Dynamic 3D Locus Organization and Its Drivers Underpin Immunoglobulin Recombination

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.

The 3D enhancer network of the developing T cell genome is controlled by SATB1

“…Since the recruitment of RAG proteins is based on the epigenetic status of a gene locus (Maman et al, 2016; Teng et al, 2015), we hypothesized that the altered TCR organization, due to missing SATB1-mediated loops, together with the disrupted recruitment of chromatin modifying complexes interacting with SATB1 (Fujii et al, 2003; Jangid et al, 2014; Kumar et al, 2005; Purbey et al, 2009; Yasui et al, 2002), would be critical contributors for the defective TCR arrangement in the Satb1 cKO. Moreover, the impact of the 3D organization of TCR and BCR, necessary for proper VDJ recombination was recently highlighted (Peters, 2021; Rogers et al, 2021).…”

High Throughput Characterization of V(D)J Recombination Signal Sequences Redefines the Consensus Sequence

“…RSS quality has been challenging to predict based on DNA sequence, since endogenous RSSs are embedded in a range of chromatin environments that can affect selectivity of individual RSSs by RAG1/2 (34,35). Examples of sequence-independent effects on RSS utilization include nucleosome density and positioning relative to the RSSs (36); the presence of certain histone marks, such as H3K4me3, that serve as a chromatin docking site for full length RAG2 (37); and 3-dimensional chromatin organization that can impact RAG1/2 access to RSSs (38).…”

The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination

“…The role of these sub-pathways in the onset of pro-B cell lymphomas in NHEJ/p53-deficient animals also remains to be investigated. Finally, antigen receptor loci relocate from the nuclear periphery to permissive euchromatin in the nuclear interior before V(D)J recombination ( Rogers et al, 2021 ). This subnuclear relocation likely provides specific local chromatin environments that might influence downstream DSB repair events ( Mitrentsi et al, 2020 ).…”

“…Contraction and spatial reorganization of antigen receptor loci during V(D)J recombination rely largely on the formation of chromatin loops through a cohesin-dependent extrusion process ( Bossen et al, 2012 ; Ebert et al, 2015 ; Ba et al, 2020 ; Hill et al, 2020 ; Dai et al, 2021 ; Davidson and Peters, 2021 ), as well as transcription and subnuclear relocation ( Rogers et al, 2021 ). Remarkably, this mechanism poises the loci for recombination independently of RAG, but also endows RSS-bound RAG with the ability to scan chromatin for a partner RSS, providing directionality and spatial restriction to RAG activity within the chromatin loop domain ( Figure 2Bi ) ( Lin et al, 2018 ; Zhang et al, 2019 ; Ba et al, 2020 ; Hill et al, 2020 ; Dai et al, 2021 ).…”