BRD2 promotes antibody class switch recombination by facilitating DNA repair in collaboration with NIPBL.

Efficient repair of DNA double-strand breaks in the Ig heavy chain gene locus is crucial for B-cell antibody class switch recombination (CSR). The regulatory dynamics of the repair pathway direct CSR preferentially through nonhomologous end joining (NHEJ) over alternative end joining (AEJ). Here, we demonstrate that the histone acetyl reader BRD2 suppresses AEJ and aberrant recombination as well as random genomic sequence capture at the CSR junctions. BRD2 deficiency impairs switch (S) region synapse, optimal DNA damage response (DDR), and increases DNA break end resection. Unlike BRD4, a similar bromodomain protein involved in NHEJ and CSR, BRD2 loss does not elevate RPA phosphorylation and R-loop formation in the S region. As BRD2 stabilizes the cohesion loader protein NIPBL in the S regions, the loss of BRD2 or NIPBL shows comparable deregulation of S-S synapsis, DDR, and DNA repair pathway choice during CSR. This finding extends beyond CSR, as NIPBL and BRD4 have been linked to Cornelia de Lange syndrome, a developmental disorder exhibiting defective NHEJ and Ig isotype switching. The interplay between these proteins sheds light on the intricate mechanisms governing DNA repair and immune system functionality.

Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition.

Antibody class switch recombination (CSR) is a locus-specific genomic rearrangement mediated by switch (S) region transcription, activation-induced cytidine deaminase (AID)-induced DNA breaks, and their resolution by non-homologous end joining (NHEJ)-mediated DNA repair. Due to the complex nature of the recombination process, numerous cofactors are intimately involved, making it important to identify rate-limiting factors that impact on DNA breaking and/or repair. Using an siRNA-based loss-of-function screen of genes predicted to encode PHD zinc-finger-motif proteins, we identify the splicing factor Phf5a/Sf3b14b as a novel modulator of the DNA repair step of CSR. Loss of Phf5a severely impairs AID-induced recombination, but does not perturb DNA breaks and somatic hypermutation. Phf5a regulates NHEJ-dependent DNA repair by preserving chromatin integrity to elicit optimal DNA damage response and subsequent recruitment of NHEJ factors at the S region. Phf5a stabilizes the p400 histone chaperone complex at the locus, which in turn promotes deposition of H2A variant such as H2AX and H2A.Z that are critical for the early DNA damage response and NHEJ, respectively. Depletion of Phf5a or p400 blocks the repair of both AID- and I-SceI-induced DNA double-strand breaks, supporting an important contribution of this axis to programmed as well as aberrant recombination.

The dystonia gene THAP1 controls DNA double-strand break repair choice.

The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.

53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.

Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse.

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.

DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity.

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.

Collateral DNA damage produced by genome-editing drones: exception or rule?

In the recent issue of Nature Biotechnology, Frock et al. (2015) developed an elegant technique to capture translocation partners that can be utilized to determine off-target regions of genome-editing endonucleases as well as endogenous mutators at nucleotide resolution.

Identification of DNA cleavage- and recombination-specific hnRNP cofactors for activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is essential for antibody class switch recombination (CSR) and somatic hypermutation (SHM). AID originally was postulated to function as an RNA-editing enzyme, based on its strong homology with apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC1), the enzyme that edits apolipoprotein B-100 mRNA in the presence of the APOBEC cofactor APOBEC1 complementation factor/APOBEC complementation factor (A1CF/ACF). Because A1CF is structurally similar to heterogeneous nuclear ribonucleoproteins (hnRNPs), we investigated the involvement of several well-known hnRNPs in AID function by using siRNA knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated disruption. We found that hnRNP K deficiency inhibited DNA cleavage and thereby induced both CSR and SHM, whereas hnRNP L deficiency inhibited only CSR and somewhat enhanced SHM. Interestingly, both hnRNPs exhibited RNA-dependent interactions with AID, and mutant forms of these proteins containing deletions in the RNA-recognition motif failed to rescue CSR. Thus, our study suggests that hnRNP K and hnRNP L may serve as A1CF-like cofactors in AID-mediated CSR and SHM.

Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination.

Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR.

Chromatin reader Brd4 functions in Ig class switching as a repair complex adaptor of nonhomologous end-joining.

