A single 3' alpha hs1,2 enhancer in the rabbit IgH locus

Multiple cis-acting elements including the intronic enhancer and the 3'alpha enhancer (3'alphaE) regulate expression of the Ig heavy chain genes during B cell development. A 3'alphaE is composed of DNase I-hypersensitive sites, hs1,2, hs3a,b, and hs4, found 3' of the murine Calpha gene as well as 3' of both human Calpha genes, Calpha1 and Calpha2. Rabbits have 13 Calpha genes, and we tested whether a 3'alphaE is associated with each of these genes. To identify 3'alphaE regions we developed a rabbit hs1,2 probe and used this to search for enhancer homologues of human hs1,2 in a genomic fosmid library. We identified a single hs1,2 fragment 8-kb downstream of Calpha13, the presumed 3'-most Calpha gene. We also identified and partially sequenced a new Calpha gene, Calpha14, located 6 kb upstream of Calpha13. Genomic Southern blot analysis confirmed that the rabbit genome contains only one hs1,2 enhancer region. We tested the enhancer activity of the hs1,2 with the SV40, V(H), and Ialpha promoters using the luciferase reporter gene in transient transfection assays and found that it significantly enhanced the activity of SV40 and V(H) promoters and slightly enhanced an Ialpha promoter. We conclude that the rabbit has a single hs1,2 enhancer that resides at the 3' end of the IgH gene cluster and may constitute one of the cis-elements regulating the expression of IgH genes.