Polymorphisms in the Paan-AG promoter influence NF-κB binding and transcriptional activity

摘要

The human leukocyte antigen-G (HLA-G) gene encodes a protein that is highly expressed at the human maternal–fetal interface during pregnancy and may be critical to the survival of the semiallogenic fetus. A unique feature of this gene is a 13-bp deletion in the proximal promoter that renders it unresponsive to transactivation by the nuclear factor-κB (NF-κB). We previously showed that the proximal promoter of Paan-AG, the functional homologue of HLA-G in the olive baboon (Papio anubis), is intact. We cloned the promoters of two putative Paan-AG alleles (AG1 and AG2) and identified a number of regulatory elements including two κB sites. In the current study, binding and activity of the two κB elements in each putative allele were assessed by electrophoretic mobility shift and supershift assays. Functional activity was determined using luciferase reporter assays. The κB1 and κB2 elements in AG1 bound NF-κB with similar affinity. In contrast, the κB1 element of AG2 bound NF-κB with a much higher affinity than AG-1 κB1 (a 30-fold increase), whereas κB2 did not bind. Mutagenesis analysis showed that the difference in binding intensities was due to two nucleotides in the 3′ end of κB1. Similarly, failure of AG2 κB2 binding was a result of the last nucleotide in the 3′ end that differed from the consensus; mutating this nucleotide to match the consensus reestablished binding. Functional activity of the two putative alleles also differed; AG1 luciferase activity was consistently lower than that of AG2. Mutating the last two nucleotides in the 3′ end of AG1 κB1 resulted in increased luciferase activity to levels comparable to that of AG2. Overall, these results show that in vitro variations in the promoter region may influence transcription of Paan-AG.