A short proximal promoter and the distal hepatic control region-1 (HCR-1) contribute to the liver specificity of the human apolipoprotein C-II gene - Hepatic enhancement by HCR-1 requires two proximal hormone response elements which have different binding specificities for orphan receptors HNF-4, ARP-1, and EAR-2

We have identified the regulatory elements, some of the factors and potential regulatory mechanisms which determine the tissue specificity of the human apoC-II gene. The -545/+18 apoC-II promoter directs high levels of expression of the reporter CAT gene in cells of hepatic origin (NepG2), low levels of expression in cells of intestinal origin (CaCo-2) and basal expression in HeLa cells, Deletion analysis identified negative regulatory elements within the -545/-388 region and positive regulatory elements within the -388/-55 region. Linkage of different apoC-II promoter segments to the hepatic control region-1 (HCR-1) enhanced the promoter activity 2.5-11-fold in HepG2 cells but did not affect its activity in CaCo-2 or COS-1 cells. DNase I footprinting analysis using rat liver nuclear extracts identified five protected regions within the -545/+18 apoC-II promoter as follows: CIIA (-74/-44), CIIB (-102/-81), CIIC (-159/-116), CIID (-288/-265), and CIIE (-497/-462), Elements CIIB and CIIC: contain hormone response elements. CIIB is recognized by hepatic nuclear factor-4 (HNF-4) but not ARP-I or EAR-2, whereas CIIC is recognized by ARP-1 and EAR-2 but not by HNF-4, HNF-4 transactivated the apoC-II promoter or the apoC-II promoter linked. to the HCR-1 in COS-1 cells, A double mutation in elements CIIB and CIIC that eliminated binding of HNF-4 or ARP-1 and EAR-2, respectively, to these sites abolished the enhancer activity of HCR-1. The combined data suggest that the apoC-II: promoter/HCR-1. cluster can direct expression In cells of hepatic origin and that optimal enhancer activity requires synergistic interactions between factors bound Let the distal HCR-1 and nuclear receptors bound to the two proximal hormone response elements.