Biased dA/dT somatic hypermutation as regulated by the heavy chain intronic iEμ enhancer and 3′Eα enhancers in human lymphoblastoid B cells

Abstract

Somatic hypermutation (SHM) in immunoglobulin gene (Ig) variable (V) regions is critical for the maturation of the antibody response. It is dependent on the expression of activation-induced cytidine deaminase (AID) and translesion DNA polymerases in germinal center B cells as well as Ig V transcription, as regulated by the Ig heavy chain (H) intronic enhancer (iEµ) and the 3′ enhancer (3′Eα) region. We analyzed the role of these cis elements in SHM by stably transfecting Ramos human lymphoblastoid B cells with a rearranged human IgH chain VD (diversity) J (joining) DNA construct containing a V H promoter at the 5′ end and C H 1 and C H 2 exons of Cγ1 at the 3′ end. In this construct, mutations preferentially targeted dA/dT basepairs in the RGYW/ WRCY hotspot. Most of the dA/dT mutations and accompanying dC/dG mutations were transitions. Deletion of iEµ resulted in decreased SHM which could be partially restored by insertion of the IgH hs1,2 enhancer. Other two 3′Eα enhancers, hs3-hs4, did not significantly increase the mutation frequency, but further strengthened the dA/dT bias. The frequency and spectrum of the mutations were independent of the genomic integration of the transgene or V gene transcription level. Thus, we have established a novel in vitro system to analyze SHM and identify the role of multiple cis-regulatory elements in regulating dA/dT biased SHM. This model system will be useful to further address the role of other cis-regulating elements and recruited trans-acting factors in expressing the modalities of SHM.

The Biochemistry of Somatic Hypermutation

“…This general idea is supported by studies in which the intronic enhancer has been manipulated in other ways (153). For example, in Ramos cells, when the core Eμ enhancer is deleted in an ectopic H chain gene, there is a twofold reduction in SHM.…”

Allele *1 of HS1.2 Enhancer Associates with Selective IgA Deficiency and IgM Concentration

“…Because 3ЈRR in humans is duplicated it can be hypothesized there is a coordinated activity for the two 3Ј RRs during Ig maturation. In addition, they are considered relevant for germline transcription of constant regions during B cell maturation (24). When the switch involves the duplicated constant regions (␥-2, ␥-4, , ␣-2) the 3ЈRR-1 will be deleted and the entire region could be regulated in the next steps of maturation or in memory cell and plasma cell by the presence of 3ЈRR-2 alone (22).…”

Nurse shark T‐cell receptors employ somatic hypermutation preferentially to alter alpha/delta variable segments associated with alpha constant region

“…In mice and humans, V(D)J recombination in Ig heavy chain (IgH: IgM/IgW) V regions is controlled by regulatory elements upstream of the IgH constant (C) region exons (between the V and D clusters) [43,44]. However, AID accessibility to the IgHC locus is controlled by transcriptional enhancers located in the 3' regulatory region (RR) downstream of the IgHC exons [43,45,46], and SHM requires both the transcription of IgH V regions and the upregulation of AID expression [46]. It is likely that these same transcriptional enhancers regulate SHM of nurse shark TCR, allowing AID access to TCRα chain but limiting access in other TCR chains, evidenced by the greater use of SHM by TCR αδV segments associated with TCRα than with TCRδ constant regions.…”