Cooperative Binding of Upstream Stimulatory Factor and Hepatic Nuclear Factor 4 Drives the Transcription of the Human Apolipoprotein A-II Gene

• Published in: The Journal of biological chemistry Vol. 274; no. 3; pp. 1216 - 1225
• Main Authors: Ribeiro, A, Pastier, D, Kardassis, D, Chambaz, J, Cardot, P
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 15.01.1999
• Subjects: Animals; Apolipoprotein A-II - genetics; Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cell Line; Chromatography, Affinity; COS Cells; DNA - metabolism; DNA-Binding Proteins; Helix-Loop-Helix Motifs; Hepatocyte Nuclear Factor 4; Humans; Molecular Sequence Data; Nuclear Proteins - metabolism; Phosphoproteins - metabolism; Promoter Regions, Genetic; Rats; Transcription Factors - metabolism; Transcription, Genetic; Transcriptional Activation; Upstream Stimulatory Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.3.1216

The activity of the human apoA-II promoter is controlled by a synergistic interaction of the distal enhancer and the proximal promoter. An important role in apoA-II promoter activity is exerted by a transcription factor, designated CIIIB1, which binds to the proximal element AB and the distal elements of the enhancer, K and L. In the present communication we establish that CIIIB1 corresponds to the previously described factor, upstream stimulatory factor (USF) using the following criteria. ( a ) Purification of CIIIB1 by affinity chromatography provided a heat-stable protein with an apparent molecular mass of 45 kDa that cross-reacted with anti-USF1 and -USF2a antibodies; ( b ) CIIIB1 bound to the elements AB, K, and L was supershifted by these antibodies; ( c ) the heterodimer USF1/2a is the predominant form that corresponds to CIIIB1. Cotransfection experiments in HepG2 cells established the functional significance of USF in apoA-II transcription. It was found that the minimal promoter AB was transactivated by USF2a. In addition, all three E-box motifs present in elements AB, K and L are necessary for maximum transactivation by USF2a. A dominant negative form of USF2a inhibits the activity of apoA-II promoter. The USF1/2a heterodimer, which is naturally expressed in the liver, is as efficient as the USF2a homodimer in the transactivation of apoA-II promoter/enhancer constructs. Cotransfection experiments in COS-1 cells showed that hepatic nuclear factor 4 (HNF-4) synergized with USF2a in the transactivation of the apoA-II promoter. In addition, we showed that HNF-4 and USF2a bind to the enhancer cooperatively. This may account for the transcriptional synergism observed between USF and HNF-4 in the transactivation of the apoA-II promoter.