Class switch recombination (CSR) is a B cell-specific genomic alteration induced by activation-induced cytidine deaminase (AID)-dependent DNA break at the immunoglobulin heavy-chain locus, followed by repair. Although chromatin-associated factors in promoting AID-induced DNA break have been widely reported, the involvement of chromatin adaptors at the repair phase of CSR remains unknown. Here, we show that the acetylated histone reader Brd4 is critical for nonhomologous end-joining (NHEJ) repair of AID- and I-SceI-induced DNA breaks. Brd4 was recruited to the DNA break regions, and its depletion from the chromatin caused CSR impairment without affecting the DNA break generation. Inhibition of Brd4 suppressed the accumulation of 53BP1 and uracil DNA glycosylase at the switch regions, perturbed the switch junctional microhomology, and reduced Igh/c-myc translocation. We conclude that Brd4 serves as a chromatin platform required for the recruitment of repair components during CSR and general DNA damage.

Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase.

Activation-induced cytidine deaminase (AID) is essential to class-switch recombination (CSR) and somatic hypermutation (SHM) in both V region SHM and S region SHM (s-SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair (BER) complex, is required for CSR. Strikingly, however, UNG deficiency causes augmentation of SHM, suggesting involvement of distinct functions of UNG in SHM and CSR. Here, we show that noncanonical scaffold functions of UNG regulate s-SHM negatively and CSR positively. The s-SHM suppressive function of UNG is attributed to the recruitment of faithful BER components at the cleaved DNA locus, with competition against error-prone polymerases. By contrast, the CSR-promoting function of UNG enhances AID-dependent S-S synapse formation by recruiting p53-binding protein 1 and DNA-dependent protein kinase, catalytic subunit. Several loss-of-catalysis mutants of UNG discriminated CSR-promoting activity from s-SHM suppressive activity. Taken together, the noncanonical function of UNG regulates the steps after AID-induced DNA cleavage: error-prone repair suppression in s-SHM and end-joining promotion in CSR.

Accumulation of the FACT complex, as well as histone H3.3, serves as a target marker for somatic hypermutation.

Somatic hypermutation (SHM) requires not only the expression of activation-induced cytidine deaminase, but also transcription in the target regions. However, how transcription guides activation-induced cytidine deaminase in targeting SHM to the Ig genes is not fully understood. Here, we found that the "facilitates chromatin transcription" (FACT) complex promotes SHM by RNAi screening of transcription elongation factors. Furthermore, FACT and histone H3.3, a hallmark of transcription-coupled histone turnover, are enriched at the V(D)J region, 5' flanking sequence of the Sµ switch region and the light chain Jκ 5 segment region in the Ig loci. The regions with the most abundant deposition of FACT and H3.3 were also the most efficient targets of SHM. These results demonstrate the importance of histone-exchanging dynamics at the chromatin of SHM targets, especially in Ig genes.

The histone chaperone Spt6 is required for activation-induced cytidine deaminase target determination through H3K4me3 regulation.

H3K4me3 plays a critical role in the activation-induced cytidine deaminase (AID)-induced DNA cleavage of switch (S) regions in the immunoglobulin heavy chain (IgH) locus during class-switch recombination (CSR). The histone chaperone complex facilitates chromatin transcription (FACT) is responsible for forming H3K4me3 at AID target loci. Here we show that the histone chaperone suppressor of Ty6 (Spt6) also participates in regulating H3K4me3 for CSR and for somatic hypermutation in AID target loci. We found that H3K4me3 loss was correlated with defects in AID-induced DNA breakage and reduced mutation frequencies in IgH loci in both S and variable regions and in non-IgH loci such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and small nucleolar RNA host gene 3 (SNHG3). Global gene expression analysis revealed that Spt6 can act as both a positive and negative transcriptional regulator in B cells, affecting ∼5% of the genes that includes suppressor of Ty4 (Spt4) and AID. Interestingly, Spt6 regulates CSR and AID expression through two distinct histone modification pathways, H3K4me3 and H3K36me3, respectively. Tandem SH2 domain of Spt6 plays a critical role in CSR and H3K4me3 regulation involving Set1 histone methyltransferase. We conclude that Spt6 is a unique histone chaperone capable of regulating the histone epigenetic state of both AID targets and the AID locus.

The DSIF subunits Spt4 and Spt5 have distinct roles at various phases of immunoglobulin class switch recombination.

