Senataxin and RNase H2 act redundantly to suppress genome instability during class switch recombination
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This paper will be of interest to the audience in the fields of genome stability and B lymphocyte biology for highlighting the role of R loop metabolism in maintaining genome integrity during antigen gene diversification. Although RNA:DNA hybrids and R loops have been described at the immunoglobulin (Ig) loci long ago, their contribution to Ig heavy chain (Igh) class switch recombination and Igh locus integrity have not been fully elucidated yet. Overall, the experiments and results generally support this conclusion; however, several aspects of the model put forward are highly speculative in the current form.
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Abstract
Class switch recombination generates distinct antibody isotypes critical to a robust adaptive immune system, and defects are associated with autoimmune disorders and lymphomagenesis. Transcription is required during class switch recombination to recruit the cytidine deaminase AID—an essential step for the formation of DNA double-strand breaks—and strongly induces the formation of R loops within the immunoglobulin heavy-chain locus. However, the impact of R loops on double-strand break formation and repair during class switch recombination remains unclear. Here, we report that cells lacking two enzymes involved in R loop removal—senataxin and RNase H2—exhibit increased R loop formation and genome instability at the immunoglobulin heavy-chain locus without impacting its transcriptional activity, AID recruitment, or class switch recombination efficiency. Senataxin and RNase H2-deficient cells also exhibit increased insertion mutations at switch junctions, a hallmark of alternative end joining. Importantly, these phenotypes were not observed in cells lacking senataxin or RNase H2B alone. We propose that senataxin acts redundantly with RNase H2 to mediate timely R loop removal, promoting efficient repair while suppressing AID-dependent genome instability and insertional mutagenesis.
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eLife assessment
This paper will be of interest to the audience in the fields of genome stability and B lymphocyte biology for highlighting the role of R loop metabolism in maintaining genome integrity during antigen gene diversification. Although RNA:DNA hybrids and R loops have been described at the immunoglobulin (Ig) loci long ago, their contribution to Ig heavy chain (Igh) class switch recombination and Igh locus integrity have not been fully elucidated yet. Overall, the experiments and results generally support this conclusion; however, several aspects of the model put forward are highly speculative in the current form.
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Reviewer #1 (Public Review):
In this study Zhao et al. investigated the effect of defective R loop removal during Class Switch Recombination (CSR). Using conditional deletion of RNaseH2b in combination with a Senataxin germline knock-out, the authors showed that combined loss of these enzymes, which participate in R loop removal in mouse B cells, is accompanied by an increase of RNA:DNA hybrid formation at the Sµ region and results in AID-dependent Igh locus instability. No changes were detected in germline transcription, AID expression or recruitment, and surprisingly CSR efficiency was unaffected in these cells. Altogether, these observations led the authors to conclude that persistent R loop formation predisposes B cells to increased genome instability at the Igh locus without affecting CSR. In addition, the authors reported that …
Reviewer #1 (Public Review):
In this study Zhao et al. investigated the effect of defective R loop removal during Class Switch Recombination (CSR). Using conditional deletion of RNaseH2b in combination with a Senataxin germline knock-out, the authors showed that combined loss of these enzymes, which participate in R loop removal in mouse B cells, is accompanied by an increase of RNA:DNA hybrid formation at the Sµ region and results in AID-dependent Igh locus instability. No changes were detected in germline transcription, AID expression or recruitment, and surprisingly CSR efficiency was unaffected in these cells. Altogether, these observations led the authors to conclude that persistent R loop formation predisposes B cells to increased genome instability at the Igh locus without affecting CSR. In addition, the authors reported that ablation of Senataxin, individually or in combination with RNaseH2, correlates with an increase in insertional/deletional repair at CSR junctions at the expense of blunt joining events. Based on these findings, they suggested a potential link between AID-induced lesions in the absence of efficient R loop removal and the use of A-EJ repair during CSR.
Overall, the study contains many interesting observations in reference to AID-induced DNA damage, Igh locus instability, and S region break processing and repair under conditions of persistent R loop formation. As such, the manuscript has the potential to contribute insights to the biology of R loops' metabolism and their contribution to CSR. However, there are major conceptual and technical concerns in reference to the data and their interpretation:
Key experiments in reference to R loop formation, AID and RNA-Pol II recruitments show high inter-experimental variability. Because of this point, and the unexpected finding of increased AID-dependent Igh genomic instability and mutational load in the absence of any effect on GL transcriptional status, AID recruitment and CSR, the model put forward by the authors is speculative in its current form.
The proposed link between persistent R loop formation and insertional/deletional repair is somewhat not supported by the fact that R loop phenotype is only detected in the double-KO cells, but altered junction profiles are observed in both Setx-/- and double-KO cells.
The involvement of the A-EJ pathway is postulated only on the basis of the analysis of CSR junctions, but no evidence is provided regarding the recruitment (or lack of) of key A-EJ and cNHEJ factors. This is one of the most interesting points of the study but it has not been fully developed.
