DNA binding specificity and transactivation properties of SREBP-2 bound tomultiple sites on the human apoA-II promoter

DNase I footprinting of the apoA-II promoter using sterol regulatory elemen t binding protein-2 [(SREBP-2 (1-458)] expressed in bacteria identified fou r protected regions, designated AIIAB (-64 to -48), AIICD (-178 to -154), A IIDE (-352 to -332) and AIIK (-760 to -743), which bind SREBP-2 and contain either palindromic or direct repeat motifs, Potassium permanganate and dim ethyl sulfate interference experiments using the AIIAB region as probe show ed that the nucleotides of a decameric palindromic repeat RTCAMVTGMY and tw o 5' T residues participate in DNA-protein interactions, SREBP-2 transactiv ated the intact (-911/+29) apoA-II promoter 1.7-fold and truncated apoA-II promoter segments which contain one, two or three SREBP-2 sites 11- to 17-f old in HepG2 cells, Transactivation of a promoter construct containing the binding site AIIAB and the apoA-II enhancer, which includes the binding sit e AIIK, was abolished by mutations in element AIIAB, An SREBP-2 mutant defe ctive in DNA binding caused a dose-dependent repression of the apoA-II prom oter activity. Repression was also caused by an SREBP-2 mutant which lacks the N-terminal activation domain (residues 1-93) but binds normally to its cognate sites, In contrast, a double SREBP-2 mutant which lacks both the DN A binding and the activation domains has no effect on the apoA-II promoter activity, Overall, the findings suggest that SREBP-2 can transactivate the apoA-II promoter by binding to multiple sites, Furthermore, the repression caused by the DNA binding deficient mutants results from squelching of posi tive activator(s) which appear to recognize the activation domain of SREBP- 2.