15 E.J. Schaefer (ed.), High Density Lipoproteins, Dyslipidemia, and Coronary Heart Disease, DOI 10.1007/978-1-4419-1059-2_2, © Springer Science+Business Media, LLC 2010 Abbreviations C/EBP CAAT/enhancer binding protein CAT Chloramphenicol acetyl transferase EGR-1 Early growth response factor-1 FXR Farnesoid X receptor HNF-4 Hepatocyte nuclear factor-4 HDL High density lipoprotein HRE Hormone response element LRH-1 Liver receptor homolog-1 LXRs Liver X receptors PPARa Peroxisome proliferator-activated receptor a PLTP Phospholipid transfer protein RORa Retinoic acid receptor-related orphan receptor a RXRa Retinoid X receptor a SHP Small heterodimer partner SP1 Specificity protein 1 SR-BI Scavenger receptor class B type I SREBP Sterol regulatory element binding protein WT Wild type Transcriptional Regulation of the Human ApoA-I Gene in Cell Culture and in ApoA-I Transgenic Mice Role of hormone nuclear receptors, SP1 and of a common enhancer on the transcriptional regulation of the human apoA-I gene in cell cultures. Earlier studies established that there is a linkage and common regulatory mechanism of the apoA-I/apoCIII/apoA-IV gene cluster (Fig. 1a). In this cluster, the distal regulatory region of the apoCIII promoter is an enhancer that increases the strength of the neighboring promoters in vitro [1–4]. When the apoCIII enhancer is joined with the proximal apoA-I promoter, then the activity of the promoter increases over tenfold [1–4]. The proximal apoA-I promoter contains two hormone response elements (HREs) that bind orphan and ligand-dependent nuclear receptors [5, 6]. The apoCIII enhancer also has two HREs which differ in their receptor specificity, and also has three SP1 binding sites [1, 7] (Fig. 1a). Systematic in vitro mutagenesis of different sites of the promoter/enhancer cluster and determination of the promoter activity by CAT assays showed that mutations in the HRE, and the SP1 binding sites affect the activity of the apoA-I promoter/enhancer cluster in HepG2 cells [1–7]. Mutation in the proximal HREs reduced the strength of the promoter/ enhancer to 6% of the WT control, whereas mutations in the HRE (I 4 ) of the enhancer reduced the activity of the promoter enhancer cluster to 18% of the WT control (Fig. 1b). Finally, the individual mutations in the SP1 binding site reduced the activity of the promoter enhancer cluster to 40–60% of the WT control (Fig. 1b). The receptor specificity of the HREs of the apoA-I promoter and the apoCIII enhancer were determined by DNA binding gel electrophoresis assays. These analyses established that both HREs present in the proximal apoA-I promoter bind HNF-4, other orphan receptors, and a variety of ligand- dependent nuclear receptors with different affinities [6] (Fig. 1c). One of the HREs of the enhancer binds also HNF-4, other orphan nuclear receptors, and ligand-dependent nuclear receptors with different affinities, whereas the other HRE does not bind HNF-4, binds other orphan receptors, and different combinations of ligand-dependent nuclear receptors with different affinities [7] (Fig. 1c). Transcriptional regulation of the human apoA-I gene in transgenic mice. To validate the conclusions drawn by the in vitro experiments, we generated a variety of transgenic mouse lines which express the WT A-I/CIII cluster or the same cluster with the mutations in the HREs and the binding sites of SP1 and other transcription factors. In these constructs, the apoCIII gene was replaced by the CAT gene (Fig. 2). V.I. Zannis (*) Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA e-mail: vzannis@bu.edu Regulation of ApoA-I Gene Expression and Prospects to Increase Plasma ApoA-I and HDL Levels Vassilis I. Zannis, Adelina Duka, Konstantinos Drosatos, Despina Sanoudou, Georgios Koukos, Eleni Zanni, and Dimitris Kardassis