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 2 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 po tential regulatory mechanisms which determine the tissue specificity o f the human apoC-II gene. The -545/+18 apoC-II promoter directs high l evels of expression of the reporter CAT gene in cells of hepatic origi n (NepG2), low levels of expression in cells of intestinal origin (CaC o-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 a poC-II promoter segments to the hepatic control region-1 (HCR-1) enhan ced the promoter activity 2.5-11-fold in HepG2 cells but did not affec t its activity in CaCo-2 or COS-1 cells. DNase I footprinting analysis using rat liver nuclear extracts identified five protected regions wi thin the -545/+18 apoC-II promoter as follows: CIIA (-74/-44), CIIB (- 102/-81), CIIC (-159/-116), CIID (-288/-265), and CIIE (-497/-462), El ements CIIB and CIIC: contain hormone response elements. CIIB is recog nized by hepatic nuclear factor-4 (HNF-4) but not ARP-I or EAR-2, wher eas CIIC is recognized by ARP-1 and EAR-2 but not by HNF-4, HNF-4 tran sactivated 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 tha t eliminated binding of HNF-4 or ARP-1 and EAR-2, respectively, to the se sites abolished the enhancer activity of HCR-1. The combined data s uggest that the apoC-II: promoter/HCR-1. cluster can direct expression In cells of hepatic origin and that optimal enhancer activity require s synergistic interactions between factors bound Let the distal HCR-1 and nuclear receptors bound to the two proximal hormone response eleme nts.