Chromatin re-organisation regulates V(D)J recombination
B lymphocytes are cells of the immune system that produce antibodies (immunoglobulins) that recognize and inactivate foreign antigens such as bacteria. To cope with the enormous numbers of foreign antigens encountered in an individual's lifespan, these cells must produce millions of different antibodies. Recombination or ‘shuffling’ of genes in the immunoglobulin heavy chain (IgH) locus is the first step in generating this huge repertoire.
The IgH locus contains 200 variable (V) genes, 12 diversity (D) genes, and 4 joining (J) genes, which are recombined (cut and pasted) together in many different combinations; a D and a J gene are spliced together first, followed by a V gene (Figure 1).
Only one of each gene type is used in an individual cell, and the resulting DNA sequence codes for a unique IgH, which is expressed with an Ig light chain as a unique highly specific antibody in each cell.
Our aim is to understand the chromatin remodelling mechanisms that open up the IgH locus in B cells to enable V(D)J recombination. Its chromatin structure (DNA wrapped around nucleosomes composed of histone proteins) is highly compacted and inaccessible in non-B cells. It must be unfolded to give access to the enzymes RAG1 and RAG2 that catalyse the DNA breaks (cutting) of VDJ rearrangement. If the locus isn't opened up enough, too few antibodies are made, resulting in immunodeficiency. Conversely if it’s opened up excessively, or not closed down after recombination, inappropriate DNA breaks may occur, which may be repaired incorrectly causing B cell lymphomas.
We are particularly interested in the role of non-coding RNA in this process. Large-scale sequence analysis has revealed that the majority of RNAs in the genome are non-coding ie do not produce protein. Further, 20% of RNAs come from the non-coding strand (antisense transcripts). There is intense interest in the function of these transcripts. They may regulate activation of large chromatin domains that are differentially expressed in different cell lineages, as shown for the b-globin locus.
We and others have previously shown that non-coding RNA transcripts are generated from individual V genes prior to recombination, although it is still unknown if they play a directive role.
Recently we have shown that antisense transcription, both genic and intergenic (between genes) occurs throughout the V region prior to V to DJ recombination. It is then switched off after recombination. The transcripts appear large and may cover several genes. Our work suggests that this transcription process drives through the entire large closed V region and opens up the chromatin into a poised state to facilitate further remodelling and recombination at the V genes. It may then be downregulated to prevent inappropriate recombination.
This is the first example of antisense transcription that is not associated with transcriptional repression, and suggests antisense intergenic transcription may remodel closed chromatin to regulate recombination of other antigen receptor loci and transcription of other large multigene developmentally regulated loci. We are currently investigating how widespread this process is and how it is regulated.
Updated 22 August, 2011