A far-downstream hepatocyte-specific control region directs expression of the linked human apolipoprotein E and C-I genes in transgenic mice

Abstract

The human apolipoprotein (apo) E and apoC-I genes are located 5 kilobases apart in the same transcriptional orientation on chromosome 19, and they are expressed at high levels in the liver with lower levels of expression in selected other tissues. Analysis of a series of overlapping human apoE and apoC-I genomic fragments in transgenic mice revealed that the expression of these transgenes in the liver requires a common cis-acting regulatory domain. This hepatic control region (HCR) was localized to a 764-base pair region that is located about 18 kilobases downstream of the apoE promoter and about 9 kilobases downstream of the apoC-I promoter. All the transgenic animals that had been prepared with a construct that contained this region had relatively high levels of transgene expression in the liver, whereas constructs that lacked this region showed no expression in the liver. In situ hybridization studies showed that the HCR directed apoE and apoC-I transgene expression in hepatocytes. When the HCR from the apoE/C-I gene locus was ligated proximal to a human apoA-IV gene fragment, which is not normally expressed in the liver, the resulting apoA-IV/HCR fusion construct was expressed at high levels in the liver, indicating that the HCR could direct high level liver expression of a heterologous promoter/gene construct. Expression of the apoE transgene in the liver and kidney, and perhaps other tissues, required the presence of a nonspecific proximal enhancer element in the apolipoprotein E gene promoter, located between 161 and 141 bp relative to the transcription initiation site. However, the proximal apoE gene promoter, including this enhancer element, contained no sequences capable of directing hepatocyte expression in the absence of the HCR. Thus, the far-downstream HCR appears to contain all of the sequences necessary for determining high level liver-specific gene expression.