Subcloning identifies a 232-bp fragment between positions 7524 and 7755 as sufficient for full enhancer activity. Several of the six footprinted elements on this segment may cooperate functionally, since subclones of this region show decreased or no cell-type-specific enhancer function.
Gloss
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B
Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge.
Chong
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T
Bernard
H U
HU
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D027383:Papillomaviridae / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014779:Virus Replication
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D003850:Deoxyribonuclease I