BAP1/ASXL complex modulation regulates epithelial-mesenchymal transition during trophoblast differentiation and invasion
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Abstract
Normal function of the placenta depends on the earliest developmental stages when trophoblast cells differentiate and invade into the endometrium to establish the definitive maternal-fetal interface. Previously, we identified the ubiquitously expressed tumour suppressor BRCA1-associated protein 1 (BAP1) as a central factor of a novel molecular node controlling early mouse placentation. However, functional insights into how BAP1 regulates trophoblast biology are still missing. Using CRISPR/Cas9 knockout and overexpression technology in mouse trophoblast stem cells, here we demonstrate that the downregulation of BAP1 protein is essential to trigger epithelial-mesenchymal transition (EMT) during trophoblast differentiation associated with a gain of invasiveness. Moreover, we show that the function of BAP1 in suppressing EMT progression is dependent on the binding of BAP1 to additional sex comb-like (ASXL1/2) proteins to form the polycomb repressive deubiquitinase (PR-DUB) complex. Finally, both endogenous expression patterns and BAP1 overexpression experiments in human trophoblast stem cells suggest that the molecular function of BAP1 in regulating trophoblast differentiation and EMT progression is conserved in mice and humans. Our results reveal that the physiological modulation of BAP1 determines the invasive properties of the trophoblast, delineating a new role of the BAP1 PR-DUB complex in regulating early placentation.
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This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on November 8 2020, follows.
Summary
The manuscript from Perez-Garcia et al. follows up on a prior study by the same authors in which they identified the tumor suppressor BAP1 as a regulator of mouse placentation and trophoblast stem cells (TSCs) (Perez-Garcia et al., Nature, 2018). In their preceding work the authors showed that CRISPR-mediated knockout of BAP1 in TSCs results in upregulation of key stem cell markers Cdx2 and Esrrb and biased differentiation towards trophoblast giant cells at the expense of the syncytiotrophoblast lineage. Here the authors have expanded on these observations by demonstrating that BAP1 modulates the epithelial-to-mesenchymal transition (EMT) in TSCs and that a similar phenotype can be obtained by genetic …
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on November 8 2020, follows.
Summary
The manuscript from Perez-Garcia et al. follows up on a prior study by the same authors in which they identified the tumor suppressor BAP1 as a regulator of mouse placentation and trophoblast stem cells (TSCs) (Perez-Garcia et al., Nature, 2018). In their preceding work the authors showed that CRISPR-mediated knockout of BAP1 in TSCs results in upregulation of key stem cell markers Cdx2 and Esrrb and biased differentiation towards trophoblast giant cells at the expense of the syncytiotrophoblast lineage. Here the authors have expanded on these observations by demonstrating that BAP1 modulates the epithelial-to-mesenchymal transition (EMT) in TSCs and that a similar phenotype can be obtained by genetic deletion of Asxl1/2. Declining protein levels of BAP1 during differentiation of human TSCs into extravillous trophoblast suggest that the role of BAP1 may be conserved in humans. As the molecular mechanisms of trophoblast development, including EMT and invasive behaviors of trophoblast giant cells in the mouse and extravillous trophoblast cells in human are only beginning to be understood, this study provides an important advance.
This is a well-written and technically sound study that clarifies the role of BAP1 in trophoblast development. Overall, the work presented is very important to the fields of EMT and trophoblast stem cell biology, and it warrants publication in eLife in principle. However, the claims in the abstract, the model in Figure 5, and the conclusions in the discussion are not well-supported. Therefore, additional experimental work will be essential for the manuscript to become suitable for publication in eLife.
Essential Revisions
There was consensus that the current manuscript lacks functional data to demonstrate conservation of BAP1/ASXL1/2 function in human TSCs. These are crucial claims in the abstract that are not supported, and some elements of these claims are necessary for the manuscript to have impact beyond the previous Nature 2018 publication. Currently, the studies in human TSCs are purely observational (Fig. 6D-E). The authors should employ genetic approaches to interrogate whether the functions of BAP1 in TSC self-renewal and differentiation are truly conserved between mouse and human.
The main takeaway from Figure 2 is that BAP1 is dispensable for mouse TSC maintenance and that BAP1 knockout results in increased expression of stem cell markers Cdx2 and Esrrb. Both of these findings were previously reported in the authors' 2018 paper (see Fig. 4b in the Nature paper). Therefore, the statement that "BAP1 deletion does not impair the stem cell gene regulatory network" is not surprising and the authors should state clearly that these experiments confirm their prior observations.
The overexpression data in Figure 4 is difficult to interpret. Vector transduced TSCs show a tight, epithelial morphology (Figure 3A), whereas the NT-sgRNA control cells look like they are undergoing EMT (Figure 4C). Why does the introduction of the NT-sgRNA induce EMT characteristics? Bap1 sgRNA1 cells seem less epithelial than the Vector transduced cells. Do NT-sgRNA TSCs have less BAP1 than Vector transduced TSCs?
Moreover, all the data in Figure 4 are based on a single sgRNA that could activate BAP1 expression. To exclude off target effects, the authors should confirm the effect of BAP1 overexpression using another sgRNA or cDNA overexpression system.
The authors need to examine the gene expression data more closely as well as the functional consequences of BAP1 overexpression on TSC proliferation and differentiation. In particular it would be important to compare the list of DEG in BAP1 KO and overexpression condition. Are they mirror-image or are there differences? For example, Zeb2 expression is strongly upregulated in BAP1 mutant line but not significantly altered in cells overexpressing BAP1. This should be discussed.
In the abstract, the authors state that BAP1 function during trophoblast development is dependent on its binding to Asxl1/2/3. However, the data presented in this manuscript do not address whether BAP1 and Asxl1/2/3 are indeed part of the same complex in TSCs. Furthermore, the fact that Asxl1/2 KO increases expression of syncytial genes (Fig. 5) does not provide direct evidence of functional synergy between these proteins and BAP1. This conclusion could be strengthened by demonstrating that Asxl1 and BAP1 indeed have a protein-protein interaction in TSCs and/or by deleting the BAP1 binding domain in Asxl1/2. It would also be instructive to examine whether the phenotype of BAP1 overexpression in TSCs (e.g. gain of epithelial features and reduced invasiveness) is dependent on Asxl1. This could be examined by overexpressing BAP1 in Asxl1-deficient TSCs.
In some cases, experiments are carried out to "confirm" and "corroborate" hypotheses rather than test them. For example, the similarity between the gene expression signature of Bap1 mutant murine TSCs is and Bap1 mutant melanocytes and mesothelial cells is shown and emphasized. One wonders how unique is this similarity? Is Bap1 expression modulation observed in other EMT processes during development or in cancer? This should be explored and discussed.
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