Purified apolipoprotein B gene regulatory factor-3 is DNA topoisomerase I

Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located between position s -128 and +122 [Chuang, S. S., & Das, H. K. (1996) Biochem. Biophys. Res. Commun. 220, 553-562]. A negative cis-acting element (+20 to +40) is locate d in the first nontranslated exon of the human apoB gene, and apoB regulato ry factor-3 (BRF-3) interacts with this. In this paper, we report the purif ication and characterization of BRF-3 from rat liver nuclear extracts. BRF- 3 has been purified to apparent homogeneity by DEAE-cellulose, heparin-agar ose, and DNA-specific affinity chromatography. Purified BRF-3 produced two polypeptide bands with apparent molecular masses of 70 kDa and 67 kDa in SD S/PAGE as detected by silver staining. Both 70-kDa and 67-kDa proteins have been found to hybridize specifically with labeled double-stranded oligonuc leotide containing BRF-3 binding site in a South-Western blot. Double-stran ded oligonucleotide containing mutations in the BRF-3 binding site was foun d to abolish DNA binding by these two proteins. Amino acid sequences of try ptic peptides derived from affinity purified 70-kDa and 67-kDa rat BRF-3 pr oteins were found to have 100% sequence homologies with DNA topoisomerase I . These data suggest that the 70-kDa and 67-kDa forms of BRF-3 are derived by proteolytic cleavage of topoisomerase I, and therefore, topoisomerase I may; play an important role in transcriptional regulation of apoB.