Biochemical and mutational analysis of the polyomavirus core promoter: involvement of nuclear factor-1 in early promoter function.
The polyomavirus enhancer is separated from the early RNA initiation sites by a 120 bp promoter region. To identify the core promoter elements, we introduced base-substitution mutations within the potential elements in the vicinity of the RNA initiation site. Three of these mutants, two with mutations within a putative nuclear factor-1 (NF-1) binding site and the other within the TATA box, exhibited reduced promoter activity by about threefold in the mouse NIH 3T3 cell line. The activity of the other three mutants was either little affected or remained unchanged. Mobility shift assays using specific competitors and antibodies against NF-1 demonstrated the binding of a protein of the NF-1 family at a site adjacent to the TATA box, suggesting a role for NF-1 binding in early promoter function. The effect of these mutations was also evaluated in undifferentiated mouse embryonal carcinoma (F9) cells in the presence of an additional mutation (F441) at nucleotide position 5233. This additional mutation creates a strong binding site for a transcription factor, TEF-1, and helps the virus to grow in this cell line. While the TATA box and the GC box mutants behaved qualitatively in a similar fashion, the NF-1 motif now played a minor role in F9 cells. Western blot experiments demonstrated low levels of NF-1 protein in this cell line. The NF-1 motif partially overlaps a T-antigen binding motif and this motif is not involved in T-antigen-mediated regulation of the early promoter. Our results suggest that a protein of the NF-1 family binds to the core promoter and is important for early transcription in vivo. We further demonstrate that undifferentiated F9 cells contain a very low level of NF-1 and the F441 mutant possibly follows a different mechanism for promoter function in these cells.
Shivakumar
C V
CV
Department of Molecular Biology, University of Texas Health Science Center at Tyler 75710.
Das
G C
GC
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011120:Polyomavirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012367:RNA, Viral; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human