PAAN-AG PROXIMAL PROMOTER, IN CONTRAST TO THAT OF HLA-G, IS INTACT AND FUNCTIONAL

The human leukocyte antigen (HLA)-G gene and its putative baboon homologue, Paan-AG, encode proteins that are highly expressed at the maternal-fetal interface during pregnancy and may be critical to survival of the semi-allogenic fetus. Both genes are similar in the coding region but differ significantly in the proximal promoter. HLA-G has a 13 bp deletion in the proximal promoter that renders it unresponsive to transactivation by the nuclear factor-kB (NF-kB) and interferong. In contrast, the proximal promoter of Paan-AG is intact. The promoters of two previously identified putative Paan-AG alleles (AG1 and AG2) were sequenced and shown to contain two kB binding sites each. In the current study, binding and activity of the two kB elements in each putative allele were assessed by electrophoretic mobility shift and Supershift assays. Functional activity was determined using luciferase reporter assays. The kB1 and kB2 elements in AG1 bound NF-kB with similar affinity. In contrast, the kB1 element of AG2 bound NF-kB with a much higher affinity than AG-1 kB1 whereas kB2 did not bind. Mutagenesis analysis showed that the difference in binding intensities was due to two nucleotides in the 3′ end of kB1. Similarly, failure of AG2 kB2 binding was a result of the last nucleotide in the 3′ end that differed from the consensus; mutating this nucleotide to match the consensus re-established 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 kB1 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 as reported recently for HLA-G, and provide further evidence of the potential usefulness of the baboon as a model for in vivo HLA-G studies. Supported by NIH grant HD39878 (JSH). (poster)