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: μ, γ1, γ2a, ε, and α. For the most part, these binding sites lie 5′ of C_H-associated tandem repeats. Binding sites for the same B cell lineagespecific proteins have also been defined in the region 3′ of Cα, 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: Sa-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 Sγ2a (5′Sγ2a-176) and 3′ of Ca (3′α-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′ Sγ2a-176 and for 3′α-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′Sγ2a-176 or 3′α-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, preB, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3′α-enhancer and segments of the Ig H gene cluster.