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Reviewer #2 (Public Review):
Here, the authors aim to address the role of R loops in CSR. Though implicated in CSR since decades, R loops remain enigmatic regarding their true function at the Igh locus during CSR. In particular, its role in AID targeting to S regions remains debated with no direct evidence supporting this claim. In this study, the Barlow lab sheds interesting new light on what R loops may be doing during CSR. They study the response to elevated R loop levels which they achieve using single or double KO of SETX (a helicase that can unwind R loops) and RNaseH2 (which can cleave R loops). In this system, R loop removal is deficient and the effect on CSR and genome instability can be assessed. This is a fresh approach which allows the authors to draw new insights into R loop biology. Overall, the results support the …
Reviewer #2 (Public Review):
Here, the authors aim to address the role of R loops in CSR. Though implicated in CSR since decades, R loops remain enigmatic regarding their true function at the Igh locus during CSR. In particular, its role in AID targeting to S regions remains debated with no direct evidence supporting this claim. In this study, the Barlow lab sheds interesting new light on what R loops may be doing during CSR. They study the response to elevated R loop levels which they achieve using single or double KO of SETX (a helicase that can unwind R loops) and RNaseH2 (which can cleave R loops). In this system, R loop removal is deficient and the effect on CSR and genome instability can be assessed. This is a fresh approach which allows the authors to draw new insights into R loop biology. Overall, the results support the conclusions that the timely removal of R loops is not necessary for optimal CSR but is necessary to maintain genome stability. But there are some experiments that need to be done to solidify this conclusion.
The major findings are that the increase in steady-state R loops in dKO cells does not appear to affect CSR frequency although small increase in mutation is observed. However, in dKO cells, there is a significant increase in gross chromosomal aberrations (translocations and fusions) as well as increased usage of alternative end-joining during CSR. Thus, surprisingly, increased DNA damage and increased reliance on alternative end-joining do not appear to reduce CSR which would have been expected based on many previous studies. Thus, they conclude that R loop removal by SETX and RNaseH2 is necessary to enhance the usage of classical end-joining repair pathways that are more efficient and less prone to genome instability.
The major weakness here is the lack of a proper characterization of B cell development in the mice. They use Cd19-cre which acts earlier in B cell development in the bone marrow and hence it is important to know whether B cell populations were skewed or otherwise influenced by the early KO of Setx and Rnaseh2. Along these lines, gene expression analysis is necessary to know whether the single and double KO (both naïve and activated) splenic B cells have undergone differential expression in DNA repair pathways or other pathways that could impinge upon CSR and contribute to the DNA repair phenotype they observe.
There is no western blot analysis to show how well RNASEH2 is depleted. Cd19-cre is known to have variable effects hence it is unclear whether efficient deletion was obtained in mature B cells.
One puzzling finding is that R loops were increased only in the S-mu but not the S-gamma1 region although both form R loops. Some thoughts on this would be useful for the readers since this implies that R loop resolution at S-gamma1 is independent of both enzymes.
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Reviewer #3 (Public Review):
Zhao et al. investigate how RNA:DNA hybrids/R loops that are generated during class switch recombination (CSR) due to the transcription activity at the switch regions in the IgH locus affect the outcome of CSR. Specifically, the authors used primary B cells lacking the helicase senataxin and RNaseH2 to interrogate the changes of R loop levels in the switch regions during CSR. Consistent with the known activities of these two proteins in R loop resolution, the authors find increased R loop formation in the double deficient cells. The effect of senataxin and RNaseH2 double deficiency on R loop processing appear to be restricted to the donor switch region Sm but not the acceptor switch regions. Importantly, senataxin and RNaseH2 function redundantly in resolving R loops in activated B cells as inactivation of …
Reviewer #3 (Public Review):
Zhao et al. investigate how RNA:DNA hybrids/R loops that are generated during class switch recombination (CSR) due to the transcription activity at the switch regions in the IgH locus affect the outcome of CSR. Specifically, the authors used primary B cells lacking the helicase senataxin and RNaseH2 to interrogate the changes of R loop levels in the switch regions during CSR. Consistent with the known activities of these two proteins in R loop resolution, the authors find increased R loop formation in the double deficient cells. The effect of senataxin and RNaseH2 double deficiency on R loop processing appear to be restricted to the donor switch region Sm but not the acceptor switch regions. Importantly, senataxin and RNaseH2 function redundantly in resolving R loops in activated B cells as inactivation of individual genes does not affect R loop levels. Aberrant R loop resolution has been implicated in defected DNA double strand break (DSB) repair and productive CSR involves the generation and repair of DSBs between the recombining switch regions. Surprisingly, CSR to several Ig isotypes is not affected in Setx-/-, RNaseH2b-/- and the double knockout cells when compared to WT cells. The double knockout cells, in contrast to Setx-/-, RNaseH2b-/- and WT cells, do accumulate more chromosomal abnormalities, including AID-dependent IgH DSBs. The authors went on to conduct a series to show that in the activated double knockout primary B cells, cell proliferation, germline transcription, AID expression, the association of activated RNA pol II and AID with switch chromatin all appear comparable to WT or single deficient cells; therefore ruling out that the defects in these events cause chromosomal abnormalities observed in the activated Setx-/-: RNaseH2b-/- primary B cells and consistent with normal CSR in these cells. Lastly, the authors determine the switch junction sequences and found that in the activated Setx-/-: RNaseH2b-/- primary B cells, insertions and C to T mismatches are increased, suggesting a deviation from normal DSB processing in these cells that eventually lead to increased usage of alternative end joining during CSR.
The experiments conducted are well done and support the conclusion that the loss of senataxin and RNaseH2 leads to an increase in genome stability in the setting of IgH class switch recombination. The aberrant accumulation of R loops is very subtle at the switch region in the activated Setx-/-: RNaseH2b-/- primary B cells. Could this be due to RNaseH1 activity? How do the authors reconcile the increase in un-repaired switch DSBs without an impact on IgH CSR?
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