A novel octamer regulatory element in the VH11 leader exon of B-1 cells.
B-1 cells (CD5 B cells) represent an initial fetal wave of B cell lymphopoiesis. B-1 cells have fundamental properties that are unique from conventional B cells, including a restricted Ab repertoire. We investigated the mechanism for the overrepresentation of one such Ig H chain variable-region gene, VH11, by murine B-1 cells. We postulated that a cis-regulatory element contributed to the use of VH11. We observed that the DNA encoding the leader peptide of VH11 was atypically A/T rich and thus was a candidate for nuclear protein binding. By electrophoretic mobility shift analysis, we found that the VH11 leader DNA specifically bound to three protein complexes present in the nucleus of the B-1 cell line AJ9. Of these bands, one was ubiquitous for all cells examined (lymphoid and nonlymphoid); another band was present only in B cells, and the third band was specific for B-1 cells that expressed VH11 or VH12. In addition to its binding properties, the VH11 leader sequence also displayed modest tissue-specific enhancer activity. By DNA footprint analysis, all three protein complexes were found to bind to an octamer motif embedded within the VH11 leader DNA. To identify the octamer-binding proteins, a panel of octamer-specific Abs was used. We found that the ubiquitous band was Oct-1, and the B cell-specific band was Oct-2. The B-1 cell-specific nuclear binding protein was neither Oct-1 nor Oct-2, but may be a novel POU domain protein. We hypothesize that the VH11 leader octamer site may target this gene for preferential rearrangement and/or expression and therefore would be a contributing factor in the increased use of this gene by B-1 cells.
Goodglick
L
L
Department of Pathology and Laboratory Medicine, Jonsson Comprehensive Cancer Center, University of California Los Angeles School of Medicine, USA.
Felsher
D W
DW
Neshat
M S
MS
Braun
J
J
D000818:Animals; D016175:B-Lymphocyte Subsets / Q000276:immunology*; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods; D005091:Exons / Q000235:genetics*; D051863:Host Cell Factor C1; D051379:Mice; D008969:Molecular Sequence Data; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D004268:DNA-Binding Proteins; C497693:Hcfc1 protein, mouse; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; C494166:Pou2f1 protein, mouse; C489206:Pou2f2 protein, mouse; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase