Synergistic interactions between transcription factors control expression of the apolipoprotein AI gene in liver cells.

Abstract

The gene coding for apolipoprotein Al (apoAl), a plasma protein involved in the transport of cholesterol and other lipids in the plasma, is expressed predominantly in liver and intestine. Previous work in our laboratory has shown that different cis-acting elements in the 5'-flanking region of the human apoAl gene control its expression in human hepatoma (HepG2) and colon carcinoma (Caco-2) cells. Hepatocyte-specific expression is mediated by elements within the -256 to -41 DNA region relative to the apoAl gene transcription start site (+1). In this study it was found that the -222 to -110 apoAI gene region is necessary and sufficient for expression in HepG2 cells. It was also found that this DNA region functions as a powerful hepatocyte-specific transcriptional enhancer. Gel retardation and DNase I protection experiments showed that HepG2 cells contain proteins that bind to specific sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within this enhancer. Site-directed mutagenesis that prevents binding of these proteins to individual or different combinations of these sites followed by functional analysis of these mutants in HepG2 cells revealed that protein binding to any one of these sites in the absence of binding to the others was not sufficient for expression. Binding to any two of these sites in any combination was sufficient for only low levels of expression. Binding to all three sites was essential for maximal expression. These results indicate that the transcriptional activity of the apoAl gene in liver cells is dependent on synergistic interactions between transcription factors bound to its enhancer.The accumulation and utilization of cholesterol by tissues are dependent on a dynamic balance between the mechanisms that determine the rates of de novo cholesterol synthesis, the rates of synthesis and hydrolysis of stored pools of cholesteryl esters, and the rates of uptake and removal of cholesterol from cells by plasma lipoproteins (reviewed in references 4 and 20). Removed cholesterol binds to a species of high-density lipoprotein (HDL) particles containing primarily apolipoprotein AI (apoAI). After its esterification by lecithin:cholesterol acyltransferase (an enzyme activated by apoAI), cholesterol is transported to the liver, where it is excreted either directly or in the form of bile acids (reviewed in references 3, 20, and 24). The critical role of HDL and apoAI in cholesterol homeostasis, and in particular in preventing deposition of excessive amounts of cholesterol in coronary and other arteries, is exemplified by epidemiological and genetic evidence indicating a strong correlation between decreased HDL and apoAI plasma levels and the development of atherosclerotic heart disease (reviewed in references 3, 39, and 51). Thus, the recent observation that there is a direct correlation between apoAI plasma levels and hepatic apoAI mRNA concentrations (54, 55) suggests that factors controlling expression of the apoAI gene in liver could play an important role in tissue cholesterol a...

Transcription efficiency of human apolipoprotein A‐I promoter varies with naturally occurring A to G transition

“…A CAT is a slightly stronger promoter than its G counterpart (72.4% vs. 62% of awtylation). Altion factors identified [21,23]. The deletion studies have most identical results were obtained when the two other shown previously [13] that the apo A?…”

Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

“…To construct pREϫ4-E4T and pREϫ4-MLT, a double-stranded oligonucleotide containing four tandemly repeated copies of the HNF4␣-responsive element from the apolipoprotein A-I enhancer element (TGAACCCTTGACCCCTGC) (50,51) was introduced between the PstI and BamHI sites of pG5-E4T and pG5-MLT (52). The BamHI site is located 13 or 27 bp upstream of the TATA boxes of the adenovirus major late (AdML) or E4 core promoter, respectively.…”

Effects of ketoacidosis on rat apolipoprotein A1 gene expression: a link with acidosis but not with ketones

“…Hep G2 liver cells and Caco-2 intestinal cells were transfected with the plasmid pA1·474.CAT, which carries the full sequence of rat apoAI gene promoter, along with the CAT reporter gene (Widom et al 1991, Romney et al 1992, Taylor et al 1996, Mooradian et al 1997, Harnish et al 1998, Murao et al 1998. Both of these cell lines are suitable for these studies since they express the endogenous apoA1 gene (see below).…”