Histone H3.3 Hira chaperone complex contributes to zygote formation in mice and humans
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Abstract
Elucidating the underlining mechanisms underpinning successful fertilisation is imperative in optimising IVF treatments, and may lead to a specific diagnosis and therefore potential treatment for some infertile couples. One of the critical steps involves paternal chromatin reprogramming, in which compacted sperm chromatin packed by protamines is removed by oocyte factors and new histones, including histone H3.3, are incorporated. This step is critical for the formation of the male pronucleus, without which the zygote contains only 1 pronucleus (1PN), in contrast to normally fertilised zygotes with two-pronuclei (2PN). 1PN zygotes are a frequently observed phenomenon in IVF treatments, therefore aberrant mechanism of action controlling paternal chromatin repackaging may be an important cause of abnormal fertilisation. Hira is the main H3.3 chaperone that governs this protamine-to-histone exchange. In this study, we investigated the maternal functions of two other molecules of the Hira complex, Cabin1 and Ubn1 in the mouse. Loss-of-function Cabin1 and Ubn1 mouse models were developed: their zygotes displayed abnormal 1PN zygote phenotypes, similar to the phenotype of Hira mutants. We then studied human 1PN zygotes, and found that the Hira complex was absent in 1PN zygotes which were lacking the male pronucleus. This result confirms that the role of the Hira complex in male pronucleus formation has coherence from mice to humans. Furthermore, rescue experiments showed that the abnormal 1PN phenotype derived from Hira mutants could be resolved by overexpression of Hira in the mouse oocytes. In summary, we have provided evidence of the role of Hira complex in regulating male pronucleus formation in both mice and humans, that both Cabin1 and Ubn1 components of the Hira complex are equally essential for male pronucleus formation, and that this can be rescued. We present a proof-of-concept experiment that could potentially lead to a personalised IVF therapy for oocyte defects.
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###Reviewer #3:
The role of histone chaperone Hira during the formation of paternal pronucleus has been well documented in both mouse (Lin et al, 2014; Inoue and Zhang, 2014; Nashun et al, 2015), and in Drosophila (Loppin et al, 2005). The histone chaperone Hira is known to act in a protein complex with Ubn and Cabin 1 (Tang et al, 2012). The authors built on their previous findings (Lin et al, 2014) and assessed the effect of the Ubn and Cabin 1 oocyte deletion during the fertilisation. Not surprisingly, the observed phenotypes more or less recapitulated the observation made using Hira deletion. In this sense, the findings are not novel. It has also been previously shown that deletion of Hira leads to the removal of the whole complex (Nashun et al, 2015).
The authors add some potentially interesting observations using 1PN (aberrant) …
###Reviewer #3:
The role of histone chaperone Hira during the formation of paternal pronucleus has been well documented in both mouse (Lin et al, 2014; Inoue and Zhang, 2014; Nashun et al, 2015), and in Drosophila (Loppin et al, 2005). The histone chaperone Hira is known to act in a protein complex with Ubn and Cabin 1 (Tang et al, 2012). The authors built on their previous findings (Lin et al, 2014) and assessed the effect of the Ubn and Cabin 1 oocyte deletion during the fertilisation. Not surprisingly, the observed phenotypes more or less recapitulated the observation made using Hira deletion. In this sense, the findings are not novel. It has also been previously shown that deletion of Hira leads to the removal of the whole complex (Nashun et al, 2015).
The authors add some potentially interesting observations using 1PN (aberrant) human zygotes. Although the observed lack of Hira complex components in these zygotes could be interesting, the causality is not established.
Beyond the statements above, there are major issues that would need to be addressed:
Validation and characterisation of the ko/kd models: Ubn1 knockdown using morpholinos: Fig S1C - lots of protein remains present in the nucleus, Hira Zp3Cre driven oocyte specific knockout - how much Hira protein is left in the zygote?
H3.3 staining to document the deletion of the complex: Figs1E - not obvious what the authors are trying to say here? How is H3.3 signal quantified? Only paternal signal should be affected by the KO ?? The same is true for Fig2D - no signal is obvious even in the control.
Presence of Cabin1 in the zygote - pre-extraction needs to be carried out (Fig 2C)
Fig S2: overexpression of Hira : is there a significant difference between the Hira signal in control (het) and KO zygote?? It does not appear so, which undermines the whole knockout study. The same is true for the quantification of H3.3 . What should the quantification of GFP signal demonstrate?
5.) The authors say that they developed a conditional KO for Hira in the main text. But they haven't verified the Hira deletion after Cre expression (by IF or PCR)
"Data not shown" in the text. The authors say that their new hiraF/F, zp3 females are sterile but they don't show it.
The authors never show anti-ubn1, cabin1 staining on HiraKO.
Language: the text needs editing. There a number of statements that are wrong: Hira (or any other component of the complex) does not incorporate into chromatin - the complex associates with chromatin to incorporate histones (there are several other examples of similar statements).
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###Reviewer #2:
A high proportion of in vitro fertilized eggs yield zygotes with 1 pronucleus (1PN) instead of the normal 2PN. The authors previously showed that maternal Hira is important for H3.3 deposition on the male pronucleus; and that the loss of Hira leads to a high proportion of 1PN zygotes.
