Structure of the Hepatic Control Region of the Human Apolipoprotein E/C-I Gene Locus

Abstract

The specificity of expression in the liver of the human apolipoprotein (apo) E/C-I gene locus is determined by a hepatic control region (HCR) that is located 15 kilobases downstream of the apoE gene. DNase I footprint studies of this sequence using nuclear extracts identified a region of the HCR that is enriched in nuclear proteinbinding sites. Nuclease analysis of chromatin revealed liver-specific DNase I-hypersensitive sites that were associated with this region, and additional liver-specific nuclease-sensitive sites associated with the apoE gene were identified. The HCR domain has a limited binding affinity for the nuclear scaffold. The specific domain required for liver expression was tested by ligating subfragments of the HCR to the apoE gene and examining their activity in transgenic mice. A segment of 319 nucleotides that contained several potential regulatory sequences was required for full activity of liver-specific transcription with shorter segments yielding much lower levels of expression in the liver. All constructs that contained a fully active HCR were expressed in approximately a copy-dependent manner, suggesting that transgene expression was independent of integration position. Taken together, the properties of the HCR are consistent with its function as a locus control region for the liver-specific expression of the apoE gene.Human apolipoprotein (apo) 1 E, a 299-amino-acid glycoprotein of M r ϭ 35,000 (1), is a major component of various plasma lipoprotein classes, including chylomicron remnants, very low density lipoproteins, and high density lipoproteins (2, 3). It facilitates the redistribution of cholesterol from peripheral tissues to the liver and is required for the receptor-mediated uptake of chylomicron remnants (2, 4). Although apoE is produced by many different tissues, more than 90% of the circulating apoE in human plasma comes from liver hepatocytes (2). Receptor binding defective variants of apoE having the E2 phenotype are associated with type III hyperlipoproteinemia and premature atherosclerosis (2). A commonly occurring apoE variant, the E4 allele, has been linked to an enhanced occurrence of the neurofibulary tangles and plaque deposits characteristic of Alzheimer's disease (5-7).The human apoE gene is 3.6 kb in length (8,9) and is located at the 5Ј end of a 45-kb cluster of apolipoprotein genes on chromosome 19, all of which have the same transcriptional orientation (10). The apoC-I gene (4.7 kb) is located 5.3 kb downstream of the apoE gene and a 4.4-kb apoC-IЈ pseudogene is located 7.5 kb still further downstream. The apoC-II gene (3.3 kb) is found about 16 kb downstream of the apoC-IЈ pseudogene. Each of these genes contains four exons, with the introns located in similar intragenic positions, suggesting that this gene family evolved from a common ancestor (1, 9).Expression of the apoE gene in the liver was shown initially by Simonet et al. (11,12) to require the presence of a distal downstream tissue-specific enhancer. Subsequent studies by Simonet et al. (12) demonstra...

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

“…This 580-nucleotide region or a minimum of 319-nucleotide region containing the first three footprints are sufficient to direct hepatic expression of the apoE gene in transgenic mice (14). The present study showed that this 319-base pair region enhanced 2.5-fold the strength of the Ϫ545/ϩ18 apoC-II promoter in HepG2 cells and had no effect in CaCo-2 or COS-1 cells in the absence of HNF-4.…”

“…an HRE which may bind different members of the hormone receptor family including HNF-4 (14). To test whether the HREs present in the proximal promoter and/or the HCR-1 contribute to the HNF-4 mediated transcriptional enhancement, we performed co-transfection experiments using the reporter construct HCR-1/(Ϫ545/ϩ18)CII CAT and HNF-4 expression plasmid.…”

“…Studies using transgenic mice have provided evidence for the existence of common regulatory regions within the 45 kb that control the tissue-specific expression of the apoC-I and apoE genes (13-15). These regions were designated hepatic control region-1 and -2 (HCR-1 and HCR-2), respectively (13,14).…”

“…Studies using transgenic mice have provided evidence for the existence of common regulatory regions within the 45 kb that control the tissue-specific expression of the apoC-I and apoE genes (13-15). These regions were designated hepatic control region-1 and -2 (HCR-1 and HCR-2), respectively (13,14).The objective of the current study was to identify the promoter elements which confer tissue specificity and assess the potential role of HCR-1 in the regulation of the human apoC-II gene. We demonstrate that the Ϫ545/ϩ18 5Ј-intergenic sequence located between the apoC-IV and apoC-II genes is a very strong hepatic promoter.…”

Long Terminal Repeats Are Used as Alternative Promoters for the Endothelin B Receptor and Apolipoprotein C-I Genes in Humans

“…The LTR was also inserted at a distance (see "Experimental Procedures") in constructs with the apoC-I promoter where the LTR was removed, to test for the possibility that the LTR acts as an enhancer of the native promoter. The expression in liver is completely dependent on a distal HCR (27), and we saw no promoter activity of the apoC-I constructs without the presence of this HCR. The results of the transfections of HepG2 (liver) cells with a variety of apoC-I constructs are shown in Fig.…”