Structure, function and regulation of IgH 3' enhancers

Abstract

Expression and rearrangement of the murine immunoglobulin heavy chain (IgH) locus are regulated by an intronic enhancer, Emu, and a 3' regulatory region, consisting of four enhancers (Calpha3'E, 3'alphaE(hs1.2), hs3 and hs4). Genomic Southern and DNA sequence analysis of the 3' regulatory region revealed that a ∼25kb segment of DNA commencing at Calpha3'E and terminating at hs3 possessed a quasi-dyad symmetry, with 3'alphaE(hs1,2) at its center. Additionally, Calpha3'E, like 3'alpha(hs1,2) and hs3, but unlike hs4 which lies outside this dyad structure, exhibited DNase I hypersensitivity and transcriptional activity in plasma cell lines. These results led to a model in which the dyad structure of the 3' regulatory region was involved in co-ordinate regulation of the enhancers Calpha3'E, 3'alphaE(hs1,2) and hs3.;To test this hypothesis and assess the function of the 3' IgH enhancers in their native context, I utilized a naturally occurring deletion of two of these enhancers, hs3A and hs1,2 in a pre-B cell line, 70Z/3. mu expression in 70Z/3 was found to be comparable to that in a pre-B cell line with an intact 3'' region, indicating that hs3A and hs1,2 were not essential for mu expression at this stage. Furthermore, a 70Z/3-plastnacytoma fusion hybrid exhibited substantially elevated levels of mu RNA and protein relative to its pre-B parent and comparable to that observed in a number of mu-producing spleen cell hybridomas. Additionally, the hybrid cell line underwent spontaneous class switching in culture to IgG1 at a frequency comparable to most hybridomas. These results indicated that Calpha3'EE/hs3A and hs1,2 were not essential for high levels of IgH expression or for spontaneous class switching in a plasma cell line and implied a redundancy of enhancer function in the IgH locus at this stage.;Further analysis of the enhancer elements also led to the identification of a transcription factor Yin-Yang 1 (YY1) binding site in hs3, that is regulated in response to LPS, an in vitro switching stimulus. Mutation of the YY1 binding site in hs3 resulted in a 30--50% decrease in the activity of hs3 in a plasma cell line.;These studies have led to a more detailed delineation of the structure of the mouse 3' regulatory region, its role in influencing the various processes involved in heavy chain production, and identification of a new regulator of the 3' enhancers.