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).
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,转染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.
Oudinetet 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.
图：小鼠IgH基因座的方案。轨迹分为varible区和恒定区。V（d）J重组靶向含有基因区段的多个簇的可变区：V（可变的），d（多样性）和J（连接处）的段。在IgH基因座，V（d）J重组发生在两个步骤，这两个由RAG复合物（由剪刀表示）催化的：在第一步骤中，与J区段中的一个（d-J重组）的d段重组之一。在第二步骤（在此未示出）中，用预先形成的DJ段（V-DJ重组）的V区段重组之一。复合中心（RC）跨越歼簇和最前端d段。Eμ增强（紫色椭圆形）正好位于RC的下游。蓝色箭头表示不同的意义和反义转录。绿色箭头指示与RC内转录相关的表观遗传修饰。右侧面板总结了在这项工作中所采取的办法。 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.
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
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