The human apolipoprotein E gene is negatively regulated in human liver HepG2 cells by the transcription factor BEF-1.
Apolipoprotein E (apoE) is a major constituent of plasma lipoprotein that functions in lipid transport and redistribution (reverse cholesterol transport) and probably plays an important role in inhibiting the development and/or progression of atherosclerosis. While cis-acting regions involved in basal and tissue-specific control of the apoE gene have been identified by promoter mapping studies, much less is known about factors that regulate the gene. In this study, we demonstrate that the region between -94 and -84 upstream of transcriptional start site of the human apoE gene contains a binding site for the transcriptional repressor factor BEF-1, a tyrosine-phosphorylated nuclear protein that was first identified in HeLa cells. Using gel retardation assays, we show that HeLa cell-derived BEF-1 binds the apoE BEF-1 homology, and this binding can be competed with the prototype BEF-1 sequence, but not by a mutated sequence. Furthermore, we demonstrate that the apoE- producing human liver HepG2 cell produces significant levels of BEF-1, which could bind to both the prototype BEF-1 sequence and the apoE homology, and be competed equivalently with cold BEF-1 or apoE homology. To determine if BEF-1 affected the expression of apoE, we performed competition experiments using plasmids containing the intact or mutated BEF-1 homology. The introduction of the intact BEF-1 site into HepG2 cells resulted in an induction of apoE mRNA, whereas control and mutated BEF-1-containing plasmids had no significant effect. We also found that increasing the level of nuclear BEF-1 by treatment of cells with orthovanadate resulted in a reduction in the level of apoE mRNA. Overall, our data suggest that the endogenous apoE gene in the human HepG2 cell line is repressed by the trans-acting influence of nuclear factor BEF-1.
Berg
D T
DT
Department of Cardiovascular Research, Indianapolis, Indiana 46285, USA.
Calnek
D S
DS
Grinnell
B W
BW
D001057:Apolipoproteins E / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D005786:Gene Expression Regulation; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D012097:Repressor Proteins / Q000502:physiology*
D001057:Apolipoproteins E; D012333:RNA, Messenger; D012097:Repressor Proteins