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Mutations in the Insulator Protein Suppressor of Hairy Wing Induce Genome Instability

Shih-Jui Hsu, Emily C. Stow, James R. Simmons, Heather A. Wallace, Andrea Mancheno Lopez, Shannon Stroud, Mariano Labrador-San Jose

Preprint posted on 15 February 2019 https://www.biorxiv.org/content/10.1101/551002v1

Article now published in Chromosoma at http://dx.doi.org/10.1007/s00412-020-00743-8

Arrested oogenesis, eggshell patterning defects, and DNA damage accumulation by replication stress, oh my! Drosophila’s Suppressor of Hairy Wing shows that insulator proteins are not just for transcription anymore.

Selected by Maiko Kitaoka

Background

Chromatin insulators are long-range regulatory elements that mediate chromosomal contacts. These contacts allow for proper control of gene expression during development and cellular differentiation, generally preventing enhancer-promoter communication and protecting genes from silencing by heterochromatic mechanisms. These functions are mediated by specific insulator proteins, which bind the insulator DNA sequence to facilitate these DNA-DNA contacts. Insulators and their corresponding proteins have thus been implicated in 3D genome architecture, but their precise roles in mediating genome organization or integrity have not been explored.

Suppressor of Hairy wing (Su(Hw)) binds to the gypsy retrotransposon insulator in Drosophila and, while mutations in insulator proteins are usually lethal, Su(Hw) loss is remarkably not essential for viability. It’s well-known that homozygous females lacking su(Hw) are sterile since oocyte development is arrested midway through oogenesis, but the mutant phenotype has several complex components that have not been analyzed fully. In this preprint, Hsu et al further investigate these defects in oogenesis and define a more detailed role for Su(Hw) that protects the genome from excessive accumulation of DNA damage and instability.

Key findings

The authors first observed that su(Hw) mutants had irregular numbers of nurse cells in egg chambers, which were the result of incomplete or extra cell divisions or egg chamber fusions. Abnormal nurse cell number is also seen in mutants with aberrant piRNA pathway components, as well as proteins that position the microtubule organizing center (MTOC). The MTOC is critical for determination of oocyte polarity and localization of maternal mRNAs, and overall microtubule organization is important for oogenesis. Su(Hw) mutants have weaker, disorganized microtubules, suggesting that loss of Su(Hw) impairs MTOC formation and cannot form the proper microtubule network necessary to allow for egg chamber development through oogenesis. In addition, gurken (Grk) is also mislocalized in these mutants. Normally, Grk is transported along microtubules to the posterior oocyte to allow for dorsoventral axis determination before translocating to the anterior-dorsal corner of the oocyte. However, su(Hw) mutants show failure to translocate Grk, eventually leading to dorsoventral patterning defects in eggshells.

The presence of disorganized microtubules and mislocalized Grk is reminiscent of spindle genes, where cells have excessive DNA double stranded breaks (DSBs) and trigger the ATR/Chk2 DNA damage signaling pathway. The authors show that gH2Av, which marks DNA damage, is increased in su(Hw) mutants as well as su(Hw) and spo11 double mutants. Spo11 induces DSBs normally in meiosis to form crossovers and genetic recombination, so this analysis demonstrates that the DNA damage in su(Hw) mutants is not produced by the meiotic program. In addition, gH2Av persists in later stages of oogenesis in su(Hw) mutants, pointing to an accumulation of non-meiotic DNA damage. Interestingly, this accumulation of DNA damage is not due to overexpression of transposable elements in su(Hw) mutants.

DNA damage (marked by Drosophila gH2Av) persists through oogenesis in su(Hw) mutants. Orb marks the location of the oocyte. From Figure 4.

 

The increased amount of DNA damage likely activates a DNA repair pathway or checkpoint during oogenesis. Given the phenotypic connections to the ATR/Chk2 pathway, the authors examined oogenesis in su(Hw) and ATR double mutants. The loss of ATR partially recovered oocyte development with proper Grk localization and enlargement of the oocyte, suggesting that loss of Su(Hw) triggers the ATR pathway to respond to the accumulated DNA damage. Through careful double mutant analysis of chk1 and chk2 kinases downstream the ATR pathway, the authors were able to show that microtubule disorganization and Grk mislocalization are likely dependent on Chk1-mediated activity. Chk1 has been shown to respond to replication stress, so the loss of Su(Hw) is likely causing replication stress in egg chambers. H4K20 monomethylation, which is also implicated in genome instability from DNA replication mis-regulation, was also increased in su(Hw) mutant ovaries, further supporting the model that the accumulated DNA damage occurs from replication stress.

