Differential nuclear expression of enhancer A DNA-binding proteins in human first trimester trophoblast cells

In order to investigate the possible molecular regulatory mechanisms that repress classical HLA class I and stimulate nonclassical HLA-G and E-class I gene transcription in human trophoblast cells, we searched for the nuclear expression of the enhancer A DNA-binding proteins of the KBF1/NF-KB/rel family. Using both purified extravillous cytotrophoblast and villous syncytiotrophoblast from first trimester human placenta, it appeared that members of this family were present in the cytotrophoblast and absent in the syncytiotrophoblast. First, using the double stranded enhancer A DNA nucleotidic sequence that contains the palindromic KB site, known to be the binding site of the p50 subunits of KBF1-NF-KB and c-rel factors, we demonstrated, by band-shift assay, that binding activity, inhibited by addition of anti-p50 polyclonal serum, was present in cytotrophoblast as well as control maternal decidual cells, embryonic fibroblasts, and the trophoblast-derived JAR cell line. In contrast, this DNA-protein complex was undetectable in syncytiotrophoblast nuclear extracts. The specificity of this protein-DNA complex was further demonstrated by its disappearance upon competition with an excess of cold homologous nucleotidic competitor. Other nucleoprotein complexes were also detected in all nuclear extracts, including syncytiotrophoblast, that were competed out by an excess of cold enhancer A competitor DNA but were not affected by the addition of anti-p50 or anti-NF-KB sera, suggesting the presence of additional enhancer A-binding factors different from the KBF1/NF-KB/rel family. Second, using a Western immunoblot analysis, a doublet around 85 kDa was specifically stained by the same anti-p50 serum in cytotrophoblast, maternal decidual cells, embryonic fibroblasts, and the JAR nuclear extracts whereas no signal was obtained in syncytiotrophoblast. Finally, immunofluorescence cell staining using the same anti-p50 serum showed a positive staining in both cytoplasm and nucleus of cytotrophoblast and its absence in syncytiotrophoblast. We hypothesize that this enhancer A DNA-binding factor might represent the c-rel trans-acting factor, related to p50/KBF1/NF-KB proteins, and we discuss its possible relevance to the HLA class I transcription in human tissues.