Polymorphisms in the Paan-AG Promoter Influence NF-KB Binding and Transcriptional Activity

Abstract

The human leukocyte antigen-G (HLA-G) gene encodes a protein that is highly expressed at the hum an maternal-fetal interface during pregnancy and may be critical to the Sttlvival of the semiallogenic fetus. A unique feantre of this gene is a 13-bp deletion in the proximal promoter that renders it unresponsive to tran sactivat ion by the nucl ear l'actor-KB (NF-KB). We previou sly showed that the proximal promoter of Paan-AG, the functional homo­ logue of HLA-G in the olive baboon (Papio anubis), is intact. We cloned the promoters of two putative Pa cm-AG a lleles (AG I and AG2) and identifi ed a numbe r of regulatory element s including two K8 sites. In the current stud y, binding and activity of the two K8 elements in each putati ve allele were assessed by electrophoretic mobility shift and supershift assays. Functional activ ity was deter­ mined using luciferase reporter assays. The KBI and K82 elements in AG I bound NF-KB with sim ilar affinity. In contra st, the KB I element of AG2 bound NF-KB with a much highe r affinity than AG-1 K 8I (a 30-fold increase),whereas KB2 did not bind. Mutagene sis analysis showed that the difference in binding intensities was due to two nucleotides in the 3' end of K 8I. Similarly, fai lure of AG2 K82 bind ing was a result of the last nucleotide in the 3' end that differed from the consensus; mutating this nucleotide to match the consensus reestablis hed binding. Functional activity of the two putative alleles also differed; AG I luci fcrase activity was consisten tly lower than that of AG2. Mutating the last two nucleot idcs in the 3' end of AG I KB I resu lted in increased luciferase activity to levels comparable to that of AG2. Overall, these results show that in vitro variat ions in the promoter region may influence transcrip ­ tion of Paan-AG.