CHARACTERIZATION OF AN UPSTREAM REGULATORY SEQUENCE AND ITS BINDING-PROTEIN IN THE MOUSE APOLIPOPROTEIN-E GENE

The mouse apolipoprotein (ape) E gene from strain C57BL/6 was isolated from a genomic DNA library and its complete nucleotide sequence, together with 1.3 kilobase of 5' flanking DNA and 300 base pairs of the 3' flanking DNA, was determined. Regulatory sequences in the proximal 5' flanking region of the gene were identified. Using a chloramphenicol acetyltransferase transient assay system, positive and negative cis-acting sequences were mapped within 380 base pairs of the 5' flanking region of the mouse apoE gene. Two nuclear protein binding sites were identified within this region by DNase I footprinting. We have characterized one of these regions, termed mouse apoE regulatory sequence (MARS-2), which spans nucleotides -151 to -133. Gel mobility shift assays using oligonucleotides of the MARS-2 sequence having specific deletions or substitutions as probes or competitors showed that the essential sequence of MARS-2 required for nuclear protein binding consists of 16 nucleotides encompassing -151 to -136. When nuclear extracts from different cells were examined, L cells and mouse liver nuclear protein contained the highest levels of binding protein for the MARS-2 probe. This protein, termed MARS-2 binding protein, was purified from mouse liver nuclear extracts to homogeneity using gel filtration and MARS-2 oligonucleotide-specific column chromatographic procedures. The M(r) = 66 000 binding protein showed a gel mobility shift band that was identical to that of crude nuclear extracts.