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 positions -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 located in the first nontranslated exon of the human apoB gene, and apoB regulatory factor-3 (BRF-3) interacts with this. In this paper, we report the purification and characterization of BRF-3 from rat liver nuclear extracts. BRF-3 has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. Purified BRF-3 produced two polypeptide bands with apparent molecular masses of 70 kDa and 67 kDa in SDS/PAGE as detected by silver staining. Both 70-kDa and 67-kDa proteins have been found to hybridize specifically with labeled double-stranded oligonucleotide containing BRF-3 binding site in a South-Western blot. Double-stranded oligonucleotide containing mutations in the BRF-3 binding site was found to abolish DNA binding by these two proteins. Amino acid sequences of tryptic peptides derived from affinity purified 70-kDa and 67-kDa rat BRF-3 proteins 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.
Chuang
S S
SS
Department of Pharmacology, University of North Texas Health Science Center at Fort Worth, USA.
Banerjee
D
D
Das
H K
HK
D000595:Amino Acid Sequence; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D001055:Apolipoproteins B; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D004264:DNA Topoisomerases, Type I