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ARID1A regulates R-loop associated DNA replication stress

Shuhe Tsai, Emily Yun-Chia Chang, Louis-Alexandre Fournier, James P Wells, Sean W Minaker, Yi Dan Zhu, Alan Ying-Hsu Wang, Yemin Wang, David G Huntsman, Peter Stirling

Preprint posted on 16 November 2020 https://www.biorxiv.org/content/10.1101/2020.11.16.384446v1

Article now published in PLOS Genetics at https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009238

R-loop travels to the ARID1A chromatin terrain.

Selected by Sree Rama Chaitanya

Background

R-loops are non-canonical nucleic acid structures that are formed during transcription when the nascent RNA hybridizes with the complementary DNA strand displacing the non-complementary strand. Unscheduled and aberrant R-loops pose a great threat to genome stability, mainly by inducing conflicts between transcription and replication complexes. Hence, to keep R-loops at physiological levels, cells deploy proteins or enzymes involved in RNA/DNA metabolism, transcription, replication, recombination, epigenetic regulation, and DNA topology1,2. However, it is not quite clear how chromatin, where all these genomic processes occur, regulates R-loop dynamics and distribution.

BAF (BRG1/BRM-associated factor or yeast SWI/SNF complex) is a chromatin remodeling complex that displaces nucleosomes using the energy from ATP. It is composed of ~14 subunits and drives gene expression of developmental pathways. BAF also plays a crucial role in DNA damage repair and in maintaining genome stability. For example, cells with mutations in the DNA binding subunit of BAF complex – AT-rich interactive domain 1A (ARID1A or BAF250) – manifest mitotic defects, unstable telomeres, and sensitivity to DNA damaging drugs. Moreover, mutations in genes of the BAF complex (like ARID1A) are often observed in many cancers3,4. Therefore, in the current study, the authors hypothesized a role of ARID1A in regulating R-loops.

Key findings

  1. The authors depleted ARID1A using CRISPR in isogenic clear cell ovarian cancer cells (RMG-1), considering the role of ARID1A in ovarian cancers4. As ARID1A depleted cells are sensitive to replication stress (using ATR inhibitor5) they evaluated replication stress markers in these cells. They found that ARID1A deficient cells manifested exaggerated levels of phosphorylated RPA2 (RPA2-S33) and MRE11-EdU signals (see6 for the technique) compared to ARID1A proficient cells.
  2. R-loop mediated transcription-replication conflicts are a major source of replication stress. Therefore, the authors tested their presence in ARID1A deficient cells. To this end, they demonstrated higher levels of nuclear R-loops in ARID1A deficient cells (by immunofluorescence of R-loops using the S9.6 antibody). They reported similar results in HCT116 and RPE1 cells transiently depleted of ARID1A. Notably, they found that ectopic expression of RNaseH1 (an enzyme to digest R-loops) suppressed the replication stress (RPA2-S33, FANCD2 immunofluorescence), aberrant R-loop levels, and genome instability (by comet assay) in ARID1A deficient cells. Furthermore, they also found higher levels of R-loops at R-loop prone gene locales in ARID1A deficient cells (by DNA-RNA immunoprecipitation followed by qPCR based technique – DRIP). Importantly, they note that ARID1A occupies the same gene locales in published datasets.
  3. Also, they found higher transcription-replication conflicts as visualized by co-localization of PCNA (replication) and RNA polymerase II (transcription) in ARID1A depleted cells (by proximity ligation assay). These conflicts were suppressed under transient inhibition of transcription (using Flavopiridol), suggesting their co-transcriptional nature. Thus, they suggest that ARID1A deficiency induces R-loop mediated replication stress and genome instability in cells.
  4. They then evaluated the global replication and transcription defects in ARID1A deficient cells. They found that ARID1A deficient cells exhibited higher levels of stalled or collapsed forks, but the replication forks seem to travel faster than control cells (by DNA fiber assay). Surprisingly, they report that transient inhibition of transcription (using Flavopiridol and Triptolide) increased the replication fork speed in ARID1A proficient cells but not in deficient cells. They also found global transcriptional inhibition in ARID1A depleted cells (via EU staining). Thus, they suggest that transcription anomalies observed in ARID1A deficient cells could impact the replication dynamics.
  5. Next, they investigated the role of Topoisomerase IIα (TOP2A), an interactor of BAF complex7 and a regulator of DNA topology induced by transcription-replication conflicts. In this regard, they demonstrated that chemical inhibition of TOP2A (using Etoposide) increased nuclear R-loops levels and RPA2-S33 in both ARID1A proficient and deficient cells. Then they found that co-localization of TOP2A with R-loops was impeded by loss of ARID1A (by proximity ligation assay). However, they found that TOP2A association with chromatin or other BAF subunits was not impacted by ARID1A loss (as assayed by biochemical fractionation and co-immunoprecipitation). Albeit, BAF complex association with nascent DNA was challenged by the loss of ARID1A (by BRG1-EdU co-staining). Interestingly, TOP2A recruitment to R-loop prone gene locales was impeded by the loss of ARID1A. Thus, the authors suggest that ARID1A could govern specific recruitment of TOP2A and BAF complex to R-loop forming gene locales.
Schematic model. Under normal conditions, ARID1A-containing BAF complexes recruit TOP2A to chromatin where it can prevent the formation of transcription-replication conflict inducing R-loops. Upon loss of ARID1A, TOP2A is not appropriately recruited to chromatin, resulting in increased R-loop burden, altered replication and transcription dynamics, increased incidence of transcription-replication conflicts, and DNA replication stress. The model figure was created with BioRender.com. Taken directly from Tsai S et. al., 2020 under a CC-BY 4.0 international license.

