Polymorpidsms in PAAN-AG promoter influences NF-KB binding and transcription activity in HEK293 cells

Abstract

The human leukocyte antigen-G (HLA-G), a protein highly expressed at the human maternal-fetal interface during pregnancy , is thought to be critical for the survival of the semi-allogenic fetus. Current evidence suggest that HLA-G programs immune cells at the maternal-fetal interface into immunosuppressive phenotypes, but definitive proofremai elusive since in vivo experiments in humans are not possible due to ethical concerns. In the search for an appropriate animal model, we have identified the olive baboon (Papio anubis) as a potential candidate. This primate expresses an HLA-G-like protein termed Paan-AG in the placenta. Preliminary data shows that Paan-AG gene shares many characteristics with HLA-G , including limited polymorphism , alternative splicing of the rnRNA, and restricted tissue exPression of the protein. Restricted tissue expression suggested that the two genes might share tissue-specific reL atory elements. We previously identified a number of putative regulatory elements in the proximal promoters of two Paan-AG alleles, 5'UTAG-1 (AG1) and 5'UTAG-2 (AG2). The objective ofthe current study was to assess binding of the transcription factor NF-KB to Paan-AG KB elements and determine the effects of binding on Paan-AG promoter activity. Both alleles contained two KB elements, KB 1 and KB2. Binding was assessed using electrophoretic mobility shift assays and functional activity using luciferase reporter assays. NF-KB bound both KB 1 and KB2 element in the AGI allele. In contrast, only KBl of the AG-2 allele bound to NF-KB; KB2 did not bind . The AG2 KBl element bound NFKB with a stronger affinity compared to AG-1 KB 1. Mutagenesis studies showed that the difference in bi·- -ling was due to two nucleotide differences in the 3' end ofKBl. The functional activity of the two alleles also d1 .-.ed; AG2 consistently showed higher luciferase activity compared to AG 1. Mutating the last two nucleotide s in tr 3' end of KB 1 resulted in an increase of luciferase activity to levels comparable to that of AG2. Overall , these results suggest that variations in the proximal promoter may influence transcription rates 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)