Identification and characterization of a DNA binding protein, NF-HB (BSAP), a regulatory element in the immunoglobulin heavy chain locus

Abstract

One approach to understand the molecular mechanisms that control Ig H chain gene class switching has been to utilize the DNA sequences involved in the process for selection of key trans-acting protein factors. By using electrophoretic mobility shift assays (EMSA), we have identified a B-cell lineage specific nuclear binding factor (which we have termed NF-HB) that interacts with several sequences within the H chain cluster. Most of the binding sites reside 5{dollar}\sp\prime{dollar} of tandem repeats in regions approximating start sites for germline transcripts, presumed intermediates in class switching. Several binding sites were also located 3{dollar}\sp\prime{dollar} of the H chain gene cluster both within and flanking the 3{dollar}\sp\prime\alpha{dollar}-enhancer. These observations suggest a role for NF-HB in promoting physical interaction between the 3{dollar}\sp\prime\alpha{dollar}-enhancer and the H chain gene cluster. We have shown that NF-HB is indistinguishable from BSAP (Pax 5), an early B cell specific transcriptional activator, based on the following criteria: (1) indistinguishable UV-crosslinked size; (2) related binding specificity indicated by cross competition of DNA binding sites; (3) identical partial proteolytic patterns on EMSA.;Our experiments have been suggestive of specific roles for NF-HB/BSAP and its binding sites in Ig H gene class switching. Multiple protein complexes have been formed with different DNA segments, in which both NF-HB and particular DNA segment specific ubiquitous proteins are involved. Importantly, these ubiquitous DNA-binding proteins and their interaction with NF-HB to form heteroprotein complexes are induced during LPS stimulation of cultured spleen cells. The kinetics of complex formation, maximally induced at {dollar}\sim{dollar}2 days, approximates that of class switching. Furthermore, we have shown that a NF-HB site 5{dollar}\sp\prime{dollar} of S{dollar}\gamma{dollar}2a is the same as that identified by Rothman et al to be required for the induction of {dollar}\varepsilon{dollar} germline transcripts in response to LPS and IL-4. An NF-HB binding site, originating from the region 5{dollar}\sp\prime{dollar} of S{dollar}\gamma{dollar}2a, can functionally substitute for the {dollar}\varepsilon{dollar}-associated site. These results imply that NF-HB/BSAP plays a significant--non-isotype specific--role in {dollar}\varepsilon{dollar} class switching. By inference, NF-HB may be involved in production of germline transcripts originating from other C{dollar}\sb{lcub}\rm H{rcub}{dollar} genes.