Abstract
A functional adaptive immune system must generate enormously diverse antigen receptor (AgR) repertoires from a limited number of AgR genes, using a common mechanism, V(D)J recombination. The AgR loci are among the largest in the genome, and individual genes must overcome huge spatial and temporal challenges to co-localize with optimum variability. Our understanding of the complex mechanisms involved has increased enormously, due in part to new technologies for high resolution mapping of AgR structure and dynamic movement, underpinning mechanisms, and resulting repertoires. This review will examine these advances using the paradigm of the mouse immunoglobulin heavy chain (Igh) locus. We will discuss the key regulatory elements implicated in Igh locus structure. Recent next generation repertoire sequencing methods have shown that local chromatin state at V genes contribute to recombination efficiency. Next on the multidimensional scale, we will describe imaging studies that provided the first picture of the large-scale dynamic looping and contraction the Igh locus undergoes during recombination. We will discuss chromosome conformation capture (3C)-based technologies that have provided higher resolution pictures of Igh locus structure, including the different models that have evolved. We will consider the key transcription factors (PAX5, YY1, E2A, Ikaros), and architectural factors, CTCF and cohesin, that regulate these processes. Lastly, we will discuss a plethora of recent exciting mechanistic findings. These include Rag recombinase scanning for convergent RSS sequences within DNA loops; identification of Igh loop extrusion, and its putative role in Rag scanning; the roles of CTCF, cohesin and cohesin loading factor, WAPL therein; a new phase separation model for Igh locus compartmentalization. We will draw these together and conclude with some horizon-scanning and unresolved questions.
Highlights
Reviewed by: Michael Steven Krangel, Duke University Medical Center, United States Richard L
The immunoglobulin heavy chain (Igh) locus undergoes large-scale contraction to bring VH and DH genes in spatial proximity for VH to DJH recombination [29, 30, 34]. This was measured by a significant reduction in the distance between FLUORESCENT IN SITU HYBRIDIZATION (FISH) probes positioned on opposite ends of the locus in pro-B cells compared with pre-pro-B cells
Enormous progress has been made in three key areas: description of Igh locus conformation; understanding the underlying mechanisms that organize the locus; understanding how the RAG recombinase achieves recombination signal sequences (RSSs) synapsis
Summary
V(D)J recombination is catalyzed by the recombinase complex, comprising recombination-activating genes 1 and 2 (RAG1-RAG2), that cleaves recombination signal sequences (RSSs) flanking antigen receptor (AgR) genes [1]. The Igh locus undergoes large-scale contraction to bring VH and DH genes in spatial proximity for VH to DJH recombination [29, 30, 34] This was measured by a significant reduction in the distance between FISH probes positioned on opposite ends of the locus in pro-B cells compared with pre-pro-B cells. DNA looping was subsequently visualized with three-color FISH, which showed that distal VH genes frequently loop closer to the CH region than proximal VH genes that are closer in linear sequence [33] These early FISH studies introduced pro-B cells lacking the RAG recombinase as a model to study Igh locus interactions. It was proposed that the DHJH region resides in a cavity surrounded by distant VH genes and that the viscous nuclear environment causes VH genes to bounce back and forth rapidly until a specific synapsis is established by the RAG machinery, aligning with the equal opportunity model above [37]
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