In vivo footprint analysis of the HLA-DRA gene promoter: cell-specific interaction at the octamer site and up-regulation of X box binding by interferon gamma.
Analysis of the major histocompatibility complex class II gene promoter DRA has previously identified at least five cis-acting regions required for maximal expression. We have examined the DRA promoter for protein-DNA interactions in the intact cell, which may mediate transcriptional activation. Using in vivo genomic footprinting we identified interactions in B-cell lines at the octamer site and the Y, X1, and X2 boxes. Class II antigen expressing T-cell lines maintained contacts identical to B-cell lines, while class II-negative T-cell lines exhibited no interactions. In lymphoid cell lines, the octamer site is occupied and required for maximal expression. This is most likely due to the presence of the lymphoid-specific OTF-2 factor. In contrast, the class II-positive nonlymphoid glioblastoma cell line does not exhibit interactions at the octamer site despite the presence of the ubiquitous OTF-1 factor and an open binding site. Thus, the DRA promoter discriminates against OTF-1 activation at the level of DNA binding in the glioblastoma line. Interferon gamma induces class II expression in this glioblastoma cell line and, in parallel, up-regulates X1 and X2 box protein-DNA interactions, while all other interactions remain unchanged. These results suggest that interferon gamma functions on a poised promoter by altering weak, nonproductive interactions at the X boxes to strong interactions. These findings provide direct in vivo evidence to strongly suggest that the modulation of X1 and X2 interactions is an important constituent of the interferon gamma induction pathway.
Wright
K L
KL
University of North Carolina Lineberger Comprehensive Cancer Center, Chapel Hill.
Ting
J P
JP
D001402:B-Lymphocytes; D001483:Base Sequence; D001665:Binding Sites; D002051:Burkitt Lymphoma; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D005802:Genes, MHC Class II / Q000187:drug effects; D005910:Glioma; D006684:HLA-DR Antigens / Q000235:genetics*; D059805:HLA-DR alpha-Chains; D051863:Host Cell Factor C1; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D008969:Molecular Sequence Data; D050811:Octamer Transcription Factor-1; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D006684:HLA-DR Antigens; D059805:HLA-DR alpha-Chains; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; D009838:Oligodeoxyribonucleotides; C494165:POU2F1 protein, human; D014157:Transcription Factors; D007371:Interferon-gamma; D015500:Chloramphenicol O-Acetyltransferase