The immunoglobulin heavy chain 3ʼ enhancer : coupling of basic-Helix-Loop-Helix proteins to the regulation of enhancer function

Abstract: During B lymphocyte development, the transcriptional activity of the IgH locus is subject to spatial and temporal changes. One cis-control element, the IgH 3' enhancer, has been implicated in regulating functional events in late B cell development. This study aims to delineate the regulation of IgH 3'enhancer function, as well as to reveal the extent of its contribution to elevating expression of IgH genes. A 3' enhancer-dependent beta-globin transgenic reporter construct demonstrated enhancer function to be restricted to large, in vivo activated B lymphocytes. In small, resting splenic B cells, enhancer activity can be induced either with LPS, or by ligand-receptor-dependent stimulation of IgM and CD40 receptors. Hence, the IgH 3' enhancer is susceptible to activation via distinct stimulatory pathways. In addition, activation of enhancer function appears to be coupled to the machinery that triggers B cell growth and differentiation. A second transgenic study, utilising rearranged IgH genes revealed that the IgH 3' enhancer is capable of boosting expression levels of the transgene, both at the RNA and protein levels, suggesting possible synergism between this enhancer and the Emy enhancer/VH promoter during later stages of B cell differentiation. Biochemical analyses identified a cluster of protein binding sites within the domains (A, B, C) of the enhancer. Whilst some bind lineage restricted factors, others are recognition sites for ubiquitous DNA-binding proteins. One of these sites is the E3 motif located within Domain C. This domain displays ubiquitous activity when displaced from the lymphoid restricted Domains A and B, and the E3 motif is likely, in part, to contribute to this activity. The E3 site contains the core CACGTG sequence and has been shown to bind the basic -helix-loop-helix (bHLH) protein USF, both in cell lines and in splenic B cell extracts. Further investigation revealed that this E-box could be transactivated by a member of the bHLH/PAS family, the Arnt (Aryl-hydrocarbon receptor nuclear translocator) protein. The data indicate that the E3box is a target for Arnt transactivation and suggests a role for this factor in the transcriptional regulation of E box containing genes. Furthermore, Arnt was shown to form complexes with the adenovirus major late promoter USF site (ML). Surprisingly, the 3'enhancer E3 core sequence (CACGTG), although identical to the ML motif, does not bind vaccinia virus expressed Arnt. Sequence alignment revealed that the triplet immediately 3' to the core sequence is necessary for Arnt-E box binding. Substituting the 3'enhancer E3 triplet with a ML triplet resulted in Arnt binding to this converted ML-like E3 site. Interestingly, in COS cells an additional slow migrating complex was detected, and may explain why, despite the absence of Arnt binding to the E3 box, an E3 dependent reporter gene could be transcriptionally potentiated in the presence of co-transfected Arnt. This novel complex could be a putative heteromeric partner for Arnt and may thus contribute to the regulation of transcriptional activity of E boxes in many different genes including the immunoglobulin genes.