ATR or Chk1 loss can rescue Grk (green) localization and oocyte development. From Figure 6.

 

Lastly, the authors examine a somatic tissue, dividing neuroblasts, to determine whether Su(Hw) has a role in general or ovary-specific genome maintenance. Metaphase spreads from neuroblasts showed several different types of chromosomal aberrations and were less severe in heterozygotes, suggesting that Su(Hw) is important for chromosomal integrity in somatic tissue as well as female germline cells.

This comprehensive work in examining Su(Hw)’s roles in genome integrity appreciates the full complexity of the su(Hw) mutant phenotype. It’s clear that the insulator protein Su(Hw) has functions in oogenesis beyond its classical role in transcriptional regulation, providing a novel mechanistic avenue to examine genome stability through architectural proteins.

Questions for the authors

Is the DNA repair pathway compromised in su(Hw) mutants so that it also becomes inefficient and unable to keep up with the accumulating DNA damage?

The polyploidy and endoreplication of nurse cells means that some regions are underreplicated during oogenesis – are these regions in particular more damaged than other euchromatic and fully replicated regions?

Is it possible that the entire genome is more prone or susceptible to DNA damage without Su(Hw) and how might the loss Su(Hw) mediate this vulnerability?

Tags: atr, chromatin, dna damage, drosophila, fruit flies, genome instability, insulators, oogenesis

Posted on: 20 March 2019

doi: https://doi.org/10.1242/prelights.8950

Read preprint (1 votes)

Author's response

Mariano Labrador-San Jose shared

Is the DNA repair pathway compromised in su(Hw) mutants so that it also becomes inefficient and unable to keep up with the accumulating DNA damage?

ML: We think lack of su(Hw) is the main source of DNA damage. However, it is certainly possible that insulator proteins such as Su(Hw) may play a role in DNA damage repair. Mammalian CTCF has been found to play a direct role in damage repair1 and my lab is currently exploring the possibility that DNA repair pathways may also be impaired in su(Hw) mutants.

The polyploidy and endoreplication of nurse cells means that some regions are underreplicated during oogenesis – are these regions in particular more damaged than other euchromatic and fully replicated regions?

ML: Yes, wildtype endoreplicating cells accumulate stalled replication forks and DNA damage at the borders of underreplicated regions. Diploid cells respond to DNA damage and underreplication by activating checkpoints to repair damage before the cell cycle moves forward. Endocycling cells do not activate checkpoints because they do not undergo the complete cell cycle. Additionally, endocycling cells survive with accumulating genotoxic stress by inhibiting proapoptotic pathways2.

Is it possible that the entire genome is more prone or susceptible to DNA damage without Su(Hw) and how might the loss Su(Hw) mediate this vulnerability?

ML: Yes, this is certainly a possibility. Specifically, we observe that su(Hw) mutant diploid cells are more likely to accumulate chromosome aberrations, even with no exogenous source of DNA damage applied. Unpublished work in our lab suggests the absence of Su(Hw) causes replication stress by misregulation of the mechanisms that control the transition from early replication to late replication domains, as replication forks pass through euchromatin-heterochromatin boundaries. On average, 11.9% of cells from su(Hw)e04061 mutant brains display chromosome aberrations, indicating that checkpoint activation and DNA damage repair are overwhelmed by replication stress in a significant fraction of replicating cells (Figure 9B and C).

  1. Lang, Fengchao et al. “CTCF prevents genomic instability by promoting homologous recombination-directed DNA double-strand break repair”Proceedings of the National Academy of Sciences of the United States of America 114,41 (2017): 10912-10917.
  2. Mehrotra, Sonam et al. “Endocycling cells do not apoptose in response to DNA rereplication genotoxic stress” Genes & development 22,22 (2008): 3158-71.

 

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