About the Project
B cells have the unique ability to produce a tailored and highly specific antibody response against a virtually limitless range of antigens. This is possible due to diversification of antibody genes (also known as immunoglobulin heavy- (IgH) and light-chain loci) via DNA deletion-recombination mechanisms and high-rate point mutation. As B cells differentiate into antibody secreting cells, the IgH locus rearranges by a mechanism known as class switch recombination (CSR). CSR replaces the exon encoding the constant region of the IgH chain (CH exon), which determines the antibody isotype (e.g. IgG) and hence effector function. At the molecular level, the CSR mechanism depends on transcription of long non-coding RNA (switch RNA) initiated upstream of each CH exon undergoing recombination. Due to the G-richness and highly repetitive nature of switch RNA sequences, the transcript can stably hybridize to the template DNA strand thereby forming a so-called R-loop structure. Alternatively, switch RNA can fold in G-quadruplex structures (G4 RNA). Both G4 RNA and R-loop structures have been implicated in targeting the activity of the mutagenic enzyme activation-induced cytidine deaminase (AID) to initiate CSR. Downstream of AID-induced mutation, CSR relies on the generation of DNA double-strand breaks (DSBs) and DNA repair by non-homologous end-joining (NHEJ) proteins. Failure in CSR results in immunodeficiency disorders characterized by defective production of switched antibody isotypes (hyper-IgM syndromes), while inappropriate targeting of AID to non-Ig hotspots (off-target genes) leads to the development of B cell lymphoma. Notably, the incidence of certain B cell lymphoma subtypes increases with age.
We study how RNA binding proteins (RBPs) regulate DNA recombination at Ig loci. In particular, we are interested in the role of DEAD-box RNA helicases, a large family of RBPs that utilize ATP hydrolysis to remodel RNA structure or RNA-protein interactions. Previously we have shown that DEAD-box RNA helicase 1 (DDX1) binds to G4 RNA and converts it into R-loops, thus targeting AID to the IgH locus and promoting CSR (Ribeiro de Almeida et al, 2018). This PhD project aims to understand how this novel mechanism of R-loop formation plays a role downstream of AID targeting. Our current hypothesis is that DDX1 participates in DNA repair of AID-induced IgH DSBs. Importantly, DSB formation at AID off-targets and aberrant DNA repair are drivers of B cell lymphomagenesis. Therefore, a related question is whether G4 RNA- and DDX1-dependent R-loops are implicated in AID targeting elsewhere in the genome. To address these questions we take a complementary approach, using conditional knock-out mouse models, ex vivo cellular systems and in vitro assays. The methods employed include in vivo immunizations, multi-parameter flow-cytometry, CRISPR-Cas9 genome editing, protein co-immunoprecipitation followed by mass-spectrometry, genome-wide analyses of RBP targets or DNA recombination events, and in vitro assays for protein-RNA interactions or RNA helicase activity. The successful candidate will be trained in state-of-the-art molecular and cellular biology techniques combined with the analysis of B cell activation in vivo, to tackle how DDX1 regulates IgH CSR and AID off-target activity. In addition, the project will allow the PhD candidate to develop skills in bioinformatics analysis of the data generated from experimental approaches.
References
Ribeiro de Almeida C, Dhir S, Dhir A, Moghaddam AE, Sattentau Q, Meinhart A, and Proudfoot
NJ*. RNA helicase DDX1 modulates G-quadruplex and R-loop formation to promote IgH Class
Switch Recombination. Molecular Cell. 2018 May 17;70(3):1-13.