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

Multiple cis-acting elements including the intronic enhancer and the 3'alph a enhancer (3'alphaE) regulate expression of the Ig heavy chain genes durin g B cell development. A 3'alphaE is composed of DNase I-hypersensitive site s, hs1,2, hs3a,b, and hs4, found 3' of the murine C alpha gene as well as 3 ' of both human C alpha genes, Cal and C alpha2, Rabbits have 13 C alpha ge nes, and we tested whether a 3'alphaE is associated with each of these gene s. 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 l ibrary, We identified a single hs1,2 fragment 8-kb downstream of C alpha 13 , the presumed 3'-most C alpha gene. We also identified and partially seque nced a new C alpha gene, C alpha 14, located 6 kb upstream of C alpha 13. G enomic Southern blot analysis confirmed that the rabbit genome contains onl y one hs1,2 enhancer region. We tested the enhancer activity of the hs1,2 w ith the SV40, V-H, and I alpha 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 I alpha pro moter. We conclude that the rabbit has a single hs1,2 enhancer that resides at the 3' end of the IgH gent cluster and may constitute one of the cis-el ements regulating the expression of IgH genes.