The Drosophila maternal-effect gene abnormal oocyte ( ao ) does not repress histone gene expression
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Abstract
The abnormal oocyte ( ao ) gene of Drosophila melanogaster is a maternal-effect lethal gene previously identified as encoding a transcriptional regulator of core histones. However, background genetic mutations in existing ao mutant strains could compromise their utility in manipulating histone levels. To distinguish the true ao phenotype from background effects, we created two new ao reagents: a CRISPR/Cas9-mediated knockout of the ao allele for genetic and molecular analyses and an epitope-tagged ao allele for cytological experiments. Using these reagents, we confirm previous findings that loss of ao causes maternal-effect lethality, which can be rescued by either a decrease in the histone gene copy number or by Y chromosome heterochromatin. Our data indicate that ao genetically interacts with the heterochromatin, as previously suggested. However, contrary to a prior study, we find neither Ao localization to histone genes nor ao repression of core histone transcript levels. Thus, the molecular basis for ao -associated maternal-effect lethality remains unknown.
Article Summary
A series of foundational papers established that abnormal oocyte ( ao ), a euchromatic maternal-effect lethal gene, interacts with heterochromatin and the histone multigene cluster to dictate embryonic viability in D. melanogaster. An earlier report argued that ao encodes a protein that localizes to and represses histone gene expression, thereby connecting histone gene overexpression with ao mutant maternal-effect lethality. Using new reagents for genetics and cytology, we recapitulate findings that ao encodes a maternal-effect lethal gene, whose loss is ameliorated by excess heterochromatin or loss of histone genes. However, we find that ao does not affect histone gene expression. Thus, how ao loss causes maternal-effect lethality remains unknown.
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Referee #3
Evidence, reproducibility and clarity
In this manuscript, Takenaka et al revisits the role of the maternal-effect gene abnormal oocyte (ao) in the regulation of canonical histone gene expression in Drosophila. The authors generated both a knockout allele of ao and a V5-tagged ao allele using CRISPR/Cas9. They show that (i) loss of ao results in maternal-effect lethality; (ii) Ao protein is localised to histone locus bodies; (iii) an increased dosage of AO heterochromatin partially rescues maternal-effect lethality. These results are consistent with previous reports. However, this study convincingly demonstrates that ao is not required to suppress histone expression, …
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Referee #3
Evidence, reproducibility and clarity
In this manuscript, Takenaka et al revisits the role of the maternal-effect gene abnormal oocyte (ao) in the regulation of canonical histone gene expression in Drosophila. The authors generated both a knockout allele of ao and a V5-tagged ao allele using CRISPR/Cas9. They show that (i) loss of ao results in maternal-effect lethality; (ii) Ao protein is localised to histone locus bodies; (iii) an increased dosage of AO heterochromatin partially rescues maternal-effect lethality. These results are consistent with previous reports. However, this study convincingly demonstrates that ao is not required to suppress histone expression, which is in contrast to previous suggestions.
The manuscript is well structured and written. The conclusions are supported by the data. However, I have a few comments that may help to improve the manuscript:
- Localisation of the Ao-V5 protein: Ao-V5 co-localises with Mxc, a marker for the histone locus body. The authors state that some Ao-V5 puncta are without Mxc colocalisation. As far as I can see, it looks more like the other way around - Ao-V5 co-localises with Mxc, but there are some Mxc-marked HLBs without Ao-V5, which would imply that Ao-V5 does not have any other additional genomic loci as suggested. Therefore, I think a quantification of the existing images would be beneficial.
- qPCR analysis of histone gene expression in ao mutant ovaries shows that histone H1 RNA is slightly upregulated, while all other canonical histones remain unchanged compared to control. However, Western blot analysis shows a significant decrease in histone H3 protein levels (~50%), which the authors describe as 'slightly decreased'. This result needs to be considered in more detail, as it would suggest that Ao is required to maintain (at least) histone H3 protein levels?
- Some of the supplementary figures are not referenced in the main text (only in the figure legends).
- Ao deletion (Figure S1): I suggest including the Sanger sequencing results (as mentioned in the text) to demonstrate that neighbouring gene sequences remain unaffected.
Referees cross-commenting
I also think that all reviews are consistent with only a few suggestions for improving the manuscript.
Significance
Strength: This paper shows that ao is not a histone gene repressor, as previously thought, which is an important finding. It also describes new alleles of ao, which are valuable tools for the Drosophila research community.
Limitations: The study lacks insight into the actual molecular role of ao. The finding that H3 protein levels are considerably reduced in ao ovaries could be further investigated.
Advances and audience: As described above, this paper extends our knowledge of the role of ao by showing that it is not a histone gene repressor, which is of interest to the scientific community working in the fields of chromatin and development.
