What is the function of antisense intergenic transcription in V(D)J recombination?

Lead Research Organisation: Babraham Institute
Department Name: Nuclear Dynamics

Abstract

One of the ways in which the immune system fights infections is by using B lymphocytes, white blood cells that make antibodies. These antibodies attack and remove the many foreign agents such as bacterial toxins that the body encounters. Since there are literally millions of different possible invading proteins that the immune system may have to deal with, B cells of higher species, including mice and man, have evolved a way of making millions of different antibodies. They are made by cutting and pasting together one each of three different kinds of gene segments: V, D and J, to make an immunoglobulin protein. There are several D and J genes and 200 V genes, thus many different combinations can be made and this process, called V(D)J recombination, together with other associated processes, ensures that the immune system produces a sufficient diversity of antibodies to fight infection. The enormous DNA locus that contains all of these genes must be kept shut down in most cells since the DNA cutting and pasting involved can be very damaging to cells because the gene segments can paste to DNA sequences on other chromosomes by mistake, leading to chromosomal translocations that can cause cancerous B cell lymphomas. Equally it must be opened up efficiently in B cells to allow the cutting and pasting enzymes access to all the genes to generate a diverse repertoire. If some of the genes are not opened up, this can lead to immunodeficiency since there is a limited choice of gene segments to make antibodies. We have discovered that just before V(D)J recombination, RNA transcripts are made through the large antibody DNA locus. They are highly unusual because they do not make protein and they are made from the opposite strand of DNA to the genes themselves. It has recently been shown by genome mapping studies that this type of 'opposite strand' or 'antisense' transcription occurs throughout the genome, but its function is unknown. The key aim of our work is to discover the function of this non-coding RNA transcription in V(D)J recombination. This will also contribute to our understanding to its role in the rest of the genome. The only way to do this unambiguously is to stop this transcription in mouse B cells to determine what effect it has on V(D)J recombination and production of antibodies, and also how it achieves such effects. We plan to do this in a mouse model, since all the processes associated with making antibodies are very similar in mouse and human B cells. We have developed a technique to visualise by fluorescent microscopy what happens at each DNA locus in individual B cells. Overall this work will tell us what function this large-scale non-coding RNA transcription has in V(D)J recombination. This work will (i) help us to understand how B cells make antibodies and (ii) may also identify molecules or processes that are involved in human disease, such as immunodeficiency and lymphomas. Further studies would then be possible to develop diagnostic tests and treatments for these diseases. This research will contribute to the BBSRC's aims of understanding fundamental mechanisms of gene regulation and normal healthy development, and of improving quality of life.

Technical Summary

V(D)J recombination, the process by which multiple gene segments are recombined together to generate a diverse antigen receptor repertoire, is tightly regulated by differential chromatin opening. How this is achieved in these large multigene loci is poorly understood. We have discovered that antisense intergenic transcription occurs throughout the immunoglobulin heavy chain (Igh) locus in regions poised for V(D)J recombination. We have proposed that this transcription remodels chromatin for VH-to-DJH recombination. The aim of this project is to test this hypothesis by asking: What is the function of antisense intergenic transcription in V(D)J recombination? Our objectives are: (1) To interrupt antisense intergenic transcription in the mouse Igh DJ region in vivo to determine whether it is required for Igh D to J recombination. (2) To analyse the effects of inhibiting transcription on events preceding D to J recombination, to determine how antisense transcription achieves its effects on D to J recombination. (3) To remove antisense transcripts produced prior to D to J recombination, in vitro, to determine the role of these transcript products in D to J recombination separately from the process of transcription. We will achieve these objectives firstly by 'knocking-in' transcription termination cassettes into the DJ region , downstream of two major antisense transcription start sites we have identified. We will determine the effects of transcription loss on D to J recombination using RNA-FISH analysis of primary transcripts in single cells, and DNA-FISH and PCR-based V(D)J recombination assays. If loss of transcription inhibits D to J recombination, we will analyse associated processes including nuclear relocation and histone modification, by DNA-immunoFISH and CHIP, respectively, to determine how antisense transcription exerts its effects. Similar strategies will be used to determine the effects of in vitro loss of antisense transcripts.
 
Description We have removed a non-coding RNA transcript which we had previously discovered from the mouse immunoglobulin heavy chain locus D region by gene-targeting and have determined the effect of this deletion on D to J recombination.
Exploitation Route Our findings suggest that the non-coding transcription that we were studying is normally required for D to J recombination. Others have integrated these findings with other information to build a model of the epigenetic features that contribute to D to J recombination.
Sectors Healthcare

 
Description To increase understanding of VDJ recombination
Sector Healthcare
 
Description NIH 
Organisation National Institutes of Health (NIH)
Country United States 
Sector Public 
PI Contribution The postdoc on the BSBRC grant visited the NIH lab to do experiments for a publication and teach them RNA FISH
Collaborator Contribution Provided mouse models
Impact Publication in Cell 2008
Start Year 2008
 
Description Babraham Institute 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 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
 
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