Conclusion and perspective

R-loops lie at the nexus of interlinked genomic processes like transcription, replication, and recombination, etc. This makes R-loops unique; because cells do not seem to have a dedicated ‘R-loop regulating system’ but rather swiftly exploits the trans-acting factors available in the local chromatin milieu. While some factors prevent aberrant R-loops by sequestering the RNA or DNA from binding with each other (e.g., splicing factors), some could resolve them by unwinding or digesting the R-loops (e.g., helicases or nucleases). Chromatin remodelers could fall into a different class of R-loop regulators. For example, the histone chaperone complex (FACT) regulates R-loops by remodeling the nucleosomes during transcription8. Maybe, ARID1A (and BAF) fits into this piece of the puzzle.

R-loops and transcription-replication conflicts are major endogenous sources of genome instability. Here, the authors report that the DNA binding subunit (ARID1A) of a multisubunit chromatin remodeler (BAF) suppresses aberrant R-loops that could otherwise trigger tumorigenic events. But how exactly ARID1A suppresses R-loops is left for future research. Understanding the design principles of R-loops is crucial to design novel therapeutic strategies for cancer or neurodegenerative diseases.

 Acknowledgments

I am thankful to all the authors for their support, especially Peter Stirling for taking the time in an often busy schedule to comment on the preLight.

References

  1. https://doi.org/10.1016/j.cell.2019.08.055
  2. https://doi.org/10.1016/j.trecan.2019.08.006
  3. https://dx.doi.org/10.1101%2Fcshperspect.a026930
  4. https://doi.org/10.1056/nejmoa1008433
  5. https://doi.org/10.1038/ncomms13837
  6. https://doi.org/10.1083/jcb.201709121
  7. https://doi.org/10.1038/nature12146
  8. https://doi.org/10.1101/gad.234070.113
  9. https://doi.org/10.1101/2020.09.01.277103
  10. https://dx.doi.org/10.1158%2F2159-8290.CD-14-0849

Tags: arid1a, chromatin, dna damage, genome instability, replication stress, rloops

Posted on: 5 December 2020 , updated on: 21 April 2021

doi: https://doi.org/10.1242/prelights.25754

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1. The authors report that loss of ARID1A leads to deleterious R-loops and downregulation of global transcription. This is conceivable considering that R-loops/DNA damage could inhibit transcription. Yet, it is contradicting considering the co-transcriptional nature of R-loops. It would be interesting to hear the authors’ comments on this.

2. Surprisingly, ARID1A KO cells survived even after accumulating transcription defects and genome instability. Do the authors know if these cells exhibit any cell cycle defects?

3. The authors show that ARID1A depleted cells have increased replication fork speed. This seems ‘similar’ to the phenotype described in a study that shows an increase in the number of ongoing forks at centromeres9. What do the authors think about this?

4. Your data suggests that ARID1A works with BAF to suppress R-loops (Fig.3E). Although speculative at this moment, how do the authors think ARID1A suppresses aberrant R-loops? (also considering7).

5. How do the authors reconcile their data considering the role of ATR and PARP inhibitors in a background ARID1A deficiency5,10?

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