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.
Ogami
K
K
Department of Medicine Housman Medical Research Center, Boston University, Medical Center, Massachusetts 02118.
Kardassis
D
D
Cladaras
C
C
Zannis
V I
VI
D000818:Animals; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents; D016172:DNA Fingerprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011401:Promoter Regions, Genetic; D051381:Rats; D015534:Trans-Activators
D053305:Apolipoprotein C-III; D001056:Apolipoproteins C; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D015534:Trans-Activators; C068859:nuclear factor CIIIB1