The immunoglobulin heavy chain 3 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 tospatial and temporal changes. One cis-control element, the IgH 3' enhancer, has been impli-cated in regulating functional events in late B cell development. This study aims to delineatethe regulation of IgH 3'enhancer function, as well as to reveal the extent of its contribution toelevating expression of IgH genes.A 3' enhancer-dependent beta-globin transgenic reporter construct demonstraeed enhancer func-tion 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 stimulationof IgM and CD40 receptors. Hence, the IgH 3' enhancer is susceptible to activation via distinctstimulatory pathways. In addition, activation of enhancer function appears to be coupled to themachinery that triggers B cell growth and differentiation. A second transgenic study, utilisingrearranged IgH genes revealed that the IgH 3' enhancer is capable of boosting expression levelsof the transgene, both at the RNA and protein levels, suggesting possible synergism betweenthis 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 forubiquitous 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 resticted DomainsA 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 inves-tigation 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 transcriptionalregulation of E box containing genes.Furthermore, Arnt was shown to form complexes with the adenovirus major late promoter USFsite (ML). Surprisingly, the 3 'enhancer E3 core sequence (CACGTG), although identical to theML motif, does not bind vaccinia virus expressed Arnt. Sequence alignment revealed that thetriplet immediately 3' to the core sequence is necessary forArnt-E box binding. Substituting the3'enhancer E3 triplet with a ML triplet resulted in Arnt binding to this converted ML-like E3site. Interestingly, in COS cells an additional slow migrating complex was detected, and mayexplain why, despite the absence of Arnt binding to the E3 box, an E3 dependent reporter genecould be transcriptionally potentiated in the presence of co-transfected Arnt. This novel com-plex could be a putative heteromeric partner for Arnt and may thus contribute to the regulationof transcriptional activity of E boxes in many different genes including the immunoglobulingenes.ISBN 91-628-1895-3
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