Double-strand breaks in facultative heterochromatin require specific movements and chromatin changes for efficient repair

Background

Every day our genome suffers a significant amount of damage, including DNA double-strand breaks (DSBs). This type of damage can be repaired by a few different mechanisms, distinguished by their ability to faithfully repair DNA and restore the sequence that existed prior to the damage. The choice of repair pathway for DNA double-strand breaks has an impact on the appearance of DNA mutations, which can be the onset of pathologies such as cancer. Of the various existing repair mechanisms, non-homologous end-joining (NHEJ) and homologous recombination (HR) are the ones most solicited by cells. DSB repair by NHEJ can be unfaithful and therefore lead to nucleotide loss, whereas HR is a faithful process that uses a homologous sequence as a template during repair (such as the sister chromatid) (Scully et al., 2019).

In recent decades, numerous scientific articles have described how repair pathways and their choice are regulated within different nuclear compartments and chromatin environments, such as transcribed genes (Long et al., 2021; Machour & Ayoub, 2020), nucleoli (Lesage et al., 2021), telomeres (Doksani, 2019), centromeres (Scelfo & Fachinetti, 2023) and constitutive heterochromatin (Kendek et al., 2021). However, it remains unclear how DSB repair mechanisms are regulated within facultative heterochromatin domains and what their consequences are for the integrity of these chromatin domains.

In this preprint (Marieke R. Wensveen et al., 2023), the Janssen laboratory has used Drosophila as a model to induce and study DSBs within euchromatin and facultative heterochromatin domains (polycomb bodies), characterized by the presence and enrichment of methylation of lysine 27 (lys27) of histone H3 (H3K27me3) (Figure 1A). More specifically, the authors integrated the DR-white reporter system (Figure 1B) (Janssen et al., 2016; Do et al., 2014) into euchromatin and facultative heterochromatin loci, in order to (1) monitor DSBs’ mobility within these domains, (2) measure the levels of H3 methylation specific to facultative heterochromatin (H3K27me3) and (3) identify the repair mechanisms at work within these domains. These approaches enabled the authors to reveal that DSBs induced within facultative heterochromatin are rapidly excluded from these domains to be repaired by homologous recombination (HR). Remarkably, the authors show that the movement of DSBs away from these domains requires the demethylation of lys27 of histone H3 orchestrated by the histone demethylase dUtx.

 

Figure 1. (A) Cartoon illustrating the models and strategies employed by the authors to study the dynamics and repair of facultative heterochromatic DSBs in Drosophila. (B) Simplified cartoon depicting the DR-white reporter system integrated in different genomic loci and its use in studying DNA repair in Drosophila tissues. (Inspired by and adapted from (Janssen et al., 2016; Marieke R. Wensveen et al., 2023))

 

Key findings

Studying DNA repair in facultative heterochromatin in Drosophila

With the aim to study the repair of DSBs in facultative heterochromatin in Drosophila tissues, the authors introduced the DR-white reporter system in two loci localized in euchromatin and three loci in facultative heterochromatin. As anticipated, heterochromatic insertions showed an enrichment for the histone mark H3K27me3 when compared to euchromatic insertions. Furthermore, monitoring γH2Aν levels by ChIP and immunofluorescence in larvae (third instar) and in imaginal discs, respectively, showed that I-SceI can induce DSBs in both euchromatin and facultative heterochromatin loci to the same extent. Finally, the analysis of the repair products by Sanger sequencing – followed by TIDE analyses – revealed that all DR-white insertions employed NHEJ (67-83%) and HR (17-37%) to the same extent.

 

DSBs induce damaged facultative heterochromatin exclusion from polycomb bodies and lead to H3K27me3 reduction

To study the dynamics of facultative heterochromatic DSBs induced by the endonuclease I-SceI or ionizing radiation, the authors performed live-cell imaging experiments during which they tracked DSB breaks using the fluorescently-tagged repair factor Mu2, which binds the histone mark γH2Aν. In a remarkable way, the vast majority of Mu2-tagged DNA breaks moved outside the polycomb bodies (facultative heterochromatin) within 5 to 10 minutes (Figure 2A). In addition, the authors found that H3K27me3 levels decrease in facultative heterochromatic breaks and that this is orchestrated by the histone demethylase dUtx (Figure 2B).

 

Figure 2. (figure panels from (Marieke R. Wensveen et al., 2023)). (A) Live-cell imaging in third instar larval disc to follow DNA breaks (Mu2-eYFP, green) induced within the DR-white reporter system integrated in facultative heterochromatin (polycomb bodies, ph-p-mCherry, purple). (B) ChIP-qPCR of H3K27me3 to monitor its levels in response to DSBs induced within the DR-white reporter system integrated within the euchromatin (light blue) or the facultative heterochromatin (purple), either in wild-type or dUtx-depleted cells.