In this manuscript, the increase in 1PN zygotes after fertilization was confirmed following deletion of Hira in mouse oocytes. The effect could be rescued upon microinjection of Hira RNA. The authors also depleted the other Hira protein complex subunits, Cabin1 and ubinuclein-1. The 1PN phenotype was again seen. Human 1PN zygotes were finally shown to lack HIRA on the abnormal pronucleus.
This is an interesting observation that is definitely worth the investigation. The lack of HIRA components on the abberant pronucleus in 1PN human zygotes …
###Reviewer #2:
A high proportion of in vitro fertilized eggs yield zygotes with 1 pronucleus (1PN) instead of the normal 2PN. The authors previously showed that maternal Hira is important for H3.3 deposition on the male pronucleus; and that the loss of Hira leads to a high proportion of 1PN zygotes.
In this manuscript, the increase in 1PN zygotes after fertilization was confirmed following deletion of Hira in mouse oocytes. The effect could be rescued upon microinjection of Hira RNA. The authors also depleted the other Hira protein complex subunits, Cabin1 and ubinuclein-1. The 1PN phenotype was again seen. Human 1PN zygotes were finally shown to lack HIRA on the abnormal pronucleus.
This is an interesting observation that is definitely worth the investigation. The lack of HIRA components on the abberant pronucleus in 1PN human zygotes is an important find. However, because the authors had already shown that the loss of Hira correlates with a high proportion of 1PN in mice, the experiments (though respectable) provide limited novelty as is.
Main concern:
- Unless there are reasons to believe that there are Hira-independent Cabin1 and ubinuclein-1 functions in oocytes, their depletion only serves to confirm the role of Hira and its relation to the 1PN phenotype. The rescue experiment and human data is important, but again serves as confirmation on the role of HIRA without further mechanistic insights.
Perhaps novelty could come through a deeper exploration on Hira levels in oocytes and what differentiates 'poor quality' oocytes that lead to 1PN from normal ones. For example, does maternal Hira RNA and protein levels increase with maturation? Are HIRA levels lower in poor quality oocytes? Is there a step in the IVF procedure that affects Hira levels and/or changes on the paternal chromatin?
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###Reviewer #1:
This study establishes the role of additional members of the Histone chaperone HIRA complex in male pronucleus formation in mouse. Genetic inactivation of maternal Ubn1 and Cabin1 affects histone deposition following protamine removal on the fertilizing sperm nucleus in a way similar to maternal Hira mutants. However, the study does not provide new insights about the way these factors function or cooperate during paternal chromatin assembly. Analysis of aberrant human zygotes revealed a correlation between the lack of male pronucleus and the absence of maternal HIRA. Although the data are generally convincing, the manuscript does not sufficiently acknowledge earlier work. Notably, the rescue experiment which is presented as a "proof of principle" for future human therapy is not entirely original.
Substantive concerns:
The…
###Reviewer #1:
This study establishes the role of additional members of the Histone chaperone HIRA complex in male pronucleus formation in mouse. Genetic inactivation of maternal Ubn1 and Cabin1 affects histone deposition following protamine removal on the fertilizing sperm nucleus in a way similar to maternal Hira mutants. However, the study does not provide new insights about the way these factors function or cooperate during paternal chromatin assembly. Analysis of aberrant human zygotes revealed a correlation between the lack of male pronucleus and the absence of maternal HIRA. Although the data are generally convincing, the manuscript does not sufficiently acknowledge earlier work. Notably, the rescue experiment which is presented as a "proof of principle" for future human therapy is not entirely original.
Substantive concerns:
The authors present the (partial) rescue experiment of maternal Hira KO (oocyte injection of Hira mRNA) as an original experiment that serves as a proof of principle for future therapy. However, a very similar rescue experiment of Hira KD oocytes was successfully performed by Inoue & Zhang, NSMB, 2014, a work that is not cited in the manuscript.
The authors used PLA to detect interactions between the Hira complex proteins in mouse zygotes. However, it is not clear from the images in Fig. 1C how the specific interactions are actually appreciated. The foci seem to be everywhere and not particularly in the male pronucleus shown in the insets.
The occurrence of 1 PN human zygotes is intriguing but the origin of this defect is unknown. It could reflect a more general problem than the sole lack of Hira expression. In this context, overcoming male pronuclear formation by re-expression of Hira seems to represent a hazardous therapeutic strategy.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
All three reviewers agree on the fact that the study, although interesting, does not appear sufficiently novel regarding the already established role of Hira complex in sperm chromatin remodeling in mouse and other animals. In addition, although the reviewers were intrigued by the observation that 1PN human zygotes lack HIRA, the origin and timing of this defective expression are not established. The reviewers share the feeling that these experiments do not really bring novel insights about the regulation of …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
All three reviewers agree on the fact that the study, although interesting, does not appear sufficiently novel regarding the already established role of Hira complex in sperm chromatin remodeling in mouse and other animals. In addition, although the reviewers were intrigued by the observation that 1PN human zygotes lack HIRA, the origin and timing of this defective expression are not established. The reviewers share the feeling that these experiments do not really bring novel insights about the regulation of HIRA levels in mammalian oocytes.
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