Nuclear organisation of V(D)J recombination and genomic integrity

Lead Research Organisation: Babraham Institute
Department Name: Nuclear Dynamics

Abstract

One of the ways in which the immune system fights infections is by making antibodies, which attack and remove the many foreign agents such as bacterial toxins that the body encounters. Since there are literally millions of different invading proteins, B cells have evolved a way of making millions of different antibodies. One each of three different kinds of gene segments V, D and J are cut and pasted together to make an immunoglobulin (antibody) protein, using two specialized enzymes called Recombinase Activating Gene 1 and 2 (Rag 1 and 2). There are several D and J and 200 V genes, thus many different combinations can be made and this process, called V(D)J recombination, ensures that the immune system produces a sufficient diversity of antibodies to fight infection. However this process can go wrong. Defects in the Rag enzymes cause immunodeficiency and in some cases the reason is unclear. Conversely, the Rag enzymes can cut and paste the immunoglobulin genes to an incorrect DNA sequence on another chromosome, causing a chromosomal translocation, the principal cause of lymphomas. Further, DNA byproducts called excision circles can be reinserted into the genome in aberrant locations, also causing lymphomas. It is unknown how the V, D and J genes find each other in the nucleus, or how the Rag enzymes find them. Understanding this is key to understanding normal antibody production and the mistakes that can be made. We propose that the genes find each other because they go to a specialized site within the nucleus called a transcription factory to transcribe, and that the Rag enzymes recognize and bind structures found there. We have developed novel assays to visualize the Rag enzymes and DNA excision circles in the nuclei of single cells for the first time, in combination with the genes they are cutting and pasting. We hope to discover which genes other than immunoglobulin genes are targeted by the Rag enzymes as this will provide insight into how chromosomal translocations occur. We will track where the excision circle byproducts go in the nucleus, and use large-scale genome sequencing to discover where these byproducts have reintegrated in the genome, to identify ?hotspots? of reintegration. This research will help us to understand how B cells make antibodies and may also identify molecules or processes that are involved in human diseases including immunodeficiency and lymphomas.

Technical Summary

V(D)J recombination assembles immunoglobulin and T cell receptors from multiple gene segments to generate the diverse antigen receptor repertoires which underpin the adaptive immune system. Distant gene segments are brought together, flanking double-strand DNA breaks are made by recombinase enzymes, Rag1 and Rag2, followed by non-homologous end joining (NHEJ) DNA repair. Aberrant repair of DNA breaks causes chromosomal translocations underlying B and T cell lymphomas. The intervening DNA is looped out to form excision circles. T cell excision circles can re-insert into the genome, primarily at other antigen receptor loci, a mechanism termed trans-V(D)J recombination. This can cause primary lymphomas and secondary chromosomal translocations. It is unclear how distant gene segments co-localise to undergo V(D)J recombination. Furthermore, the Rag enzymes have never been visualised, and thus their nuclear location and its impact on normal or dysfunctional V(D)J recombination are unknown. Our aim is to visualise how V(D)J recombination is organised within the nucleus and how its byproducts are controlled to prevent aberrant genomic re-integration. We propose that distal genes are brought to a shared RNA Polymerase II transcription factory because they are transcribing, and that recombination occurs co-transcriptionally in specialised ?transcription-recombination? factories containing Rag enzymes. Furthermore, we propose that co-transcription may underpin recombination-associated chromosomal translocations. This hypothesis is supported by high frequency Igh non-coding RNA transcription, and by the discovery that Rag2 binds histone H3 trimethylated at lysine 4 (H3K4me3), associated with active promoters. We will visualise Rag1/2 nuclear localisation before, during and after V(D)J recombination, in combination with RNA PolII factories, Igh locus DNA sequences and non-coding transcripts, H3K4me3 and NHEJ components, using high-throughput single cell RNA-DNA-immunofluorescence imaging in mouse B cells. Combined with CHIP-seq, this will provide a dynamic picture of V(D)J recombination and genome-wide targets of Rags. Conversely, despite up to 4 excision circles per lymphocyte, genomic re-insertion frequency is low, but it is unknown how it is prevented. We will determine whether removal of excision circles from transcription factories protects the genome from trans-V(D)J recombination, using a novel FISH assay to visualise excision circle fate, including association with Rags and repressive nuclear compartments. We will use high-throughput metaphase analysis, and 4C (chromosome conformation capture with genome-wide sequencing) to determine frequency and location of excision circle re-insertion in B cells. These studies will further our understanding of V(D)J recombination, Rag-associated immunodeficiencies, and lymphomagenesis mediated by chromosomal translocation and trans-V(D)J recombination, and may reveal diagnostic or therapeutic targets.

