Hybridization led to a rewired pluripotency network in the allotetraploid Xenopus laevis
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eLife assessment
This paper reports fundamental findings that substantially advance our understanding of a major research question - how hybridization events influence gene regulatory programs and how evolutionary pressures have shaped these programs in response to such events. The methods, data, and analyses are solid and broadly support the claims with only minor weaknesses. This convincing work uses appropriate and validated methodology in line with the current state-of-the-art.
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Abstract
After fertilization, maternally contributed factors to the egg initiate the transition to pluripotency to give rise to embryonic stem cells, in large part by activating de novo transcription from the embryonic genome. Diverse mechanisms coordinate this transition across animals, suggesting that pervasive regulatory remodeling has shaped the earliest stages of development. Here, we show that maternal homologs of mammalian pluripotency reprogramming factors OCT4 and SOX2 divergently activate the two subgenomes of Xenopus laevis , an allotetraploid that arose from hybridization of two diploid species ~18 million years ago. Although most genes have been retained as two homeologous copies, we find that a majority of them undergo asymmetric activation in the early embryo. Chromatin accessibility profiling and CUT&RUN for modified histones and transcription factor binding reveal extensive differences in predicted enhancer architecture between the subgenomes, which likely arose through genomic disruptions as a consequence of allotetraploidy. However, comparison with diploid X. tropicalis and zebrafish shows broad conservation of embryonic gene expression levels when divergent homeolog contributions are combined, implying strong selection to maintain dosage in the core vertebrate pluripotency transcriptional program, amid genomic instability following hybridization.
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eLife assessment
This paper reports fundamental findings that substantially advance our understanding of a major research question - how hybridization events influence gene regulatory programs and how evolutionary pressures have shaped these programs in response to such events. The methods, data, and analyses are solid and broadly support the claims with only minor weaknesses. This convincing work uses appropriate and validated methodology in line with the current state-of-the-art.
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Reviewer #1 (Public Review):
In this study, the authors characterize regulatory control of embryonic genome activation in the allotetraploid, Xenopus laevis. By characterizing transcription from its L and S subgenomes, they determine that homeologous genes are differentially activated in the early embryo. It has recently been appreciated that homeologs may be differentially expressed in later embryonic development (Session and Rokhsar, Nature 2016). However, an unanswered question is whether vertebrate tetraploid genomes undergo differential induction at the onset of the major wave of zygotic genome activation (ZGA). This is a fertile area for research, that enables the study of gene regulatory network adaptations to changes in ploidy, limited by the constraints of gene dosage and an essential early developmental transition. Xenopus …
Reviewer #1 (Public Review):
In this study, the authors characterize regulatory control of embryonic genome activation in the allotetraploid, Xenopus laevis. By characterizing transcription from its L and S subgenomes, they determine that homeologous genes are differentially activated in the early embryo. It has recently been appreciated that homeologs may be differentially expressed in later embryonic development (Session and Rokhsar, Nature 2016). However, an unanswered question is whether vertebrate tetraploid genomes undergo differential induction at the onset of the major wave of zygotic genome activation (ZGA). This is a fertile area for research, that enables the study of gene regulatory network adaptations to changes in ploidy, limited by the constraints of gene dosage and an essential early developmental transition. Xenopus laevis, which recently underwent a tetraploidization event, approximately 18 million years ago, provides a very useful model embryonic system for the study of homeologous gene activation during vertebrate ZGA.
