Heteromeric transcription factor NF-Y is a novel component of the estrogen receptor transcription complex

Previous studies from our laboratory indicate that multiple interactions between estrogen-dependent and tissue-specific transcription factors are required for optimal regulation of blood coagulation factor XII (FXII) gene expression, thus contributing to maintenance of the physiological balance between circulating pro- and anti-coagulants. Computerized search of functional signal sequences on FXII promoter identified a CCAAT box overlapping the 3' half-site of the FXII estrogen response element. The CCAAT box is a widespread regulatory sequence specifically bound by the heteromeric NF-Y complex, an evolutionarily conserved transcription factor which ensures proper expression of several tissue-specific genes, including coagulation factors. Aim of this study was to investigate whether NF-Y could modulate estrogen induction of FXII gene expression through interaction with the estrogen receptor ? (ER?) transcription complex. NF-Y effect on estrogen inducibility of FXII promoter was evaluated by cotransfecting expression vectors for ER? and the NF-Y subunits (A, B and C) with a FXII promoter-CAT reporter. NF-YA significantly inhibited estrogen induction of FXII promoter both in liver and non-liver cells. No effects were elicited by single cotransfection of NF-YB and C subunits. NF-Y interference was not mediated by interaction with FXII-CCAAT box since gel retardation assays did not show any NF-Y binding to FXII promoter. Moreover, a DNA-binding deficient NF-Y mutant was still capable of altering ER? transactivation properties. Molecular dissection analysis of NF-YA revealed that the HAP2 domain, a region mediating the interaction with NF-YB and C subunits, is necessary to inhibit ER-induced FXII transcription. Competition binding experiments on a 32P-labeled FXII-ERE/CCAAT oligonucleotide using increasing concentrations of affinity-purified NF-Y trimer and a fixed amount of baculovirus-expressed ER? did not impair formation of ER-DNA complex nor showed any minimal binding of NF-Y to FXII ERE/CCAAT sequences. Protein-protein interaction between ER??and NF-Y was demonstrated in vivo by co-immunoprecipitation using anti-ER??monoclonal antibody followed by decoration with anti-NF-Y polyclonal antibodies. In conclusion, our results indicate that the heteromeric transcription factor NF-Y modulates estrogen induction of FXII gene expression by associating, through its HAP2 domain, with the ER? multiprotein transcription complex.