Publications

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Bolland DJ (2013) Robust 3D DNA FISH using directly labeled probes. in Journal of visualized experiments : JoVE

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Matheson LS (2012) Local and global epigenetic regulation of V(D)J recombination. in Current topics in microbiology and immunology

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Stubbington MJ (2013) Non-coding transcription and large-scale nuclear organisation of immunoglobulin recombination. in Current opinion in genetics & development

 
Title Rag FISH reagents and assay 
Description Development of a high-throughput fluorescence in situ hybridization assay for analysis of VDJ recombination in single cells, including cell cycle markers, Rag expression and FISH probes for the immunoglobulin loci. 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact FISH assay taught to several other groups at Babraham Institute and visiting scientists from the Laboratory of Molecular Biology Cambridge and University of Cambridge. Contributed underpinning improvements to publication in JOVE in 2013. 
 
Description Rag antibodies 
Organisation Yale University
Department Department of Immunobiology
Country United States 
Sector Academic/University 
PI Contribution We have used Rag antibodies to determine the nuclear localisation of Rag enzymes by fluourescence in situ hybridisation.
Collaborator Contribution Our collaborator, Professor David Schatz has provided antibodies to the Rag enzymes, unpublished Chip-Seq data on Rag binding targets, and intellectual input.
Impact Manuscript Sliva-Martins et al under review
Start Year 2009
 
Description Rag mutant cell lines 
Organisation Boston Children's Hospital
Country United States 
Sector Hospitals 
PI Contribution We are using cell lines containing mutant forms of Rag enzymes provided by our collaborator to determine the binding patterns of Rag enzymes in single cell nuclei by confocal microscopy
Collaborator Contribution Our collaborator, Professor Marjorie Oettinger has provided cell lines in which binding and catalytic mutants of Rag enzymes are overexpressed, and has provided expertise and useful discussion on the project
Impact No outputs as yet.
Start Year 2010
 
Description B lymphocyte development lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact 60 Cambridge University undergraduate medical, veterinary and natural science students attended the lecture on B lymphocyte development, which sparked many questions afterwards. Supervision sessions were also conducted on the topic, essays assessed, and exam questions submitted.

Numerous students asked for tutorials on the lecture, emailed queries about the topic, wrote essays, and the majority of students attempted the question on this topic when it came up in the exam. The lecture was rated by the students as 'excellent'. Several requests were made to do a summer project in our lab.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015,2016,2017
 
Description Babraham Institute Annual Schools Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact As part of Annual Schools Day, 12 GCSE and A level students spent a day in our lab, doing a small project. Many asked questions about our research and careers in biology. Positive feedback relayed after the visit by teachers.

The feedback afterwards from the students was that biological research was more interesting and scientists more normal than the students expected.
Year(s) Of Engagement Activity 2010,2011,2013,2014,2015,2016,2017,2018,2019,2020
 
Description Primary School visits 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact Visits were made to a local school. 60 Year 4 to 6 primary school children attended presentations and hands-on practical related to our science.

The teachers reported an increase in understanding and interest in biological research and what real scientists do. We are frequently requested to visit.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015,2016