Alternative end-joining results in smaller deletions in heterochromatin relative to euchromatin

Reviewer #1 (Public Review):

Summary:
In this study, the authors investigated the mechanisms to repair DSBs induced in euchromatic (Eu) or heterochromatic (Het) contexts in Drosophila. They used a previously described reporter construct that can be used to differentiate between HR, SSA, and mutagenic end joining in response to an I-SceI-induced DSB. Different sub-pathways of end joining (NHEJ, MMEJ, and SD-MMEJ) could be further distinguished by DNA sequence analysis. The main findings of the study are: (1) HR repair is more frequent in Het than in the Eu context; (2) mutagenic EJ repair is more frequent than HR in both contexts; (3) sub-pathways of mutagenic EJ are variable even within the same chromatin domain; and (4) SD-MMEJ repair is associated with larger deletions in the Eu than within the Het compartment.

Strengths:
Overall, the study is well designed and the use of the Bam promoter to drive I-SceI removes some of the variability observed in previous studies. Importantly, the observation of different repair outcomes using the same reporter integrated at different genomic sites suggests that repair is influenced by chromatin state in addition to local DNA sequence context.

Weaknesses:
The main concern I have is the use of only one Eu site versus four for the Het insertions. Given the variability observed between the Het insertions, analysis of a second Eu insertion would give more confidence that the differences observed are significant. One puzzling finding is that HR is increased when the reporter is inserted within the Het domain relative to the Eu domain, suggesting more end resection, yet deletions are smaller for the Het insertions. Bright Ddc2/ATRIP focus formation at DSBs induced in the Het domain is consistent with extensive end resection in this compartment. The authors speculate that this finding could indicate differences in the density of RPA loading or recruitment of Pol theta near ends. I recognize that measuring RPA density on single-stranded DNA would be extremely challenging, but is it known if Pol theta is recruited to DSBs within the Het domain before they move to the periphery?

Reviewer #2 (Public Review):

Summary:
The authors seek to vary the integration site of a double-strand break repair reporter and assess how the chromatin state of different reporter integration sites impacts the contribution of various DSB repair pathways.

Strengths:
It addresses repair in vivo. The reporter improves assay reliability (relative to previous fly DSB repair substrates) by inducing I-SceI within a more narrow and well-defined expression window. The authors' characterization of the spectrum of a-EJ products by sequencing is largely rigorous and thorough, and this often difficult to communicate data is presented in a clear and easily digested manner.

Weaknesses:
The use of the single euchromatic site undercuts their ability to generalize the impact of chromatin state. This concern is minor when considering repair by HR, as repair efficiency appears to vary little when comparing repair across the 4 different heterochromatic sites. Still, it is possible the single euchromatic site they used is an outlier in its sparing use of HR. The assessment of repair by alt-EJ is more problematic, though, since the character of repair appears to vary as much across the different heterochromatic sites as it does comparing a given heterochromatic site vs. the euchromatic site. For example, focusing on their central argument (decreased deletion during SD-MMEJ at heterochromatic sites), the difference between Het2 and all other sites appears to be more dramatic than the difference between Het1 and the single euchromatic site (Figure 5A, Supp Fig 2).

Reviewer #3 (Public Review):

Summary:
In this manuscript, Chiolo and colleagues adapt a Drosophila induced-DSB repair outcome assay to the spermatogonia. In order to compare the outcomes in H3K9me-rich centromeric heterochromatin with a euchromatic site they use a cross to a silencing mutant to reveal the sequence changes in the reporter, which otherwise are not expressed. The authors corroborate that homologous recombination (HR) is up-regulated in this chromatin context, consistent with prior studies. Applying sequencing to mutagenic products the authors reveal context-dependent preferences in mutagenic end joining pathways and mechanisms, although these seem less categorical in terms of hetero- and euchromatin and instead sensitive to more subtle aspects of the local chromatin landscape. One theme, however, is that the microhomologies used for synthesis-dependent end joining are nearer to the induced DSB in heterochromatin than seen for the euchromatic DSB.

Strengths:
1. The use of the mitotically active spermatogonia and transient expression of the I-SceI to induce the DSB mitigates some caveats of prior experimental approaches including the fact that the cells are universally mitotically active. This approach also enables the outcomes to be assayed in the next generation, which is necessary for reporters expressed within heterochromatin. Thus, this is a technological tool that will be useful to other groups.

2. The observations suggest that MMEJ within heterochromatin (inferred to be Pol theta-dependent) prefers to use microhomologies close to the DSB. This suggests that either DSB end resection or RPA loading/removal is modulated by chromatin context, which is a new finding.

Weaknesses:
1. The observation that HR is preferred in heterochromatin has been documented in many prior systems.

2. Although the conclusions of the authors are well-supported by the data, the study is somewhat limited in mechanistic detail and would be strengthened by additional use of the genetic tools in the model system, particularly with regard to whether the preference for using microhomologies near the DSB in heterochromatin arises due to modulation of resection or RPA loading stability (the latter is the preferred interpretation of the authors, but goes untested). Nucleosome stability, presence of HP1, etc. seem attractive.

3. Given the variability observed for EJ pathway usage at the four heterochromatic genomic sites probed in the manuscript there is some concern that a single euchromatic site may not be sufficient for rigorous comparisons. This is particularly true because there seems to be little transcription at the "euchromatic" region (Fig. S5). Given that we do not know what matters to dictate the outcomes (epigenetic modifications and/or transcriptional status), this is concerning.

4. (Minor) Some caution should be stated in comparing the HR frequency between this system (low single digits) and prior induction/tissue systems (~20%) because the time domain of cut and repair cycles is vastly different.

5. (Minor) While there are certainly strengths to using the spermatogonia system, one also wonders if it might not have some unique biology given the importance of maintaining genome integrity in this tissue (e.g. the piRNA pathways to repress transposon mobilization). A comment on this point would be welcomed.

6. (Minor) The authors argue that alt-EJ is less mutagenic as a consequence of the observed use of microhomologues closer to the DSB, but what they really mean perhaps is that less sequence is lost? A mutagenic outcome can be equally deleterious in other cases if 1, 5, or 20+ bps are lost, depending on the context.