Expression of the human apolipoprotein E gene is regulated by multiple positive and negative elements.
Apolipoprotein E (apoE), unlike the other major lipoproteins, is synthesized in a variety of tissues. We examined which regions of the human apoE gene contributed to its tissue-specific expression using HepG2 and HeLa cells as examples of expressing and nonexpressing tissues, respectively. Regions between -360 bp and -80 bp and within the first intron were shown to be necessary for full expression activity in HepG2 cells by a nuclease protection assay which demonstrated correct transcriptional initiation of the transfected constructions. To fine map the regulatory regions, we constructed a series of deletions fused to the reporter gene chloramphenicol acetyltransferase. We discovered eight regions which had a positive effect on expression and three regions that had a negative effect on expression, in both HepG2 and HeLa cells. In addition we found three regions which had a tissue-specific negative effect on expression in HeLa cells and one region with a tissue-specific positive effect in HepG2 cells. A DNase I protection assay revealed eight footprints within the proximal 5'-flanking sequence and the first intron. Seven of these footprints fell within closely defined regions with positive expression activity. Sequence analysis of these footprint elements revealed the presence of previously identified elements and two novel elements related to each other, identified here as B1 and B2. We also defined another repeated sequence, the A element; all three of the tissue-nonspecific negative regions contained this element or sequences with homology to it. In the context of a heterologous promotor, a synthetic oligonucleotide containing the B1 and B2 elements behaved like a classical enhancer, having a positive effect on expression, even when placed at a distance. This effect was neutralized by a different synthetic oligonucleotide containing an A element repeat.
Smith
J D
JD
Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, New York, New York 10021.
Melián
A
A
Leff
T
T
Breslow
J L
JL
D000818:Animals; D001057:Apolipoproteins E / Q000235:genetics*; D001483:Base Sequence; D003850:Deoxyribonuclease I / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007438:Introns; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids
D001057:Apolipoproteins E; D003850:Deoxyribonuclease I