The murine Ig 3' alpha enhancer is a target site with repressor function for the B cell lineage-specific transcription factor BSAP (NF-HB, S alpha-BP).
Using electrophoretic mobility shift assays (EMSAs) we have identified several target sites for nuclear proteins in the murine heavy chain Ig 3' alpha enhancer. Two of these sites, denoted oligo-H and oligo-K, were shown by several criteria, including cell distribution and stimulation experiments, EMSA cross-competition studies, and proteolytic clipping bandshift assays, to bind to the same protein identical to the transcription factor B cell lineage-specific activator protein (BSAP) (NF-HB, S alpha-BP). To assess the possible functional role of these BSAP binding sites in the 3' alpha enhancer, we transiently transfected a construct containing a 314-bp 3' alpha enhancer fragment upstream of a luciferase reporter gene in MOPC-315 cells, a plasmacytoma line lacking BSAP. In these cells, co-transfection with a vector expressing recombinant BSAP led to significant reduction in the activity of the 3' alpha enhancer fragment. Conversely, in the mature B lymphoma cell line CH12.LX, a cell line that expresses BSAP and has a less active 3' alpha enhancer, selective BSAP down-regulation by an antisense phosphorothioate oligonucleotide was sufficient to considerably up-regulate 3' alpha enhancer activity, as were mutations of both binding sites that prevented binding of BSAP to the 3' alpha enhancer. Our findings thus suggest that the natural loss of BSAP expression in terminally differentiated plasma cells contributes to the activation of the murine Ig 3' alpha enhancer.
Neurath
M F
MF
Mucosal Immunity Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
Strober
W
W
Wakatsuki
Y
Y
D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D005803:Genes, Immunoglobulin; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D051757:PAX5 Transcription Factor; D012097:Repressor Proteins / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D051757:PAX5 Transcription Factor; C497282:Pax5 protein, mouse; D012097:Repressor Proteins; D014157:Transcription Factors