Transcriptional regulation of the apolipoprotein B100 gene: purification and characterization of trans-acting factor BRF-2.
Apolipoprotein B100 (apoB), the only protein of low-density lipoprotein, is produced primarily in the liver and serves as a ligand for the low-density lipoprotein receptor. Hepatic cell-specific expression of the human apoB gene is controlled by at least two cis-acting positive elements located between positions-128 and -70 (H. K. Das, T. Leff, and J.L. Breslow, J. Biol. Chem. 263:11452-11458, 1988). The distal element (-128 to -85) appears to be liver specific since it shows positive activity in HepG2 cells and negative activity in HeLa cells. The proximal element (-84 to -70) acts as a positive element in both these cell lines, and two rat liver nuclear proteins, BRF-1 and C/EBP, bind to two overlapping sites (-84 to -60 and -70 to -50, respectively). By gel mobility shift assay, we have identified a rat liver nuclear protein (BRF-2) which binds to the distal element (-128 to -85) of the apoB gene. This putative trans-acting factor has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. The purified BRF-2 has an apparent molecular mass of 120 kDa and was found to specifically recognize sequence -128 to -85; BRF-2 also produced a strong hypersensitive site at nucleotide position -95 with copper-orthophenanthroline reagent. A double-stranded oligonucleotide (-128 to -85) containing a 3-nucleotide (TTC) insertion between position -95 and -94 was found to abolish DNA binding by BRF-2. This result suggests that the region surrounding the hypersensitive site -95 is important for protein-DNA interaction. By using apoB promoter fragments containing various internal deletions as templates for gel mobility shift assay, the region between -104 and -85 was identified to be crucial for binding by BRF-2. We propose that BRF-2 may play an important role in the tissue-specific regulation of apoB gene transcription.
Zhuang
H
H
Department of Microbiology and Immunology, University of Tennessee-Memphis 38163.
Chuang
S S
SS
Das
H K
HK
D000818:Animals; D001055:Apolipoproteins B / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry; D002874:Chromosome Mapping; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D001055:Apolipoproteins B; D004268:DNA-Binding Proteins; D010618:Phenanthrolines; C025205:1,10-phenanthroline