Characterization of an upstream regulatory element of the human apolipoprotein E gene, and purification of its binding protein from the human placenta.
We have previously demonstrated that expression of the human apolipoprotein (apo) E gene is controlled by multiple regulatory elements in the promoter [Paik et al. (1988) J. Biol. Chem. 263, 13340-13349; Chang et al. (1990) J. Biol. Chem. 265, 9496-9504]. To extend these studies, we have characterized an element in the apoE gene promoter that spans nucleotides -101 to -89, upstream regulatory element 3 (URE3). Transcription of promoter/marker gene constructs in vitro showed that URE3 modulates gene expression. Gel mobility shift assays of URE3 using human placental nuclear extracts detected a specific binding protein whose activity can be modulated by micromolar amounts of divalent copper and zinc. Competitive binding and gel shift assays with mutant oligonucleotides revealed critical nucleotides within URE3 required for its specific nuclear protein-binding activity. Gel filtration and oligonucleotide affinity chromatography were employed to isolate a URE3-binding protein (URE3BP) from human placental nuclear extracts. Purified URE3BP appears to be a M(r) = 300,000 protein that is composed of four equally-sized basic subunits of M(r) = 67,000. These studies indicate that URE3 is an active regulatory component of the apoE gene.
Jo
D W
DW
Department of Biochemistry, Yonsei University, Seoul, Korea.
Leren
T P
TP
Yang
Z Y
ZY
Chung
Y H
YH
Taylor
J M
JM
Paik
Y K
YK
D001057:Apolipoproteins E / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D005786:Gene Expression Regulation / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010920:Placenta / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic
D001057:Apolipoproteins E; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase