Genomic deletion of the whole IgH 3′ regulatory region (hs3a, hs1,2, hs3b, and hs4) dramatically affects class switch recombination and Ig secretion to all isotypes

Abstract

IntroductionThe immunoglobulin heavy chain locus (IgH) undergoes multiple changes along B-cell differentiation, affecting transcription and accessibility to V(D)J or class switch recombination (CSR). The iE enhancer upstream of C mostly promotes V(D)J recombination, 1 whereas IgH 3ЈRR enhancers (hs3a, hs1,2, hs3b, and hs4) have controversial roles. A role in CSR was suggested by replacing mouse hs1,2 with a neomycin resistance gene, thus affecting germline transcription and CSR to ␥2a, ␥2b, ␥3, and ⑀. 2 However, deletion of this neo cassette restored CSR. 3 hs3a, hs3b, and hs4 also proved individually dispensable for CSR. 3-5 Enhancer redundancies might explain that their individual deletion only results in minor effect. Indeed, joint hs3b/hs4 deletion impaired germline transcription and CSR to most isotypes except and ␥1. 6 Reporter genes also demonstrated synergies between 3ЈRR enhancers, 7 which altogether promote CSR into large transgenes. 8 The 3ЈRR is followed with DNase hypersensitive sites (hs5-7) lacking enhancer activity but binding CCCTC-binding factor and constituting the 3Ј locus boundary. 9 To reconcile the controversial phenotypes of focal mutations, potentially attenuated by functional redundancies, we evaluated IgH expression and CSR after deleting the whole 30-kb extent of the 3ЈRR.

Elucidation of IgH 3′ region regulatory role during class switch recombination via germline deletion

“…We confirmed that splenocytes from 3 0 RR-deficient mice showed a dramatically reduced ability to secrete IgG 1 in vitro in response to LPS þ IL4 (Fig. 1a), which likely results from both decreased CSR in B-cells and the known defect of Ig secretion in plasma cells from such mice 6 . Parallel flow cytometry experiments evaluating membrane IgG 1 expression in B-cells confirmed that the deletion of the 3 0 RR did not abolish but reduced by 490% in vitro IgG 1 CSR (Fig.…”

“…The IgG 1 class displays a special status with regard to 3 0 RR mutations, since it was the only Ig class whose expression was preserved after the first reported partial 3 0 RR alterations or truncations 17,18 . Even after complete deletion of the 3 0 RR, while CSR to all other class-switched Ig was nearly abolished, residual low-level CSR to IgG 1 was maintained 6 . We thus now take advantage of this preserved IgG 1 switching in 3 0 RR-deficient mice to explore whether it reveals additional 3 0 RR-dependent mechanistic features during CSR.…”

“…Among 5 0 elements, the intronic Em enhancer is reported as a master control element of V(D)J recombination 3,4 . The IgH 3 0 regulatory region (3 0 RR), that encompasses the four transcriptional enhancers hs3a, hs1,2, hs3b and hs4, controls m transcription in mature B-cells 5 , and is the master element controlling conventional class switch recombination (CSR) 6,7 and somatic hypermutation (SHM) 8 but without role on V(D)J recombination 9,10 . CSR is a cis-deletion or trans-recombination process that allows the C m gene to be replaced in mice with one of the downstream C g3 , C g1 , C g2b , C g2a , C e or C a gene.…”

“…However in C-NHEJ-deficient conditions, an alternative end-joining (A-EJ) pathway is left, which predominantly joins ends by using short homologies found in the proximity of broken ends (microhomology-based repair) 11,13 . The 3 0 RR is crucial for conventional CSR 6,7 but its mechanistic contribution remains enigmatic. The main conclusions drawn from knock-out mice are that the 3 0 RR plays a major role in promoting synapsis between S regions targeted by CSR 16 , and plays a partial role in promoting germline transcription of S regions initiating at I promoters 6,17,18 .…”

“…The 3 0 RR is crucial for conventional CSR 6,7 but its mechanistic contribution remains enigmatic. The main conclusions drawn from knock-out mice are that the 3 0 RR plays a major role in promoting synapsis between S regions targeted by CSR 16 , and plays a partial role in promoting germline transcription of S regions initiating at I promoters 6,17,18 . Whether the chromatin structure of S regions or the process of their repair after CSR are also dependent from the 3 0 RR is currently unknown.…”