Identification of HeLa cell nuclear factors that bind to and activate the early promoter of human polyomavirus BK in vitro.
Human polyomavirus BK (BKV), an oncogenic DNA virus, differs from other papovaviruses in the organization of the regulatory region and in tissue tropism for kidney cells. The noncoding regulatory region of the viral genome in prototype strains includes three 68-base-pair (bp) repeats, each containing a number of potential regulatory elements. Some of these signals are unique to human papovaviruses, and others are homologous to those identified in many viral and cellular genes. We evaluated the contribution of individual 68-bp repeats to the initiation of transcription from the early promoter in a HeLa cell extract and identified cis-acting elements to which human cellular factors bind to activate transcription. The early promoter with only one copy of the 68-bp repeat could accurately initiate transcription in vitro, but additional copies were required for its stimulation. DNA-binding assays and DNase I protection experiments identified six domains in the regulatory region protected by human cellular factors. Two of these footprints were located within the proximal and distal 68-bp repeats, and one was located at the late side of the repeats. These footprints were centered over a TGGA(N)5-6GCCA core and were produced by a protein of the nuclear factor 1 (NF-1) family. This protein is either identical or similar to that which binds to the high-affinity site at the origin of adenovirus DNA replication. Three other domains, two at the junctions of the 68-bp repeats and one in the late side of the repeats, were partially protected by proteins with AP-1- and Sp-1-like activities. Transcription initiation from the early promoter was drastically reduced when a complete 68-bp repeat or the NF-1 binding site was used as a competitor in the in vitro assay. However, a point mutation within the NF-1 binding site, which reduced NF-1 binding in vitro to a level comparable to that of nonspecific DNA, also eliminated its ability to compete with early transcription. The murine homolog of the AP-1 binding site had a modest effect on in vitro transcription. Our results suggest that, among the multiple HeLa cell nuclear factors, NF-1 acts as a major activator of the early promoter in vitro.
Chakraborty
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Department of Molecular Biology, University of Texas Health Center, Tyler 75710.
Das
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D001739:BK Virus / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D011120:Polyomavirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors; D014158:Transcription, Genetic; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human