title: Novel regulation of an MHC class I gene response to interferon-gamma.
abstract: The ability of IFN-gamma to increase the expression of MHC class I gene products is likely to enhance cytolytic T lymphocyte recognition of viral pathogens and tumor cells. The murine lymphoma AKR SL3-cl.F AZR (SL3-cl.F) responds aberrantly to treatment with interferon-gamma such that H-2Dk surface expression is augmented, but H-2Kk expression remains at constitutive levels. Somatic cell fusions have been used to demonstrate that the lesion responsible for this phenotype is cis-dominant, implicating a primary lesion within the SL3-cl.F H-2Kk gene. In this communication, we have used PCR to analyze the nucleotide sequence in regions of the SL3-cl.F H-2Kk promoter known to contain interferon-responsive enhancer elements. Comparison of the SL3-cl.F H-2Kk sequences to known consensus elements revealed complete identity. In order to identify the lesion responsible for the SL3-cl.F phenotype, two H-2Kk genomic clones were independently isolated from SL3-cl.F. Each clone exists as a 10.5-kbp EcoRI fragment containing the entire structural gene. The site of transcription initiation is at the center of this fragment; therefore, all regulatory elements within 5 kbp of the transcript start site which could alter steady-state message levels are included. Interestingly, IFN-gamma-augmented expression of the H-2Kk gene was restored following DNA-mediated transfection of either of these clones into fibroblast cell lines and the parental cell line SL3-cl.F. Because isolation of these clones required passage of the DNA through a prokaryotic host, which alters the pattern of DNA methylation, there was the possibility that demethylation was responsible for the newly acquired IFN-gamma-responsive phenotype. Treatment of SL3-cl.F with 5-azacytidine, which inhibits de novo methylation, did not restore IFN-gamma-augmented expression, however, thus excluding H-2Kk specific methylation as a potential mechanism. Collectively, these data demonstrate that the alteration responsible for the phenotype observed in SL3-cl.F does not involve known transcriptional regulatory elements. Potential mechanisms which might account for the mutant phenotype are discussed.
authors.affiliation: Department of Microbiology, Dartmouth Medical School, Lebanon, New Hampshire 03756.
authors.forename: D A
authors.initials: DA
authors.lastname: Schwarz
authors.forename: J E
authors.initials: JE
authors.lastname: Buhlmann
authors.forename: M R
authors.initials: MR
authors.lastname: Kuhne
authors.forename: T M
authors.initials: TM
authors.lastname: Lam
authors.forename: H D
authors.initials: HD
authors.lastname: White
authors.forename: W R
authors.initials: WR
authors.lastname: Green
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D005786:Gene Expression Regulation / Q000187:drug effects*; D005805:Genes, MHC Class I; D006183:H-2 Antigens / Q000235:genetics*; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000494:pharmacology*; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins; D014162:Transfection
chemical_list: D006183:H-2 Antigens; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D007371:Interferon-gamma; D001374:Azacitidine