Growing evidence points to the importance of MHC class I and II expression levels in an effective immune response against pathogens and tumors, illustrating the need to determine consistent patterns of differential expression levels across allelic lineages of each class I and II gene and to identify the mechanisms by which differential expression occurs. While a minimal number of peptide MHC complexes are sufficient for CTL killing in vitro, increasing density of these interactions improves the efficiency of cytotoxicity and alters the response to induce secretion of inflammatory cytokines. Higher HLA C expression levels have been associated with enhanced HIV control, greater odds of generating HLA C restricted CTL responses to the virus, and stronger immune pressure on HIV. We recently examined HLA A expression levels in a sample of 216 European Americans and observed a gradient of expression that associates with HLA A allelic lineage. A significant inverse correlation was observed between HLA A mRNA expression levels in untreated cells and the degree to which expression is increased after treatment of the cells with a DNA methyltransferase inhibitor. Deep sequencing and immunoprecipitation assays revealed allelic lineage specific methylation patterns within the HLA A promoter region where increased DNA methylation levels correlated significantly with reduced HLA A expression levels. Several mechanisms are likely to contribute to the variation in expression levels of HLA A lineages, and characterizing these mechanisms may present the potential for HLA A specific regulation of expression levels as drug targets. These data may also be useful in genetic association studies of diverse diseases. Differential HLA-C expression levels influence several human diseases, but the mechanisms responsible are incompletely characterized. HLA C expression levels vary in an allele-specific manner over a range of 7 fold in a pattern that is consistent between African and European Americans and highly reproducible across study groups. Based on the expression value characteristic for each given HLA C allotype as determined previously, we imputed HLA C expression levels for 228 European 1KG individuals who have previously been typed for HLA C. Imputed HLA C expression levels were tested as a continuous variable for association with 68,726 SNPs within the MHC using linear regression in order to identify cis acting variants that may cause (or mark) differential expression of HLA C. The peak association was centered in the HLA C promoter region, and correction for population structure did not alter the results. The top signal identified was rs2395471, which is 800 bp upstream of the transcription start site. The A vs. G frequency at rs2395471 was fairly evenly distributed across HLA C alleles. These variants were termed imputed expression quantitative trait loci (impeQTL) to distinguish them from those associating with expression levels of genes that were measured directly. ImpeQTL can only be used to identify candidates of expression modifiers in cis of the gene when its expression is imputed. Genotyping of rs2395471 in two independent cohorts where HLA C expression levels were measured directly by flow cytometry confirmed the association between this SNP and cell surface expression levels of HLA C on CD3 positive cells. rs2395471 is located in an Oct1 transcription factor consensus binding site motif where the A allele is predicted to have higher affinity for Oct1 than the G allele. Mobility shift electrophoresis demonstrated that Oct1 binds to both alleles in vitro, but decreased HLA-C promoter activity was observed in a luciferase reporter assay for rs2395471G relative to rs2395471A on a fixed promoter background. The rs2395471 variant accounts for up to 36% of the explained variation of HLA C expression. These data strengthen our understanding of HLA C transcriptional regulation and provide a basis for understanding the potential consequences of manipulating HLA C expression levels therapeutically.
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