Different binding of NF-Y transcriptional factor to DQA1 promoter variants

Polymorphism in the HLA-DQA1 promoter (QAP) sequences could influence the gene expression through a differential binding of transcriptional factors. Considering the main role played by the Y-box in the transcription, we focused on the QAP4 variants differing for a G vs A transition from the QAP Y-box consensus sequence. Electrophoretic Mobility Shift Assay using the two Y-box sequences was performed to determine whether this mutation could be reflected in an allele-specific binding of transcriptional factors. Indeed, the NF-Y specific band, recognised by supershift experiments, was clearly observed using the Y-box consensus probe but it was barely detectable with the QAP4 one. On the contrary, two other complexes were found to more strongly interact with QAP4 Y-box in comparison to the consensus sequence. The analysis of a selected panel of HLA homozygous lymphoblastoid cell lines by competitive RT-PCR and by Northern blotting revealed that the DQA1 *0401, *0501,*0601 alleles regulated by the QAP4 promoters were less expressed at the mRNA level than the DQA1* 0201 allele regulated by the QAP2.1 variant. In conclusion, these results show an evident reduction of NF-Y binding to the mutated QAP4 Y-box and a decreased mRNA accumulation of the DQA1 alleles regulated by these variants.