SREBP-1 binds to multiple sites and transactivates the human apoA-II promoter in vitro - SREBP-1 mutants defective in DNA binding or transcriptional activation repress apoA-II promoter activity

Screening of an expression human liver cDNA library resulted in the isolati on of several cDNA clones homologous to sterol regulatory element-binding p rotein-1 (SREBP-1) that recognize the regulatory element AIIAB and AIIK of the human apoA-II promoter. DNaseI footprinting of the apoA-II promoter usi ng SREBP-1 (1 to 460) expressed in bacteria identified 5 overall protected regions designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332), AIIHI (-594 to -574), and AIIK (-760 to -743). These regions contai n inverted E-box palindromic or direct repeat motifs and bind SREBP-1 with different affinities. Transient cotransfection experiments in HepG2 cells s howed that SREBP-1 transactivated the -911/29 apoA-II promoter 3.5-fold as well as truncated apoA-II promoter segments that contain 1, 2, 3, or 4 SREB P binding sites. Mutagenesis analysis showed that transactivation by SREBP was mainly affected by mutations in element AIIAB. Despite the strong trans activation of the apoA-II promoter by SREBP-1 we could not demonstrate sign ificant changes on the endogenous apoA-II mRNA levels of HepG2 cells after cotransfection with SREBP-1 or in the presence or absence of cholesterol an d 25-OH-cholesterol. An SREBP-1 mutant lacking the amino-terminal activatio n domain bound normally to its cognate sites and repressed the apoA-II prom oter activity. Repression was also caused by specific amino acid substituti ons of Leu, Val, or Gly for Lys359, which affected DNA binding. Repression by the DNA binding-deficient mutants was abolished by deletion of the amino -terminal activation domain (1 to 90) of SREBP-1. Overall, the findings sug gest that the wild-type SREBP-1 can bind and transactivate efficiently the apoA-II promoter in cell culture. SREBP-1 mutants lacking the activation do main bind to their cognate sites and directly repress the apoA-II promoter whereas mutants defective in DNA binding indirectly repress the apoA-II pro moter activity, possibly by a squelching mechanism.