Evaluation of the function of the human apolipoprotein B gene nuclear matrix association regions in transgenic mice

Abstract

The human apolipoprotein B (apoB) gene resides in a 47.5 kb DNasel-sensitive chromosomal domain in hepatic and intestinal cells, flanked by the 5' distal matrix association region (MAR) and the 3' proximal MAR. A third MAR, the 5' proximal MAR, is found only in transcriptionally active hepatic (HepG2) cells. Hepatic expression of the apoB gene requires a tissue-specific promoter (-898 to +121) and an enhancer from the second intron of the gene (+360 to +1064). A vector containing this portion of the gene linked to the beta-galactosidase reporter is sufficient for low level expression in the livers of transgenic mice. Expression in transgenic mice was increased when the promoter-enhancer beta-gal vector was flanked by MARs. The results were similar whether the 5' distal, the 5' proximal or the 3' proximal MARs were placed at both ends of the construct, or whether the construct was flanked by the 5' distal and the 3' MAR, suggesting that the apoB MARs play a role in gene expression in vivo. When the MAR-containing constructs were transiently transfected into HepG2 cells, the resulting beta-gal activities were similar to that of the construct lacking MARs, thus demonstrating that the MARs do not exhibit any enhancer activity. Recent experiments (Kalos, M., and R. E. K. Fournier. 1995. Mol. Cell. Biol. 15: 198-207) examining stable integration of some of our constructs into human and rat hepatoma transfectants suggest that in single and double copy transfectants, the apoB MARs behave as boundary "insulators", protecting the integrated transgenes against position effects regardless of their site of integration. However, multicopy transfectants are transcriptionally inactive and when the MARs are absent, expression of the transgenes drops to background levels. Our results to date with single and low-copy number transgenes do not support an insulator function for the apoB MARs, although they appear to be required to increase the levels of expression.