 

dUtx-mediated H3K27me3 reduction drives facultative heterochromatic breaks movement and repair by HR

In order to assess whether the mobility of heterochromatic breaks requires chromatin changes and supports DNA repair, the author decided to follow the dynamics of the fluorescently-tagged HR repair factor ATRIP. Interestingly, ATRIP accrual to damaged chromatin occurred within the polycomb bodies. Since this factor is known to bind RPA-coated ssDNA, these findings show that early steps of HR (resection) occur within the facultative heterochromatin domains. Furthermore, while dUtx was not required for ATRIP accrual to facultative heterochromatic DNA breaks, its depletion led to reduction in DSB mobility and HR efficiency. Together these findings point to a model in which dUtx-mediated H3K27me3 reduction regulates DSB mobility outside the polycomb bodies to support DNA repair completion by HR (late steps).

 

Conclusion

In this preprint, the Janssen lab unveils for the first time the choreography of facultative heterochromatic DNA breaks within the nuclear space. They show that these domains employ both NHEJ and HR to repair DSBs. In addition, they reveal that the early steps of HR occur within the polycomb bodies while the later steps of this repair mechanism are orchestrated by dUTX-mediated H3K37me3 reduction which drives DNA breaks mobility and exclusion outside of these domains to support repair completion by HR. These findings shed light on a new mechanism specific to facultative heterochromatin which helps to safeguard the stability of our genome.

 

What I like about this preprint

This study is of great importance since it adds a missing piece to the jigsaw puzzle that aims to map the different mechanisms coordinating chromatin dynamics and DNA repair. The use of this powerful whole-animal model and the approaches described in this preprint will undoubtedly provide new insights into the understanding of genome stability within various chromatin domains and during development.

 

Questions for the authors

Q1: When analyzing the repair products after sequencing and TIDE analysis, did you find any signatures that might correspond to other DSB repair mechanisms, such as Poltheta-dependent alternative end-joining? If so, do you observe a higher frequency of these repair products in facultative heterochromatin compared to euchromatin, as has been observed in mammals?

Q2: As indicated in the introduction of your article, the establishment and maintenance of facultative heterochromatin through H3K27me3 is crucial to silence specific developmental genes throughout development. Since DSBs can occur at any stage of development, do you think that the loss of H3K27me3 levels could affect the silencing of certain genes and the development of the organism (in the event of DSBs within polycomb bodies)? Conversely, do you think H3K27me3 levels are restored following DSB repair completion?

 

References

Do, A. T., Brooks, J. T., Le Neveu, M. K., & LaRocque, J. R. (2014). Double-Strand Break Repair Assay Determine Pathway Choice and Structure of Gene Conversion Events in Drosophila melanogaster. G3, 4(3): 425–432. doi: 10.1534/g3.113.010074

Doksani. (2019). The Response to DNA Damage at Telomeric Repeats and Its Consequences for Telomere Function. Genes, 10(4), 318. https://doi.org/10.3390/genes10040318

Janssen, A., Breuer, G. A., Brinkman, E. K., van der Meulen, A. I., Borden, S. V., van Steensel, B., Bindra, R. S., LaRocque, J. R., & Karpen, G. H. (2016). A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin. Genes & Development, 30(14), 1645–1657. https://doi.org/10.1101/gad.283028.116

Kendek, A., Wensveen, M. R., & Janssen, A. (2021). The Sound of Silence: How Silenced Chromatin Orchestrates the Repair of Double-Strand Breaks. Genes, 12(9), 1415. https://doi.org/10.3390/genes12091415

Lesage, E., Clouaire, T., & Legube, G. (2021). Repair of DNA double-strand breaks in RNAPI- and RNAPII-transcribed loci. DNA Repair, 104, 103139. https://doi.org/10.1016/j.dnarep.2021.103139

Long, Q., Liu, Z., & Gullerova, M. (2021). Sweet Melody or Jazz? Transcription Around DNA Double-Strand Breaks. Frontiers in Molecular Biosciences, 8. https://doi.org/10.3389/fmolb.2021.655786

Machour, F. E., & Ayoub, N. (2020). Transcriptional Regulation at DSBs: Mechanisms and Consequences. Trends in Genetics, 36(12), 981–997. https://doi.org/10.1016/j.tig.2020.01.001

Marieke R. Wensveen, Aditya A. Dixit, Serafin U. Colmenares, & Aniek Janssen. (2023). Double-strand breaks in facultative heterochromatin require specific movements and chromatin changes for efficient repair. BioRxiv. https://doi.org/https://www.biorxiv.org/content/10.1101/2023.12.01.569547v1

Scelfo, A., & Fachinetti, D. (2023). Centromere: A Trojan horse for genome stability. DNA Repair, 130, 103569. https://doi.org/10.1016/j.dnarep.2023.103569

Scully, R., Panday, A., Elango, R., & Willis, N. A. (2019). DNA double-strand break repair-pathway choice in somatic mammalian cells. Nature Reviews Molecular Cell Biology, 20(11), 698–714. https://doi.org/10.1038/s41580-019-0152-0

 

Acknowledgements

I thank Dr. Reinier Prosée (Prelights team) for critical reading of the manuscript.

Tags: dna double-strand breaks, heterochromatin facultative, homologous recombination

Posted on: 15 December 2023

doi: https://doi.org/10.1242/prelights.36228

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