Purification and characterization of a heat stable nuclear factor CIIIB1 involved in the regulation of the human ApoC-III gene

The apoC-III promoter region -86 to -74 is recognized by two nuclear factors designated CIIIB1 and CIIIB2 (NF-BA1) which are both activators of apoC-III gene transcription (Ogami, K., Hadzopoulou-Cladaras, M., Cladaras, C., and Zannis, V. I. (1990) J. Biol. Chem. 265, 9808-9815). In this communication we report the purification of factor CIIIB1 from rat liver nuclear extracts. The purification procedure included anion- and cation-exchange chromatography, DNA sequence-specific affinity chromatography, and heat treatment at 85 degrees C for 5 min. The ligand used for affinity chromatography was a mutated apoC-III -90 to -73 promoter sequence which binds only the CIIIB1 factor. The purified protein was identified as a poly-peptide of Mr 41,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity cross-linking. The binding site of CIIIB1, defined by DNase I footprinting and methylation interference assays, contains the octameric motif CAGGTGAC. Nucleotide substitutions within this sequence abolished the binding of the purified factor. DNase I footprinting analysis showed that purified CIIIB1 protein protects the apoA-II promoter region -65 to -48 which contains an identical octameric CAGGTGAC motif in the antisense strand suggesting that CIIIB1 may also play a role in apoA-II gene regulation.