Transcriptional activity of serum amyloid A-activating factor-1 is regulated by distinct functional modules

Serum amyloid A-activating transcription factor-1 (SAF-1) plays a major role in regulating transcription of several inflammation-responsive genes, including SAA and matrix metalloproteinase-1, that are implicated in the pathogenesis of reactive secondary amyloidosis, atherosclerosis, and arthritis. SAF-1 is a 477-amino acid protein with six zinc fingers. Its activation during inflammatory condition by a phosphorylation event that leads to an altered structure suggested possible structural modification of this protein as a leading cause of higher activity. However, no information is available regarding structural features that might regulate its activity. Here, we have characterized its functional domains, delineating activation and repression modules, DNA binding, and nuclear localization activities. Using GAL4AD chimeras and a DNA-binding assay with proteins prepared from various deletion constructs, the core DNA-binding domain of SAF-1 is mapped between amino acids 282 and 361, which contain second, third, and fourth zinc fingers. Results from several deletion and point mutants using green fluorescent protein reporter show that SAF-1 contains two independent nuclear localization signals; one is composed of a stretch of basic amino acids, and the other is a bipartite signal located within the core DNA-binding domain. SAF-1 contains several negative and positively functioning transactivation modules clustered at the two ends of this protein. Removal of any one of the terminal negative modules renders the SAF-1 protein functionally very active. These findings suggest that the terminal repression modules act in conjunction to regulate the functional activity of this protein.