Which chromatin modifications regulate the initiation of V(D)J recombination?

To be able to combat a vast number of potential infections, we need to produce a highly diverse set of antibody and T cell receptor molecules. Millions of antibodies are needed and if every cell carried a distinct gene to encode each of these antibodies, thousands of megabases of DNA would be required. Instead, antibody genes are assembled de novo from individual gene segments by a process known as V(D)J recombination: One gene segment is randomly chosen from a vast pool and joined to a gene segment from a separate pool. The huge number of different combinations results in an enormous number of antibody genes. Although this strategy has its advantages, it also carries dangers: DNA must be broken and rejoined and mistakes in this process can result in the wrong pieces of DNA being rejoined. In the most extreme cases, DNA from different chromosomes becomes joined, resulting in a chromosome translocation. The latter are a major cause of leukaemias and lymphomas.

This research aims to understand how V(D)J recombination is regulated in normal individuals to then understand how mistakes lead to leukaemias and lymphomas. More specifically, it aims to determine how the DNA breaks are targeted. This knowledge will then enable us to determine which environmental factors increase the risk of breaks at inappropriate sites in the genome that can trigger leukaemias and lymphomas.

To date, we know that sequences must be made accessible for them to be cut: DNA in the nucleus exists in a highly packaged structure, known as chromatin; this protects DNA from the initiation of recombination. However, in response to the appropriate signals, this chromatin packaging is specifically unravelled to allow recombination to occur. Sequences, known as enhancers, are thought to be important in controlling chromatin unpackaging; they regulate transcription that passes through the gene segments used in recombination. Transcription results in physical changes in chromatin by modifying the packaging proteins. This project aims to understand whether transcription itself, the modifications that it induces, or the modifications induced by enhancers target DNA breaks for the initial stages of recombination. Once the modifications that target DNA breaks are known, we will be able to determine which factors increase the risk of DNA breaks occurring in inappropriate regions of the genome and thus gain a better insight into the risk factors for leukaemias and lymphomas.

V(D)J recombination allows vertebrates to generate a highly diverse set of immunoglobulin and T cell receptor genes to combat a vast range of infections. However, since the reaction introduces breaks into DNA, it also poses a major risk to genome stability (reviewed by Roth). One way in which it causes genome instability is by mis-targeting the recombinase to cryptic recombination signal sequences (RSSs) in the genome, leading to illegitimate recombination and chromosome translocations. Hence, it is imperative that recombinase cutting is targeted to the appropriate antigen receptor loci and not to the estimated 10 million cryptic RSSs in the rest of the genome. This project addresses the fundamental question of how this targeting is brought about.
Chromatin modifications play a central role in targeting recombinase cutting but exactly which chromatin modifications are required is poorly understood. Some activating chromatin modifications are present at antigen receptor loci prior to recombination; in addition, recent studies showed that sterile transcription through the RSS is essential to trigger recombination (Abarrategui and Krangel).
We have developed a unique model system to determine which chromatin changes regulate recombinase cutting. Crucially, we found that over-expression of a single transcription factor, PIP, in pro-B cells is sufficient to trigger full recombination of the lambda light chain locus. Here, we propose to generate transgenic mice that express an inducible form of PIP in pro-B cells. This will allow us to turn on lambda recombination in ex-vivo pro-B cell cultures and, by inhibiting specific chromatin changes, we will be able to directly determine which chromatin modifications are essential for recombinase cutting.
Thus, our specific objectives are to:
1) Generate transgenic mice that over-express inducible PIP in pro-B cells.
2) Determine which chromatin modifications prime the lambda locus for recombination prior to sterile transcription.
3) Use our inducible system to address which of the chromatin changes triggered by sterile transcription are essential to activate recombination by asking:
a) Whether nucleosome remodelling associated with passage of the polymerase alone is sufficient to trigger initiation of recombination or
b) Whether the histone tail modifications caused by transcription are vital to allow recombinase cutting.
4) Investigate how the key chromatin changes for recombination are regulated.

By knowing which combination of chromatin modifications control the initiation of recombination, these studies will help to determine how mis-targeting of the recombinase to cryptic RSSs can occur, leading to leukaemogenic translocations.