From structure to function: How G quadruplexes control antibody diversification and B-cell fate – AID-G4-CSR

B cells have a remarkable capacity to alter their immunoglobulin (Ig) loci. Activated mature B cells diversify Ig genes through somatic hypermutation (SHM) and class switch recombination (CSR). SHM yields higher antibody affinity carried by positively selected cells. In parallel, negative selection operates through multiple pathways to get rid of clones with lower affinity, autoreactivity, or that have lost their B cell receptor. However, the mechanisms underlying negative selection are largely unknown.

Both SHM and CSR are initiated by the Activation-Induced cytidine Deaminase (AID) via transcription-dependent cytidine deamination of single-stranded DNA targets. However, the mechanisms underlying AID targeting to specific sequences within Ig loci are still unclear. Structured DNA such as G-quadruplexes (G4s) have emerged as important actors in gene expression. Yet, their role in antibody diversification mechanisms and B-cell fate have not been elucidated.

By using a combination of functional, genetic, mechanistic and pharmacological analyses, this program aims to achieve a significant progress in our understanding of the precise role of G4s in switch transcription, AID targeting, genuine CSR and associated illegitimate or non-classical recombination in both humans and mice. Moreover, we ambition to provide definitive genetic and functional evidence on whether AID targets the DNA directly or whether it requires RNA recruiters, and go beyond current knowledge on the relevance of illegitimate recombination promoted by G4-rich repeats for B-cell activation-induced cell death.

This highly integrated project involves two partners with solid expertise and established record in molecular biology of normal and pathological B cell development. The program is based in large part on already available mouse models, human and mouse cell lines, and on various well-characterized G4 ligands. The program has four inter-linked aims:

Aim 1: Pharmacological and epigenetic exploration of the role of G4s during CSR (Khamlichi)
By using available mouse lines in which switch transcription occurs in the sense orientation only or in the antisense orientation only, and various families of G4 ligands displaying differences in selectivity and activity, we will address the mechanisms underlying the interplay between G4s as chromatin organizers, sense and antisense switch transcription, and CSR in vivo.

Aim 2: The role of G4s and splicing in AID targeting (Khamlichi)
The role of splicing in CSR remains a mystery. Here, we will use different mutant clones of the CH12 B cell line bearing deletions that affect switch transcription elongation and/or splicing. Our underlying idea is that by enabling transcription and by blocking splicing, we can determine the efficiency of AID targeting and CSR, in the absence of intermediate splice products.

Aim 3: Regulatory role of non-classical DNA repeats and G4s at the human IgH locus (Cogné)
We have recently discovered new types of illegitimate recombination involving highly structured G4s that lead to deletion of critical sequences within the IgH locus. By using an available humanized mouse model, human cell lines, G4 ligands and tools for inhibition of G4-rich RNA processing by the RNA exosome complex, we will explore in-depth the regulatory roles of these sequences on transcription, alternative splicing, and recombination.

Aim 4. G4-repeats and the “dark side” of AID as killer of IgH expression (Cogné)
We showed that the IgH locus can undergo AID-mediated locus suicide recombination (LSR). Our working hypothesis is that such LSR contributes to B cell homeostasis and leads to death of sub-optimally activated cells. By using original mouse models, cell lines, and innovative assays, we will focus on the furtive stage before negative selection and cell death and determine how the specific genetic events involving G4-rich repeats in LSR lead to major functional consequences regarding B cell homeostasis.