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Transcription versus recombination in the generation of antibody diversity

The huge diversity of antibodies is primarily generated through a process called V(D)J recombination, which involves DNA breaks/fusions of distant gene segments on the chromosome. V(D)J recombination is initiated by a protein complex called RAG. The prevailing model stipulates that transcription of the gene segments is required for their recombination. In a study published in Nucleic Acids Research, The team of Ahmed Amine Khamlichi at the IPBS showed that recombination could occur in the absence of transcription, and that chromatin remodeling was sufficient to recruit the RAG complex and initiate recombination.

 

B lymphocytes play a crucial role in adaptive immunity through their exclusive capacity to produce antibodies. The antibody heavy and light chain loci undergo a complex and ordered series of DNA breaks/fusions called V(D)J recombination that enables a combinatorial assembly of different gene segments, ultimately leading to the genes that encode the antibody chains (see figure). 


DNA breaks at different gene segmenst are initiated by a protein complex called RAG made up of two subunits, RAG1 and RAG2. The prevailing model posits that transcription of gene segments (and specific associated epigenetic modifications) enables the recruitment of RAG2 first, followed by the assembly of the complex, and initiation of DNA cleavage thanks to the catalytic activity of the RAG1 subunit. 
The RAG complex is preferentially recruited to a small region called « recombination centre ». The centre is highly enriched in transcriptional activity and epigenetic modifications that render gene segmenets readily accessible to the RAG complex. The concept of accessibility was proposed in the mid-eighties based on the finding that transcription of certain gene segments of the immunoglobulin heavy chain (IgH) locus coincided with their recombination. This suggested that transcription was part of the regulatory mechanisms that control accessibility of the different gene segments. 


Subsequent studies on the role of transcription in accessibility led to conflicting conclusions depending on the system used (in vitro, transfected or transgenic substrates…). Thus, whether transcription is the causal factor of accessibility or whether it is a by-product of other processes that generate accessibility to the RAG complex is still unanswered. To make matters worse, deletion of the endogenous enhancers that control the transcriptional and epigenetic events in the recombination centres inhibits both transcription and recombination.   

 

Oudinet et al. adopted an alternative approach. They inserted a specific sequence upstream of the enhancer of the IgH recombination centre (called Eµ enhancer), leaving intact this element. They showed that the inserted sequence acted as a « transcriptional insulator » that blocked interactions between Eµ enhancer and its target promoters, leading to transcription inhibition within the recombination centre.  
Through a combination of genetic, functional and mechanistic studies, notably by analyzing transcription and recombination at the single-cell level, Oudinet et al. showed that recombination could occur in the recombination centre in the absence of transcription, and that chromatin remodeling (which also depends on Eµ enhancer) remained intact. Moreover, they showed that RAG1 subunit was efficiently recruited to the recombination centre in the absence of transcription and of RAG2.  
The authors propose the following model : Eµ enhancer controls the recruitment of the RAG complex into the endogenous recombination centre through at least two interwoven though distinct routes, chromatin remodeling and transcription. Each mechanism would determine which of RAG1 or RAG2 binds first. Chromatin remodeling may be more important for RAG1 binding first, whereas transcription will favor recruitment of RAG2.

 

These results may put an end to a lasting controversy and open the path to the identification of the precise enhancers’ motifs which control transcription and recombination at antigen receptor loci in B and T lymphocytes. They should shed new lights on the mechanisms involved in RAG-mediated chromosomal translocations associated with V(D)J recombination that potentially lead to leukemia and lymphoma, and more generally in RAG-mediated genomic instability in B and T lymphocytes.

Figure : Scheme of the mouse IgH locus. The locus divides into the varible region and the constant region. V(D)J recombination targets the variable region which contains multiple clusters of gene segments : V (variable), D (diversity) and J (junction) segments. At the IgH locus, V(D)J recombination occurs in two steps, both catalyzed by the RAG complex (indicated by scissors) : In the first step, one of the D segments recombines with one of the J segments (D-J recombination). In the second step (not shown here), one of the V segments recombines with the preformed DJ segment (V-DJ recombination). The recombination centre (RC) spans the J cluster and the most distal D segment. Eµ enhancer (purple oval) is located just downstream of the RC. The blue arrows indicate different sense and anti-sense transcripts. The green arrows indicate epigenetic modifications associated with transcription within the RC. The right panel summarizes the approach taken in this work. The stop signal indicates the insulator inserted between Eµ enhancer and the RC. The insertion inhibits sense and anti-sense transcription across the D and J clusters and associated epigenetic modifications, but not D-J recombination. 


Reference:

Oudinet C, Braikia FZ, Dauba A, Khamlichi AA. Recombination may occur in the absence of transcription in the immunoglobulin heavy chain recombination centre. Nucleic Acids Res. 2020 Feb 22. pii: gkaa108. doi: 10.1093/nar/gkaa108. PMID: 32086526 

Contacts:

Researcher IPBS : Ahmed Amine Khamlichi | Ahmed.Khamlichi@ipbs.fr | 05 61 17 55 22
Press IPBS : Francoise Viala | Communication@ipbs.fr | 06 01 26 52 59