Mechanisms of programmed DNA lesions and genomic instability in the immune system

Abstract

Chromosomal translocations involving antigen receptor loci are common in lymphoid malignancies. Translocations require DNA double-strand breaks (DSBs) at two chromosomal sites, their physical juxtaposition, and their fusion by end-joining. Ability of lymphocytes to generate diverse repertoires of antigen receptors and effector antibodies derives from programmed genomic alterations that produce DSBs. We discuss these lymphocyte-specific processes, with a focus on mechanisms that provide requisite DSB target specificity and mechanisms that suppress DSB translocation. We also discuss recent work that provides new insights into DSB repair pathways and the influences of three-dimensional genome organization on physiological processes and cancer genomes.

Figure 1. Repair of RAG-induced Antigen Receptor Locus DSBs by Classical Non-Homologous End Joining

(A) RAG1 and −2 (yellow and orange ovals) are targeted to participating gene segments in the context of the 12/23 rule. Triangles represent 12-RSSs (blue) and 23RSSs (white) and boxes represent potential coding segments. (B) RAG holds cleaved hairpin coding and blunt RSS ends in a post-cleavage synaptic complex (PSC) and (C) directs the reaction into C-NHEJ initiated by Ku70 and Ku80 (dark and light purple ovals) binding. (D) Coding ends require processing and N regions can be added by TdT (grey oval) whereas (E) RSS ends are directly ligated by the XRCC4 (yellow oval)/Lig4 (red oval) complex to form coding and RSS joins, respectively. Functional redundancy of DDR and XLF in this reaction is indicated by ATM, 53BP1 and H2AX ovals separated by a line from an XLF oval. Specifically, coding joins are modestly impaired in the absence of the ATM and 53BP1 DDR factors and normal in the absence of the H2AX DDR factor or XLF C-NHEJ factor. However, coding joins are severely impaired in the combined absence of XLF and any one of the three DDR factors. RSS joins are normal in the absence of any one of the DDR factors or XLF but severely impaired in the absence of ATM or 53BP1 (H2AX was not tested) and XLF. See text for details.

Figure 2. Regulation of V(D)J Recombination during B Cell Development

A) B cell development is directed by ordered assembly of IgH and IgL genes coupled with feedback mechanisms linking IgH and IgL expression to developmental progression (see text for details). B) During the D to J_H rearrangement stage in early pro-B cells, prevention of premature, un-ordered proximal V_H rearrangements to germline Ds requires the two IGCR1 CBEs, which may functionally segregate the D and J_H portion of the locus (blue rectangle). Gene segments and elements are indicated with the most proximal V_H segment (V_H81X) shown as a black rectangle. CBEs are indicated as extended pink arrowheads with vertical directi indicating relative sequence orientation. Known, robust germline V_H transcription is indicated by thick green line. while very low level germline V_H transcription is indicated by a thin dotted green line. Known or hypothesized iEμ-mediated transcriptional activation is indicated by red arrows. C) In early pro-B cells in whch the IGCR1 CBEs are inactivated (red crosses), the functional segregation of the D and J_H portion of the IgH locus extends to the proximal V_Hs, deregulating their transcription and rearrangement, especially V_H81X. Distal V_Hs are physically unavailable for recombination in these cells due to lack of locus contraction. D) In the late pro-B stage subsequent to DJ_H rearrangement, IGCR1 CBE activities must be neutralized (grey box) to allow V_H segments to enter into V_H to DJ_H “recombination centers” (also indicated by blue rectangles). IgH locus contraction occurs at this stage to bring distal V_Hs closer to already recombined DJ_Hs. At this stage both proximal (left) and distal (right) V_Hs may enter into a recombination center, drawn in this case as occurring in two individual cells although this process has not yet been dissected at this level. Red question marks indicate other speculated aspects of the process that have not been tested experimentally. See text for more details and references.

Figure 3. AID Targeting during SHM and IgH CSR

A) Organization of an expressed IgH locus with general location of V(D)J exon, iEμ, 3'IgHRR (3'RR) and C_Hs, S regions and I-region promoters (line/arrow). B) During SHM in GC B cells, AID targets transcribed IgH V(D)J exons leading to mutations (red circles) but does not necessarily target downstream S regions. C) During CSR, in activated B cells in culture, AID targets transcribed S regions leading to somatic mutations and DSBs (jagged gaps) but not adjacent V(D)J exons. AID-initiated DSBs in S regions can be joined to form intra-S deletions (ISDs) (truncated S region oval with black center) or CSR events (fused S region ovals). CSR is thought to occur by a deletional mechanism with intervening sequences deleted on an excision circle. D) Working model for transcriptional AID targeting (adapted from Basu et al., 2011). “U” refers to deaminated cytidines; RPA to replication protein A) and RNAP II to RNA Polymerase. See text for other details.

Figure 4. Pre-existing Spatial Organization of the Genome Influences Translocations

A) Cellular heterogeneity of spatial genome organization allows frequent DSBs to drive recurrent translocations. Two genomic loci (a, b) on heterologous chromosomes are illustrated as red and blue circles; white lines within them indicate DSBs; stars indicate translocations. fDSB indicates frequency of available DSBs at a given site; fsyn represents frequency of cells in the population in which two DSBs are physically juxtaposed. Comparison of left and right panels illustrates that frequent DSBs can drive recurrent translocations of two sequences that are, on average, not the most proximal in a population. B) Pre-existing spatial proximity markedly influences translocation frequency in the absence of dominant DSBs. Left, schematic of two experimental examples (Zhang et al., 2012) illustrates that a targeted DSB (gap after red arrowhead) in a given chromosome translocates much more frequently along the same chromosome than to other chromosomes. Right, translocation frequencies from a DSB on a given chromosome in ATM-deficient, IR-treated, and G1-arrested transformed pro-B lines are highest to sequences very proximal to the DSB. The curves are based on actual data (from Zhang et al., 2012), shown in schematic form on the left panel, and represent inversional cis translocations from the DSB site, with primer orientation indicated by red arrowhead on chromosome ideogram. Inversional translocations (IT) clearly involve joining of two DSBs in this assay and, thus, are distinguishable from resections that also contribute substantially to breaksite-proximal junctions (Chiarle et al., 2012). Black * indicates end of Chr.2. Mb, megabases. Cen and Tel refer to “centromeric” and “telomeric” relative to the DSB. See text for more details.