Novel regulation of an MHC class I gene response to interferon-gamma.

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.