A Single 3′α hs1,2 Enhancer in the Rabbit IgH Locus

Abstract

Multiple cis-acting elements including the intronic enhancer and the 3′α enhancer (3′αE) regulate expression of the Ig heavy chain genes during B cell development. A 3′αE is composed of DNase I-hypersensitive sites, hs1,2, hs3a,b, and hs4, found 3′ of the murine Cα gene as well as 3′ of both human Cα genes, Cα1 and Cα2. Rabbits have 13 Cα genes, and we tested whether a 3′αE is associated with each of these genes. To identify 3′αE 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 Cα13, the presumed 3′-most Cα gene. We also identified and partially sequenced a new Cα gene, Cα14, located 6 kb upstream of Cα13. 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, VH, and Iα promoters using the luciferase reporter gene in transient transfection assays and found that it significantly enhanced the activity of SV40 and VH promoters and slightly enhanced an Iα 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.

Sequence analysis of 0.5 Mb of the rabbit germline immunoglobulin heavy chain locus

“…Two BACs hybridized with the two probes specific for rabbit VH1 and DH, three BACs hybridized with the Cg probe. Screening of the library with a probe specific for the recently identified rabbit heavy chain 3V enhancer region (Volgina et al, 2000) did not result in the identification of any positive BAC clone. Similarly, screening of a second library (kindly provided by Dr. Jan-Fang Cheng, Lawrence Berkeley National Laboratory) consisting of 120,804 BAC clones with an average insert size of 175 kb (corresponding to 7.0 times the rabbit genome) also did not identify BAC clones hybridizing with this 3V enhancer probe.…”

“…In the mouse, one enhancer region is found 3V of Ca, while the human locus contains two enhancer regions downstream of both, Ca1 and Ca2 (Birshtein et al, 1997;Mills et al, 1997). Rabbits have 14 Ca genes, but only a single hs1,2 enhancer element downstream of the 3V-most rabbit Ca13 gene was identified (GI: 1154561) (Knight et al, 1984;Spieker-Polet et al, 1993;Volgina et al, 2000). Sequencing of Fos15B revealed besides hs1,2 (6 kb downstream of Ca13) sequences with 92% and 84% sequence identity with human and mouse hs4, respectively.…”

“…BAC 38A2 hybridizing exclusively with the VH probe, BACs 225P18 and 219D23 hybridizing with DH and VH probes, BAC 27N5 hybridizing with the Cg probe and Fos15B (kindly provided by K. Volgina et al, 2000) were sequenced. Due to large regions of perfect repeats in Fos15B, it was not possible to get the complete sequence.…”

Evolution of human IgH3′EC duplicated structures: both enhancers HS1,2 are polymorphic with variation of transcription factor's consensus sites

“…The structure, and possibly the function, of the enhancers situated in the immunoglobulin regulative region, at the 3V end of the constant heavy chain cluster, is largely conserved from mouse to human (Mills et al, 1997), although in rabbit only one 3V enhancer is present after 13 copies of the constant heavy a gene (Volgina et al, 2000).…”

Functional Analysis of Iα Promoter Regions of Multiple IgA Heavy Chain Genes

“…Recently, Volgina et al (32) reported that the rabbit IgH locus has a single 3Ј␣ (hs1, 2) enhancer, and while this element significantly enhanced the activity of a V H promoter, it had little effect on an I␣ promoter. We suggest that neither the strength of the I␣ promoters nor the 3Ј␣ (hs1, 2) enhancer is responsible for the differential expression of IgA isotypes in vivo.…”