Class-Switch Recombination in the Absence of the IgH 3′ Regulatory Region

Abstract

The ~28 kb 3′-regulatory region (3′RR) which is located at the most distal 3′ region of the immunoglobulin (Ig) heavy (H) chain locus has multiple regulatory functions that control IgH expression, class switch recombination (CSR) and somatic hypermutation (SHM). Here, we report that deletion of the entire 3′RR in a mouse B cell line that is capable of robust cytokine-dependent CSR to IgA results in reduced but not abolished CSR. These data suggest that the 3′RR is not absolutely required for CSR, and thus is not essential for targeting AID to S regions as has been suggested. Moreover, replacing the 3′RR with a DNA fragment including only its four DNase I hypersensitive sites (lacking the large spacer regions), restores CSR to a level equivalent to or even higher than wild type cells, suggesting that the four HS sites contain most of the CSR-promoting functions of the 3′RR. Stimulated cells express abundant germline transcripts, with the presence or absence of the 3′RR, providing evidence that the 3’RR has a separate role in promoting CSR, that is unique from enhancing S region transcription.

The Chromatin Reader ZMYND8 Regulates Igh Enhancers to Promote Immunoglobulin Class Switch Recombination

“…The Igh locus contains two enhancer elements, Eμ and 3′RR, that are located at 5′ of Sμ and 3′ of Cα, respectively ( Birshtein, 2014 , Perlot and Alt, 2008 ). The 3′RR is a prototypical super-enhancer ( Pinaud et al., 2011 , Whyte et al., 2013 ), which is required for GLT induction and CSR in mature B lymphocytes and, to a lesser extent, in CH12 cells ( Issaoui et al., 2018 , Kim et al., 2016 ). As expected from the genome-wide distribution ( Figures 4 A and 4B), ZMYND8 associates with the Igh locus in activated CH12 and B cells and it binds the 5′ Eμ and 3′RR enhancers ( Figures 4 C and 5 A).…”

Duplication of a germline promoter downstream of the IgH 3′ regulatory region impairs class switch recombination

“…More importantly, the normal levels of CSR to IgG2b in TGFβ-activated B cells argue against such scenario. In this context, reduced CSR to IgA, in the presence of normal levels of Sα transcripts, was also found in mouse B cells deficient for the p50 subunit of NF-κB 11 or the histone methyl-transferase Suv39h1 12 , and to some extent in 3′RR-deleted and Cohesin-deficient clones of CH12F3 B cell line 13 , 14 , as well as in some IgA-deficient patients 15 . These observations suggest that, at least for a structurally intact α constant gene, normal levels of GLT are not sufficient for efficient CSR to IgA.…”

Essential role of the initial activation signal in isotype selection upon deletion of a transcriptionally committed promoter

“…1A). Because in CH12, the non-productive allele has already undergone Sµ/Sα recombination 22,23 (Supplementary Fig. S1A), which deleted all I promoters except for the Eµ/Iµ enhancer/promoter (Fig.…”

“…g . 11,12,22,23,28 ). However, transcriptional activity of its I promoters had not yet been studied.…”