Convergent Transcription at Intragenic Super-Enhancers Targets AID-Initiated Genomic Instability

Abstract

Summary Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single stranded DNA targets. While largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-Seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting “convergent” transcription arises from antisense transcription that emanates from Super-Enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells.

Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells

“…Aggregate patterns of transcription profiles showed enrichment of reads within 1.5 kb of TSSs genome-wide (Fig. S1B, Right), consistent with previously published GRO-seq results obtained in other cell types (30,31). To perform HTGTS for DSB identification, we used a Cas9:sgRNAbased approach to introduce HTGTS bait DSBs into primary NSPCs isolated from Xrcc4 −/− p53 −/− mice, as previously described (4).…”

“…To investigate further the set of transcribed genes that contained TSS-proximal junctions in NSPCs, we performed a comparative analysis of GRO-seq transcription profiles of NSPCs and those previously generated for activated B cells (31). This analysis yielded sets of genes transcribed in NSPCs but not in B cells (class 1), genes commonly transcribed in both NSPCs and B cells (class 2), and genes transcribed in B cells but not in NSPCs (class 3).…”

“…To assess whether transcription-associated DSBs occur and form translocations in NSPCs, we first identified actively transcribed genes by performing GRO-seq (8,30,31) in Xrcc4 −/− p53 −/− NSPCs. In these analyses, a Pearson correlation coefficient analysis showed high reproducibility among three independent experiments (Fig.…”

“…Three biological replicate GRO-seq libraries per genotype (ATM −/− R26 I-SceI-GR c-myc 25xI-SceI or Xrcc4 −/− p53 −/− ) were prepared and analyzed as previously described (4,30,31). In brief, Bowtie2 (40) was used to align GRO-seq data to the mouse genome (mm9/NCBI37), HOMER (41) was used for de novo identification of transcripts, and gene transcription rates were estimated as described (31).…”

Highly sensitive and unbiased approach for elucidating antibody repertoires

“…LAM-HTGTS, like its predecessor HTGTS (17), employs a single primer for a DSB-associated bait sequence to perform linear amplification across bait-prey junctions to identify all prey sequences joined to the bait DSBs in an unbiased manner (16,18). We have used various types of DSBs as bait for LAM-HTGTS, including those generated by engineered nucleases and endogenous DSBs (17)(18)(19)(20)(21)(22). Because V(D)J recombination generates rearrangements with junctions at borders of V, D, and J segments, we can use primers for any of these gene segments as LAM-HTGTS bait to identify sites of RAG-generated DSBs, both in progenitor or precursor lymphocytes undergoing V(D)J recombination, as well as in mature lymphocytes to retrospectively identify V(D)J recombination events that occurred earlier in development.…”