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Referee #2
Evidence, reproducibility and clarity
This study by Takenaka et al. explores the role of the abnormal oocyte (ao) gene in Drosophila melanogaster. Historically, ao was thought to regulate histone gene expression, causing maternal-effect lethality when disrupted. However, the lack of critical reagents including some alleles limited further testing of this hypothesis. In this study, the authors created new CRISPR/Cas9-generated ao knockouts and an epitope-tagged allele to clarify its role. Findings confirmed ao's maternal-effect lethality, which can be rescued by reducing histone copy number or increasing Y-chromosome heterochromatin. Contrary to previous studies, they …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This study by Takenaka et al. explores the role of the abnormal oocyte (ao) gene in Drosophila melanogaster. Historically, ao was thought to regulate histone gene expression, causing maternal-effect lethality when disrupted. However, the lack of critical reagents including some alleles limited further testing of this hypothesis. In this study, the authors created new CRISPR/Cas9-generated ao knockouts and an epitope-tagged allele to clarify its role. Findings confirmed ao's maternal-effect lethality, which can be rescued by reducing histone copy number or increasing Y-chromosome heterochromatin. Contrary to previous studies, they found ao does not repress histone transcript levels, leaving its lethality mechanism unresolved. This work challenges the previously assumed role of ao in histone regulation at the level of transcription.
Major comments: Overall, this manuscript was a joy to read, and the experiments are well controlled and done and presented in a clear and precise manner.
Minor comments: My only two suggestions are if there is a way to include that ao is a conserved gene in the introduction that may broaden the readership of this manuscript. Also, there are many supplementary data panels showing Ao expression that could be condensed.
Referees cross-commenting
All the comments sound good to me.
Significance
The significance of the study lies in its challenge to the previously accepted role of ao gene in regulating histone expression. While earlier research suggested ao acted as a repressor of histone expression, this study, using new and well-controlled tools, did not find evidence supporting that role. The findings not only question the previous assumptions but also emphasize the complexity of ao's function, highlighting the need for further exploration of the mechanisms underlying maternal-effect lethality and its genetic interactions with heterochromatin and histone genes. This will contribute to a better understanding of gene regulation during early development.
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Referee #1
Evidence, reproducibility and clarity
Summary: In this manuscript Takenaka et al address the function of the gene abnormal oocyte (ao, formerly abo) in Drosophila oogenesis. Previous reports implicated ao as a negative regulator of histone biogenesis based on its localization to the histone locus body (HLB), rescue by reduction of histone copy number, and reports that histone production is increased in ao mutants. Only one of the two original ao mutants are still available and the other has been lost. The authors sought to confirm ao's mechanism of action by generating a CRISPR null allele for ao. They also generate a tagged rescue construct which allows them to …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary: In this manuscript Takenaka et al address the function of the gene abnormal oocyte (ao, formerly abo) in Drosophila oogenesis. Previous reports implicated ao as a negative regulator of histone biogenesis based on its localization to the histone locus body (HLB), rescue by reduction of histone copy number, and reports that histone production is increased in ao mutants. Only one of the two original ao mutants are still available and the other has been lost. The authors sought to confirm ao's mechanism of action by generating a CRISPR null allele for ao. They also generate a tagged rescue construct which allows them to visualize ao localization. First, they confirm that a clean deletion of ao does indeed confer maternal lethality and that their tagged construct does localize to the HLB. Next they test histone expression levels in ao null ovaries and find that histones are not, in fact, overexpressed at the RNA or protein level. They also test the RNA levels in unfertilized embryos and find that H2B is decreased rather than increased. Finally, they confirm the previous report that histone deficiency or reduced copy number reduces ao's maternal effect lethality indicating that there is a relationship between ao and the histone locus.
Major comments: I have only one suggestion and a possible extension of the work.
Suggestion: Since ao localizes to the HLB it presumably only affects the replication coupled histones. It would be very interesting to know how replication independent histone RNAs change (or do not) in ao null ovaries.
Extension: (This may be beyond the scope of this work). One wonders if the partial rescue of ao mutants by the histone deficiency/reduced histone copy number is due to a change in the localization of other HLB components in the absence of ao. To test this the authors could image Mxc in the ao null.
Minor comments: None
Significance
General assessment: Overall, I think that this is an important story that sets the record straight about the molecular function of ao, particularly because it was recently suggested as a tool to manipulate histone levels. The experiments appear to be carefully done and provide good support for the claims. The manuscript is clearly written and easy to interpret.
Advance: The main takehome of this story is that the previously described and long-standing mechanism of ao on histone biogenesis and oogenesis is flawed. This story will be the starting point for future characterization of this mutant.
Audience: Those interested in histone biogenesis, especially in flies, will be interested in this story. It does not provide an alternative molecular mechanism (nor does it claim to) which will somewhat limit the general interest. However, this story provides a vital correction to the record.
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