Interaction between the two ubiquitously expressed transcription factors NF-Y and Sp1

The regulation of the rat fatty acid synthase gene by mediators such as diet, hormones, cAMP, sterols or retinoic acid is controlled by three NF-Y binding sites. All three sites have a neighbouring Spl-binding GC-box. This NF-Y/Sp1 motif is conserved in the FAS promoters of rat, human, goose and chicken. We have previously shown cooperative binding of NF-Y and Spl to the promoter region at -500 coincident with a diet-induced DNAse I-hypersensitive site. Here, we show an in-vivo interaction of NF-YA with Spl using the yeast two-hybrid system. The interacting domains are located between amino acids 55 and 139 of the NF-Y subunit NF-YA and between amino acids 139 and 344 of Spl. In addition, we show by co-immunoprecipitation direct interaction of NF-Y subunit NF-YA with Spl in extracts of rat hepatoma cells H4IIE. Furthermore, we demonstrate by the GST pull-down assay that NF-YA interacts physically with Spl in-vitro in the absence of DNA. Therefore, NF-Y can be added to the list of transcription factors interacting with Spl. (C) 1999 Elsevier Science B.V. All rights reserved.