Class-switch recombination (CSR), induced by activation-induced cytidine deaminase (AID), can be divided into two phases: DNA cleavage of the switch (S) regions and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in a switch-proficient B cell line CH12F3-2A cells, and Spt4 and Spt5 carry out independent functions in CSR. While neither Spt4 nor Spt5 is required for transcription of S regions and AID, expression array analysis suggests that Spt4 and Spt5 regulate a distinct subset of transcripts in CH12F3-2A cells. Curiously, Spt4 is critically important in suppressing cryptic transcription initiating from the intronic Sµ region. Depletion of Spt5 reduced the H3K4me3 level and DNA cleavage at the Sα region, whereas Spt4 knockdown did not perturb the H3K4me3 status and S region cleavage. H3K4me3 modification level thus correlated well with the DNA breakage efficiency. Therefore we conclude that Spt5 plays a role similar to the histone chaperone FACT complex that regulates H3K4me3 modification and DNA cleavage in CSR. Since Spt4 is not involved in the DNA cleavage step, we suspected that Spt4 might be required for DNA repair in CSR. We examined whether Spt4 or Spt5 is essential in non-homologous end joining (NHEJ) and homologous recombination (HR) as CSR utilizes general repair pathways. Both Spt4 and Spt5 are required for NHEJ and HR as determined by assay systems using synthetic repair substrates that are actively transcribed even in the absence of Spt4 and Spt5. Taken together, Spt4 and Spt5 can function independently in multiple transcription-coupled steps of CSR.

An evolutionary view of the mechanism for immune and genome diversity.

An ortholog of activation-induced cytidine deaminase (AID) was, evolutionarily, the first enzyme to generate acquired immune diversity by catalyzing gene conversion and probably somatic hypermutation (SHM). AID began to mediate class switch recombination (CSR) only after the evolution of frogs. Recent studies revealed that the mechanisms for generating immune and genetic diversity share several critical features. Meiotic recombination, V(D)J recombination, CSR, and SHM all require H3K4 trimethyl histone modification to specify the target DNA. Genetic instability related to dinucleotide or triplet repeats depends on DNA cleavage by topoisomerase 1, which also initiates DNA cleavage in both SHM and CSR. These similarities suggest that AID hijacked the basic mechanism for genome instability when AID evolved in jawless fish. Thus, the risk of introducing genome instability into nonimmunoglobulin loci is unavoidable but tolerable compared with the advantage conferred on the host of being protected against pathogens by the enormous Ig diversification.

Histone3 lysine4 trimethylation regulated by the facilitates chromatin transcription complex is critical for DNA cleavage in class switch recombination.

Ig class switch recombination (CSR) requires expression of activation-induced cytidine deaminase (AID) and transcription through target switch (S) regions. Here we show that knockdown of the histone chaperone facilitates chromatin transcription (FACT) completely inhibited S region cleavage and CSR in IgA-switch-inducible CH12F3-2A B cells. FACT knockdown did not reduce AID or S region transcripts but did decrease histone3 lysine4 trimethylation (H3K4me3) at both the Sµ and Sα regions. Because knockdown of FACT or H3K4 methyltransferase cofactors inhibited DNA cleavage in H3K4me3-depleted S regions, H3K4me3 may serve as a mark for recruiting CSR recombinase. These findings revealed an unexpected evolutionary conservation between CSR and meiotic recombination.

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

Further evidence for involvement of a noncanonical function of uracil DNA glycosylase in class switch recombination.

Activation-induced cytidine deaminase (AID) introduces DNA cleavage in the Ig gene locus to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. The DNA deamination model assumes that AID deaminates cytidine (C) on DNA and generates uridine (U), resulting in DNA cleavage after removal of U by uracil DNA glycosylase (UNG). Although UNG deficiency reduces CSR efficiency to one tenth, we reported that catalytically inactive mutants of UNG were fully proficient in CSR and that several mutants at noncatalytic sites lost CSR activity, indicating that enzymatic activity of UNG is not required for CSR. In this report we show that CSR activity by many UNG mutants critically depends on its N-terminal domain, irrespective of their enzymatic activities. Dissociation of the catalytic and CSR activity was also found in another UNG family member, SMUG1, and its mutants. We also show that Ugi, a specific peptide inhibitor of UNG, inhibits CSR without reducing DNA cleavage of the S (switch) region, confirming dispensability of UNG in DNA cleavage in CSR. It is therefore likely that UNG is involved in a repair step after DNA cleavage in CSR. Furthermore, requirement of the N terminus but not enzymatic activity of UNG mutants for CSR indicates that the UNG protein structure is critical. The present findings support our earlier proposal that CSR depends on a noncanonical function of the UNG protein (e.g., as a scaffold for repair enzymes) that might be required for the recombination reaction after DNA cleavage.