A nuclear DNA-binding protein expressed during early stages of B cell differentiation interacts with diverse segments within and 3' of the Ig H chain gene cluster.
We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nuclear proteins that bind to diverse segments within and 3' of the Ig H chain gene cluster. DNA binding sites include sequences 5' of each of the following C region genes: mu, gamma 1, gamma 2a, epsilon, and alpha. For the most part, these binding sites lie 5' of CH-associated tandem repeats. Binding sites for the same B cell lineage-specific proteins have also been defined in the region 3' of C alpha, close to a recently described B cell-specific enhancer element. Cross-competition of EMSA indicates that the B cell lineage-specific nucleoprotein is indistinguishable from those described previously by others: S alpha-BP and BSAP. Because of the diverse sequences recognized by this protein, we term it NF-HB, B-lineage-specific nuclear factor that binds to Ig H gene segments. EMSA using segments 5' of S gamma 2a (5'S gamma 2a-176) and 3' of C alpha (3' alpha-88) shows multiple binding complexes, two of which are B cell lineage specific. The B cell-specific complex with fastest mobility contains only NF-HB, and the one with slowest mobility contains NF-HB together with a ubiquitous DNA-binding protein(s). The ubiquitous binding protein is different for 5' S gamma 2a-176 and for 3' alpha-88, representing the formation of protein-NF-HB complexes specific for these particular Ig DNA regions. Spleen cells show a single band upon EMSA with either 5'S gamma 2a-176 or 3' alpha-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, pre-B, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3' alpha-enhancer and segments of the Ig H gene cluster.
Liao
F
F
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
Giannini
S L
SL
Birshtein
B K
BK
D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002460:Cell Line; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D009698:Nucleoproteins / Q000502:physiology*; D051757:PAX5 Transcription Factor; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors
D015342:DNA Probes; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D009687:Nuclear Proteins; D009698:Nucleoproteins; D051757:PAX5 Transcription Factor; C497282:Pax5 protein, mouse; D014157:Transcription Factors