To characterize differential subgenome activation the authors focused on the ~ 2600 maternally-regulated genes expressed in the first wave of widespread ZGA. They treated embryos with cycloheximide at Stage 8 to prevent the translation of zygotic factors that would further alter the transcriptome. They found a majority of these maternally-regulated genes have asymmetric expression between the two homeologs, with transcription often occurring from the L or S copy alone. This is a fascinating result from which to dig deeper into gene regulatory mechanisms. To understand whether cis-regulatory networks dictate the biased L/S homeolog expression in the late blastula, the authors performed CUT&RUN to map active chromatin marks, H3K4me3 and H3K27ac. However, they found no differences in promoter sequences of homeologs that would implicate differential recruitment of specific transcription factors. Instead, they focused on distal enhancers and additionally performed ATAC-seq on Stage 8 and 9 animal cap explants. Approximately 70% of enhancers for homeolog pairs exhibited differential H3K27ac enrichment and chromatin accessibility. The authors then searched for transcription factor binding motifs that distinguished active enhancers from their inactive homeolog. They found binding sequences for OCT4 and SOX2/3 were enriched in active L enhancers and active S enhancers. To assess the role of these pluripotency factors, they used antisense morpholinos to block their translation in the early embryo. MOs were complementary to both the L and S homeologs of pou5f3.3 and sox3, but not to their paralogs that are primarily expressed zygotically; pou5f3.1 and pou5f3.2. MO knockdown of both Pou5f3.3 and Sox3 was inhibited leading to significant downregulation of 62% of activated genes compared to embryos injected with a control morpholino. They also analyzed binding to the genome of V5-tagged, injected versions of these 2 transcription factors and found some evidence for differential binding around TSS of homeolog pairs and a correlation between binding and the overall level of transcription at ZGA. Finally, they compare enhancer marks and accessibility in tetraploid X.laevis subgenomes to homologous enhancers in the diploid X.tropicalis. They conclude conservation of active enhancers with X.tropicalis and even zebrafish when considering the combined data from X.laevis L and S subgenome.
There are many strengths of this manuscript. In this interesting study, the authors identify what appears to be an evolutionary divergence of enhancers in a vertebrate tetraploid, that may underlie the differential expression of homeologs during the first major wave of ZGA. They generate CUT&RUN datasets of active chromatin marks during the early and late blastula. Additionally, they provide binding data for pluripotency factors OCT4 and SOX2/3 and demonstrate that their MO knockdown leads to reduced expression at ZGA. Their analyses identify correlations between differential homeolog expression and active or accessible chromatin. Further, they identify that active enhancers are enriched in OCT4 and SOX2/3. Enthusiasm is somewhat dampened by a lack of direct perturbation to differential subgenome activation or an understanding of the functional impacts of differential homeolog expression on subsequent development.
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Reviewer #2 (Public Review):
Hybridization events between species are known to result in substantial genomic upheaval, requiring subsequent coordination between gene copies to ensure proper control of gene expression and embryonic viability. An example of such an event happened over 18 million years ago between two frog species that resulted in Xenopus laevis-an allotetraploid that has largely retained copies of both genes from this event, known as L-alleles and S-alleles. Often, the presence of both copies presents an experimental and bioinformatic hurdle for researchers and is a feature of the biology of X. laevis that renders cross-species comparisons difficult. Phelps et al, however, take advantage of this feature of Xenopus biology and use it to their advantage to ask how the hybridization event in this species altered gene …
Reviewer #2 (Public Review):
Hybridization events between species are known to result in substantial genomic upheaval, requiring subsequent coordination between gene copies to ensure proper control of gene expression and embryonic viability. An example of such an event happened over 18 million years ago between two frog species that resulted in Xenopus laevis-an allotetraploid that has largely retained copies of both genes from this event, known as L-alleles and S-alleles. Often, the presence of both copies presents an experimental and bioinformatic hurdle for researchers and is a feature of the biology of X. laevis that renders cross-species comparisons difficult. Phelps et al, however, take advantage of this feature of Xenopus biology and use it to their advantage to ask how the hybridization event in this species altered gene regulatory architecture. They find that a handful of pluripotency genes are largely responsible for activating gene expression in the early embryo, but that L and S alleles are differentially activated in many cases. Moreover, they find extensive differences in cis-regulatory architecture between L/S alleles. Despite these differences in alleles, however, they find that their combined gene expression output is largely conserved, possibly reflecting strong selection pressures acting to maintain gene expression output at specific levels. This work represents a significant advance in how hybridization events are something greatly understudied in developmental biology-influence gene regulatory programs and how evolutionary pressures have shaped these programs in response to such events.
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