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The deubiquitinase Usp9x regulates PRC2-mediated chromatin reprogramming during mouse development

Trisha A. Macrae, Miguel Ramalho-Santos

Preprint posted on June 29, 2020 https://www.biorxiv.org/content/10.1101/2020.06.28.176412v1

Don’t stay awake for too long! USP9X regulates the deposition of repressive H3K27me3 marks to control the silencing of developmental regulatory genes

Selected by Sergio Menchero

During mouse embryo development, the transition from the naïve pluripotent state to the primed and ready to differentiate state is accompanied by a reprogramming of the chromatin landscape (Gökbuget and Blelloch, 2019). The repressive histone mark H3K27me3, deposited by Polycomb Repressive Complex 2 (PRC2), is redistributed from covering broad genic regions to mark promoters of developmental genes (Mierlo et al, 2019; Xiang et al, 2020). In this work, the authors investigated the role of the deubiquitinating enzyme USP9X during this transition and found that USP9X deubiquitinates and stabilizes PRC2. This process is key to get the H3K27me3 pattern that ensures proper lineage commitment.

The authors established an auxin-inducible degron system to degrade USP9X in embryonic stem (ES) cells. They isolate two subpopulations, USP9X-high (where USP9X has not been degraded) and USP9X-low (cells that have degraded USP9X), to assess the role of USP9X. USP9X-high cells expressed high levels of naïve ES cell markers as compared to USP9X-low cells. This is consistent with the decrease in Usp9x expression from pre- to post-implantation mouse embryos. By means of differentially expressed gene analysis between USP9X-high and low, and by ChIP-X Enrichment Analysis, the authors identified members of the PRC2 complex as putative targets of USP9X.

To assess the role of USP9X in vivo, the authors used a Sox2-Cre system to delete it in epiblast derived cells. Lack of Usp9x was embryonic lethal, and embryos displayed severe abnormalities by E9.5. RNA-seq in E8.5 mutant embryos (before evident morphological alterations) revealed upregulation of key developmental transcription factors whose expression is usually declined at this stage of development in wildtype embryos. The authors noticed that upregulated genes usually acquire the repressive mark H3K27me3 during gastrulation. They went back to the ES cells to demonstrate that USP9X-high cells showed enrichment of H3K27me3 as compared to USP9X-low.

Figure 1. Profile plot depicting the mean signal of H3K27me3 coverage in USP9X-high and USP9X-low ES cells. [Figure 3C in the preprint, made available with permission from the authors]

 

Finally, to understand the relationship between USP9X and PRC2, the authors depleted USP9X in ES cells using different approaches: auxin-inducible degron system, small molecule inhibition, and overexpression of a mutant catalytic domain. In all the cases, they found an accumulation of ubiquitinated forms of the PRC2 members SUZ12 and EZH2, which correlated with their destabilisation.

Altogether, the authors propose that in the preimplantation embryo (naïve pluripotent state), high levels of USP9X favour the deubiquitination and stabilisation of the PRC2 complex. This correlates with an accumulation of the repressive mark H3K27me3 along the genome and general lower transcription. In the transition to the gastrulating embryo, USP9X levels decrease, and that leads to the destabilisation of the PRC2 complex. Global levels of H3K27me3 are reduced and they are redistributed to repress specific developmental regulatory genes after lineage commitment.

Figure 2. Model for the Usp9x-PRC2 regulatory axis in early development. [Figure 5 in the preprint, made available with permission from the authors]

 

Why I chose this preprint and why I think this work moves the field forward:

During embryonic development, the coordinated activity of different transcriptional programs has to be regulated in a spatio-temporal manner to ensure that all the lineages are properly specified. For decades, a clear focus has been to unravel the establishment of gene regulatory networks in order to understand the relationship between genes and identify positive, negative, and gradient-based regulatory mechanisms. Now, the role of an epigenetic layer of transcriptional regulation has been incorporated into the cell fate scenario.

This work is a nice example of how the activity of a deubiquitinating enzyme, USP9X, regulates the deposition of the repressive mark H3K27me3 and how its absence affects the silencing of certain genes and leads to embryonic lethality. The deposition of different histone modifications, DNA methylation, and the activity of several chromatin modifiers can give insight into how specific genes are transcribed and silenced at the right time in the right cells.

 

Questions to the authors

  • Lack of Usp9x in E8.5 mouse embryos revealed an upregulation of developmental regulatory genes, probably due to the absence of proper H3K27me3 levels that fail to silence those genes. Did the authors also find upregulation of genes that are supposed to be switched on later?
  • The transition from high to low levels of USP9X is evident in the early postimplantation embryo. Is the onset of expression of specific developmental genes during early gastrulation also regulated by the removal of H3K27me3? Would the authors expect to detect earlier expression of gastrulation-related genes in Usp9x mutant peri-implantation embryos?

References

Gökbuget, D. & Blelloch, R. (2019). Epigenetic control of transcriptional regulation in pluripotency and early differentiation. Development 146(19): dev164772-16.

Mierlo, G. van et al. (2019). Integrative proteomic profiling reveals PRC2-dependent epigenetic crosstalk maintains ground-state pluripotency. Cell Stem Cell 24(2): 123-37. e8.

Xiang, Y. et al. (2020). Epigenomic analysis of gastrulation identifies a unique chromatin state for primed pluripotency. Nat Genet 52(1): 95-105.

Tags: chromatin remodelling, h3k27me3, lineage commitment, pluripotency transition, prc2, usp9x

Posted on: 7th August 2020 , updated on: 27th August 2020

doi: https://doi.org/10.1242/prelights.23770

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Author's response

The author team shared

Lack of Usp9x in E8.5 mouse embryos revealed an upregulation of developmental regulatory genes, probably due to the absence of proper H3K27me3 levels that fail to silence those genes. Did the authors also find upregulation of genes that are supposed to be switched on later? 
 
It would be really interesting to perform single-cell analyses to see if certain lineages or cell populations show the pattern of premature PRC2 target gene activation that you describe, which would suggest that Usp9x regulates subsets of PRC2 target genes in the post-implantation embryo. For example, it could be that stem/progenitor cells switch on PRC2 targets a bit early in Usp9x mutants and this signal is obscured in our whole-embryo analyses. 
 
In our analyses of E8.5 mutant embryos, we primarily found upregulation of genes that should decrease in expression during gastrulation. We think a lot of this has to do with the fact that gastrulation is a state of relatively low genomic H3K27me3–not only loss of Usp9x and destabilization of PRC2, but also fast cell divisions that promote passive dilution, increases in antagonists (e.g. H3K4me3), etc. We speculate that a key role of H3K27me3 re-accumulation right after gastrulation is to help turn off early post-implantation genes, aside from the canonical role of repressing genes that get switched on later in development.
 
The transition from high to low levels of USP9X is evident in the early postimplantation embryo. Is the onset of expression of specific developmental genes during early gastrulation also regulated by removal of H3K27me3? Would the authors expect to detect earlier expression of gastrulation-related genes in Usp9x mutant peri-implantation embryos? 
Yes, premature expression of PRC2 targets is exactly what we would expect to find in Usp9x-mutant blastocysts. Cell number-normalized RNA-seq revealed that Usp9x-low ES cells activate a gastrulation-like transcriptional program between 8h and 48h after auxin treatment. It would be interesting to try the in vivo correlate of our ES cell data, although this would require careful cell counting and use of spike-in controls. For the current study we focused on the developmental endpoint of Usp9x depletion. 

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