Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells.

Lyne Khair,Carol E Schrader,Richard E Baker,Erin K Linehan,Janet Stavnezer

doi:10.1371/journal.pgen.1005438

Lyne Khair, Carol E Schrader + Show 3 more

Open Access

https://doi.org/10.1371/journal.pgen.1005438

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Journal: PLoS genetics	Publication Date: Aug 11, 2015
Citations: 95	License type: CC BY 4.0

Affiliation: University of Massachusetts Chan Medical School

Abstract

Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.

Highlights

Activation-induced cytidine deaminase (AID) is required for initiation of somatic hypermutation (SHM) of Ig variable region genes and class switch recombination (CSR) of IgH genes in B cells during an immune response [1,2]
The immunoprecipitated samples were first evaluated by quantitative PCR (Fig 1A), which shows that Nbs1 binds to Sμ but not Cμ in WT B cells induced to switch, and does not bind to Sμ or Cμ in aid-/- cells
The findPeaks program from the Homer suite [43] was used to identify regions enriched in the WT chromatin immunoprecipitation (ChIP) relative to the aid-/- ChIP, and an ad hoc filtering scheme was applied to eliminate peaks with low tag numbers and/or low WT: aid-/- enrichment. 801 and 284 AID-dependent Nbs1-binding sites were identified in experiments (Exps) 1 and 2, respectively (S1 and S2 Tables)

Summary

Introduction

Activation-induced cytidine deaminase (AID) is required for initiation of somatic hypermutation (SHM) of Ig variable region genes and class switch recombination (CSR) of IgH genes in B cells during an immune response [1,2]. Both SHM and CSR are required for effective humoral immune responses, and humans (and mice) lacking AID are severely immunocompromised. If SSBs on opposite strands are sufficiently near each other, they form a double-strand break (DSB) If they are farther apart, they can still generate DSBs with the help of the mismatch repair (MMR) system, after recognition of a dU:dG mismatch by Msh2-Msh, followed by excision of one strand from a nick created by Ape1/2 [5].

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