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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1456-1469.)
© 1999 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

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

Pavlos Pissios; Horng-Yuan Kan; Satoshi Nagaoka; Vassilis I. Zannis

From the Section of Molecular Genetics, Cardiovascular Institute, Departments of Biochemistry and Medicine, Boston University Medical Center, Boston, Mass.

Correspondence to Vassilis I. Zannis, Section of Molecular Genetics, Cardiovascular Institute, Department of Medicine, Boston University Medical Center, 700 Albany St, W-509, Boston, MA 02118-2394.

Abstract—Screening of an expression human liver cDNA library resulted in the isolation of several cDNA clones homologous to sterol regulatory element-binding protein-1 (SREBP-1) that recognize the regulatory element AIIAB and AIIK of the human apoA-II promoter. DNaseI footprinting of the apoA-II promoter using 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 contain inverted E-box palindromic or direct repeat motifs and bind SREBP-1 with different affinities. Transient cotransfection experiments in HepG2 cells showed 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 SREBP binding sites. Mutagenesis analysis showed that transactivation by SREBP was mainly affected by mutations in element AIIAB. Despite the strong transactivation of the apoA-II promoter by SREBP-1 we could not demonstrate significant changes on the endogenous apoA-II mRNA levels of HepG2 cells after cotransfection with SREBP-1 or in the presence or absence of cholesterol and 25-OH-cholesterol. An SREBP-1 mutant lacking the amino-terminal activation domain bound normally to its cognate sites and repressed the apoA-II promoter activity. Repression was also caused by specific amino acid substitutions 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 suggest that the wild-type SREBP-1 can bind and transactivate efficiently the apoA-II promoter in cell culture. SREBP-1 mutants lacking the activation domain bind to their cognate sites and directly repress the apoA-II promoter whereas mutants defective in DNA binding indirectly repress the apoA-II promoter activity, possibly by a squelching mechanism.

Key Words: sterol regulatory element-binding protein-1 • apoA-II • transcriptional regulation • basic helix-loop-helix zipper factors • upstream regulatory factor

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