Functional characterization of the murine homolog of the B cell-specific coactivator BOB.1/OBF.1.
B cell-specific transcriptional promoter activity mediated by the octamer motif requires the Oct1 or Oct2 protein and additional B cell-restricted cofactors. One such cofactor, BOB.1/OBF.1, was recently isolated from human B cells. Here, we describe the isolation and detailed characterization of the murine homolog. Full-length cDNAs and genomic clones were isolated, and the gene structure was determined. Comparison of the deduced amino acids shows 88% sequence identity between mouse and human BOB.1/OBF.1. The NH2-terminal 126 amino acids of BOB.1/OBF.1 are both essential and sufficient for interaction with the POU domains of either Oct1 or Oct2. This protein-protein interaction does not require the simultaneous binding of Oct proteins to DNA, and high resolution footprinting of the Oct-DNA interaction reveals that binding of BOB.1/OBF.1 to Oct1 or Oct2 does not alter the interaction with DNA. BOB.1/OBF.1 can efficiently activate octamer-dependent promoters in fibroblasts; however, it fails to stimulate octamer-dependent enhancer activity. Fusion of subdomains of BOB.1/OBF.1 with the GAL4 DNA binding domain reveals that both NH2- and COOH-terminal domains of BOB.1/OBF.1 contribute to full transactivation function, the COOH-terminal domain is more efficient in this transactivation assay. Consistent with the failure of full-length BOB.1/OBF.1 to stimulate octamer-dependent enhancer elements in non B cells, the GAL4 fusions likewise only stimulate from a promoter-proximal position.
Zentrum für Molekulare Biologie Heidelberg (ZMBH), Federal Republic of Germany.
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P
Pfisterer
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S
Zwilling
J
J
Hess
T
T
Wirth
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D005091:Exons; D018398:Homeodomain Proteins / Q000378:metabolism; D051863:Host Cell Factor C1; D006801:Humans; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D013379:Substrate Specificity; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; C497693:Hcfc1 protein, mouse; D018398:Homeodomain Proteins; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; C091546:POU2AF1 protein, human; C494165:POU2F1 protein, human; C058435:POU2F2 protein, human; C490424:Pou2af1 protein, mouse; C494166:Pou2f1 protein, mouse; C489206:Pou2f2 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors