Maternal SMCHD1 controls both imprinted Xist expression and imprinted X chromosome inactivation
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Evaluation Summary:
This study assess the role of SMCHD1 provided by the oocyte at fertilization in the regulation of Xist expression in the embryo. They also provide preliminary analysis of the downstream effects of faulty Xist expression, on X chromosome silencing. This work has implications in epigenetics and embryonic development and aims at an audience interested in gene dosage regulation in mammals. Although of potential interest, some further analytical and experimental work is needed to understand how SMHCD1 works on the early stages of X inactivation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Embryonic development is dependent on the maternal supply of proteins through the oocyte, including factors setting up the adequate epigenetic patterning of the zygotic genome. We previously reported that one such factor is the epigenetic repressor SMCHD1, whose maternal supply controls autosomal imprinted expression in mouse preimplantation embryos and mid-gestation placenta. In mouse preimplantation embryos, X chromosome inactivation is also an imprinted process. Combining genomics and imaging, we show that maternal SMCHD1 is required not only for the imprinted expression of Xist in preimplantation embryos, but also for the efficient silencing of the inactive X in both the preimplantation embryo and mid-gestation placenta. These results expand the role of SMCHD1 in enforcing the silencing of Polycomb targets. The inability of zygotic SMCHD1 to fully restore imprinted X inactivation further points to maternal SMCHD1’s role in setting up the appropriate chromatin environment during preimplantation development, a critical window of epigenetic remodelling.
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Author Response:
We thank the reviewers for their time and comments on our manuscript.
On the partial labelling efficiency of Xist in E3.5 embryos, this is not unexpected given that the assay is allele-specific and less sensitive than standard RNA FISH. Harris et al. 2019 reported a ~80% labelling efficiency, ours appeared more variable from embryo to embryo, around 50%. However the fact that only paternal Xist is ever detected in the female Smchd1matΔ E3.5 embryos is a compelling indication that maternal Xist silencing is restored: if it weren’t, the probability of observing only paternal Xist would be 0.5^(# labelled cells) = 0.5^53=1e-16. Furthermore, in the male E3.5 embryos, we found no Xist expression at all, which contrasted with the EedmatΔ male E3.5 embryos that do not resilence maternal Xist at this stage (Harris et al. 2019). …
Author Response:
We thank the reviewers for their time and comments on our manuscript.
On the partial labelling efficiency of Xist in E3.5 embryos, this is not unexpected given that the assay is allele-specific and less sensitive than standard RNA FISH. Harris et al. 2019 reported a ~80% labelling efficiency, ours appeared more variable from embryo to embryo, around 50%. However the fact that only paternal Xist is ever detected in the female Smchd1matΔ E3.5 embryos is a compelling indication that maternal Xist silencing is restored: if it weren’t, the probability of observing only paternal Xist would be 0.5^(# labelled cells) = 0.5^53=1e-16. Furthermore, in the male E3.5 embryos, we found no Xist expression at all, which contrasted with the EedmatΔ male E3.5 embryos that do not resilence maternal Xist at this stage (Harris et al. 2019). Overall it provides strong evidence that Xist loss of imprinting at E2.75 is corrected at E3.5.
We did test differential expression for all expressed genes in our RNA-seq analysis. The results for autosomes were briefly presented in our previous manuscript (Wanigasuriya, Gouil et al. Fig6Supp1), which we now describe in more detail (8 genes significantly DE in males, 0 in females). The full results of DE analysis are in the Supplementary tables. The allele-specific analysis for the X-linked genes aims to detect small changes: in wild-type morulae, in the early stages of X inactivation, genes on the paternal X are only down by 40% on average (less than 2-fold). Therefore on average the maximum possible fold-change that we can observe in the mutants is less than 2-fold. Due to these small effect sizes, no X-linked gene passes the FDR threshold, which does not mean that there is no effect, but limits our ability to perform gene-by-gene analyses and comparisons with later developmental stages. Instead we have to increase power by looking for systematic changes across the X-linked genes in our E2.75 embryos. We recognise that this section could be clarified and elaborated on, which we have addressed in a new version of the manuscript.
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Evaluation Summary:
This study assess the role of SMCHD1 provided by the oocyte at fertilization in the regulation of Xist expression in the embryo. They also provide preliminary analysis of the downstream effects of faulty Xist expression, on X chromosome silencing. This work has implications in epigenetics and embryonic development and aims at an audience interested in gene dosage regulation in mammals. Although of potential interest, some further analytical and experimental work is needed to understand how SMHCD1 works on the early stages of X inactivation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Parental specific gene expression is called imprinting and has been described in many mammals but also in plants. It relies on specific epigenetic marks such as DNA methylation or specific histone modifications that mark the chromatin of the gametes according to the parental origin. The different actors that maintain such imprints at the chromatin level are still to be better understood.
The authors previously published that SMCHD1 is one of such factors acting during mouse early development as a pool of maternally provided proteins within the oocyte (Vanigasuriya et al; eLife 2020;9:e55529). In particular, they showed its importance in the expression of a subset of imprinted genes whose regulation is based on histone modifications but not on DNA methylation, at least during preimplantation timing. In the …
Reviewer #1 (Public Review):
Parental specific gene expression is called imprinting and has been described in many mammals but also in plants. It relies on specific epigenetic marks such as DNA methylation or specific histone modifications that mark the chromatin of the gametes according to the parental origin. The different actors that maintain such imprints at the chromatin level are still to be better understood.
The authors previously published that SMCHD1 is one of such factors acting during mouse early development as a pool of maternally provided proteins within the oocyte (Vanigasuriya et al; eLife 2020;9:e55529). In particular, they showed its importance in the expression of a subset of imprinted genes whose regulation is based on histone modifications but not on DNA methylation, at least during preimplantation timing. In the present study, they now focus on regulation of one of such genes called Xist. Xist encodes for a long non-coding RNA that is necessary for X chromosome inactivation in female, a process allowing gene dosage compensation between the sexes. Different previous and independent studies have shown that inappropriate expression of maternal Xist could lead to aberrant X chromosome inactivation in male and female embryos, and lethality in the worse cases, highlighting the importance of an accurate expression pattern for this gene.
Here, the authors observe, using two independent approaches (allelic RNA seq and allelic RNA FISH), the partial reactivation of the normally silent maternal allele of Xist in male and female morula (16-32Cell stage) (Figure 1d and Figure 3) derived from maternally depleted SMCHD1 oocytes. Based on their allelic RNA FISH assays, they conclude that abnormal biallelic expression is only transient as it is resorbed by the next developmental stage (blastocyst). Different groups demonstrated previously that in wild type mouse morula and blastocysts, between 90 and 100% of nuclei in female embryos are harboring one single Xist cloud (covering the paternal X) per nuclei. The fact that the authors find up to 50% of nuclei without any signal in their controls and up to 75% in the mutants, (when one should expect at least one cloud in 90-100% of females nuclei in any case), questions the robustness of their RNA FISH. This possibly faulty assay questions the conclusion that the imprinted default observed earlier is resolved. In other word, Xist misregulation might last longer in maternal mutant SMCHD1 than what is presently concluded in this manuscript. One other point made with this study is that the DNA methylation status of the Xist locus is not impaired in mutant, ruling out the role of SMCHD1 on DNA metylation at that genomic place in early mouse embryos.
Maternal Xist could lead to abnormal maternal X linked gene silencing in blastocysts and later, and be associated with failure of embryonic development (Inoue et al 2018). Understanding if aberrant Xist expression has any consequences at the level of the X chromosome in the maternal SMCHD1 mutant is therefore very relevant, and the authors of the present study use their allelic RNA seq data to enquire this question. They conclude that X chromosome expression is altered during preimplantation as well as much later in the placenta. Their transcriptomic analysis seem however succinct to fully understand if these changes are due to silencing of the maternal X chromosome in male and female embryos, which genes might be abnormally regulated, and if the observed defect in morula have any linked with the other developmental stage they analyzed. A deeper analysis of their allelic transcriptomic data would at least strengthen our understanding of how a transient expression of SMCDH1 would reflect on adequate X chromosome expression.
This study is a nice preliminary description of the importance of maternal pool of SMCHD1 on maternal Xist expression and X chromosome expression/ inactivation in mouse embryos. It does not cover any further understanding on the chromatin changes that might be due to loss of maternal SMCDH1 on specific genomic locus such as Xist and more generally on the molecular mechanisms at work for the transiently imprinted gene regulation. These are challenging questions, given the developmental stages studied, and is beyond the scope of this manuscript, yet it place SMCHD1 as a new interesting player.
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Reviewer #2 (Public Review):
SMCHD1 has a well-established role in the later stages of X-inactivation, but earlier functions, discoverable only from maternal SMCHD1 deletion, have never been performed. This study fills that gap, and shows roles for SMCHD1 in X-inactivation establishment and imprinting.
In this manuscript, Wanigasuriya et al investigate the role of SMCHD1 in mouse imprinted X-chromosome inactivation (XCI). Previous studies from this lab established a role for zygotic SMCHD1 in later stages of XCI, e.g. in regulating DNA methylation at a subset of X-genes. This study is distinct in that it investigates the effects of maternal SMCHD1 on XCI. Three phenotypes are noted: partial loss of maternal imprinted Xist silencing, as well as defective Xp silencing at E2.75, and defective Xp silencing in the placenta at E14.5. The …
Reviewer #2 (Public Review):
SMCHD1 has a well-established role in the later stages of X-inactivation, but earlier functions, discoverable only from maternal SMCHD1 deletion, have never been performed. This study fills that gap, and shows roles for SMCHD1 in X-inactivation establishment and imprinting.
In this manuscript, Wanigasuriya et al investigate the role of SMCHD1 in mouse imprinted X-chromosome inactivation (XCI). Previous studies from this lab established a role for zygotic SMCHD1 in later stages of XCI, e.g. in regulating DNA methylation at a subset of X-genes. This study is distinct in that it investigates the effects of maternal SMCHD1 on XCI. Three phenotypes are noted: partial loss of maternal imprinted Xist silencing, as well as defective Xp silencing at E2.75, and defective Xp silencing in the placenta at E14.5. The novelty here is uncovering roles for maternal SMCHD1 early in the XCI process, and showing that the effects of maternal SMCHD1 loss persist later, even when zygotic SMCHD1 kicks in. The manuscript is well-written, and the analyses by and large well-performed, with allele-specific RNA FISH a particular highlight.
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