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• W4387585511 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation 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. This convincing work uses appropriate and validated methodology in line with the current state-of-the-art. https://doi.org/10.7554/eLife.83952.sa0 Decision letter Reviews on Sciety eLife's review process Introduction In animals, zygotic genome activation (ZGA) is triggered after an initial period of transcriptional quiescence following fertilization of the egg, during the maternal-to-zygotic transition (MZT; Lee et al., 2014; Vastenhouw et al., 2019). In mammals, this occurs during the slow first cleavages (Svoboda, 2018), a few days removed from the subsequent induction of pluripotent stem cells in the blastocyst by a core network of factors including NANOG, OCT4, and SOX2 (Li and Belmonte, 2017; Takahashi and Yamanaka, 2016). In contrast, faster-dividing taxa including zebrafish, Xenopus, and Drosophila activate their genomes in the blastula hours after fertilization (Foe and Alberts, 1983; Kane and Kimmel, 1993; Newport and Kirschner, 1982a; Vastenhouw et al., 2019), which leads immediately to pluripotency. In zebrafish, maternally provided homologs of NANOG, OCT4, and SOX2 are required for a large share of genome activation (Lee et al., 2013; Leichsenring et al., 2013; Miao et al., 2022); thus, vertebrate embryos deploy conserved pluripotency induction mechanisms at different times during early development. Beyond vertebrates, unrelated maternal factors direct genome activation and the induction of stem cells, for example Zelda (Liang et al., 2008), CLAMP (Colonnetta et al., 2021; Duan et al., 2021), and GAF (Gaskill et al., 2021) in Drosophila, although they seem to share many functional aspects with vertebrate pluripotency factors, including pioneering roles in opening repressed embryonic chromatin and establishing activating histone modifications (Blythe and Wieschaus, 2016; Gaskill et al., 2021; Hug et al., 2017). This diversity of strategies implies that the gene network regulating pluripotency has been extensively modified over evolutionary time (Endo et al., 2020; Fernandez-Tresguerres et al., 2010), though it is unknown when and under what circumstances major modifications arose. We sought to understand how recent genome upheaval has affected the pluripotency regulatory network in the allotetraploid Xenopus laevis, by deciphering how embryonic genome activation is coordinated between its two subgenomes. X. laevis’s L (long) and S (short) subgenomes are inherited from each of two distinct species separated by ~34 million years that hybridized ~18 million years ago (Session et al., 2016; Figure 1A). A subsequent whole-genome duplication restored meiotic pairing. Despite extensive rearrangements and deletions, most genes are still encoded as two copies (homeologs) on parallel, non-inter-recombining chromosomes (Session et al., 2016). Previously, homeologs had been challenging to distinguish due to high functional and sequence similarity; however, the recent high-quality X. laevis genome assembly has made it feasible to resolve differential expression and regulation genome-wide between the two subgenomes (Elurbe et al., 2017; Session et al., 2016). Figure 1 with 1 supplement see all Download asset Open asset Identifying the first wave of genome activation across the two subgenomes. (A) The allotetraploid X. laevis genome contains two distinct subgenomes “L” and “S” due to interspecific hybridization of ancestral diploids. (B) Triptolide inhibits genome activation, as measured in the late blastula, while cycloheximide inhibits only secondary activation, distinguishing genes directly activated by maternal factors. NF = Nieuwkoop and Faber. (C) Heatmap of RNA-seq coverage over exons (left) and introns (right) of activated genes. Allopolyploidy often provokes acute effects on gene expression (Hu and Wendel, 2019; Moran et al., 2021), leading to regulatory shifts over time to reconcile dosage imbalances and incompatibilities between gene copies (Grover et al., 2012; Song et al., 2020; Swamy et al., 2021). This phenomenon has been explored primarily in plants (Adams and Wendel, 2005; Husband et al., 2013; Mable, 2004), but the extent to which this has occurred in the few characterized allopolyploid vertebrates is unclear (Chen et al., 2019a; Li et al., 2021; Luo et al., 2020). For X. laevis, there is a broad trend toward balanced homeolog expression across development and adult tissues, with a subtle bias in favor of the L homeolog that emerges after genome activation (Session et al., 2016), and an overall ontogenetic and transcriptomic trajectory similar to 48 million-years diverged diploid X. tropicalis (Harland and Grainger, 2011; Yanai et al., 2011). Initial observations in X. laevis have demonstrated differential homeologous enhancer activity in the eye (Ochi et al., 2017) and a divergent cis-regulatory landscape of histone modifications and recruitment of transcriptional machinery in the early gastrula (Elurbe et al., 2017), suggesting that embryonic genome activation is likely also asymmetric between the two subgenomes. Although Xenopus embryos have long been a model for understanding the MZT, for example (Amodeo et al., 2015; Charney et al., 2017; Chen et al., 2019b; Gentsch et al., 2019; Gibeaux et al., 2018; Gurdon et al., 1958; Kimelman et al., 1987; Newport and Kirschner, 1982a; Newport and Kirschner, 1982b; Paraiso et al., 2019; Skirkanich et al., 2011; Veenstra et al., 1999; Yanai et al., 2011), ZGA regulators have not previously been identified in X. laevis. Here, we identify maternal Pou5f3 and Sox3 as top-level regulators of X. laevis pluripotency and ZGA and elucidate the predicted enhancer architecture that differentially recruits them to homeologous gene copies between the two subgenomes. Despite differential subgenome activation, combined transcriptional output converges to proportionally resemble the diploid state, maintaining gene dosage for the embryonic pluripotency program. Results Identifying divergently activated homeologous genes At genome activation, the X. laevis pluripotency network consists of maternal regulators acting directly on the first embryonic genes (Figure 1B). To identify these genes, we performed a total RNA-seq early embryonic time course using our X. laevis-specific ribosomal RNA depletion protocol (Phelps et al., 2021; Figure 1A and B, Supplementary file 1). Subtle gene activation is observed in the blastula at Nieuwkoop and Faber (N.F.) stage 8, culminating in 4772 genes with significant activation by the middle of stage 9 (8 hours post fertilization [h.p.f.] at 23 °C) (Figure 1C, Supplementary file 2). Gene activation was detected through a combination of exon- and intron-overlapping sequencing reads deriving from nascent pre-mRNA (Lee et al., 2013) – indeed, two-thirds of these genes had substantial maternal contributions that masked their activation when quantifying exon-overlapping reads alone (Figure 1C). These genes fail to be activated in embryos treated at 1 cell (stage 1) with the transcription inhibitor triptolide (Gibeaux et al., 2018) when compared to DMSO vehicle control embryos (Figure 1B and C, Figure 1—figure supplement 1). To distinguish direct targets of maternal factors (primary activation) (Figure 1B), we then performed RNA-seq on stage 9 embryos treated with cycloheximide at stage 8, to inhibit translation of newly synthesized embryonic transcription factors that could regulate secondary activation (Harvey et al., 2013; Lee et al., 2013). A total of 2662 genes (56% of all activated genes) were still significantly activated in cycloheximide-treated embryos compared to triptolide-treated embryos, representing the first wave of genome activation in the embryo (Figure 1C, Supplementary file 1A). We analyzed subgenome of origin for activated genes and found that they are preferentially encoded as two homeologous copies in the genome (p=2.3 × 10–225, χ-squared test, 10 d.o.f.; Figure 2A). However, a majority of these genes have asymmetric expression between the two homeologs, often with transcription deriving from only the L or S copy alone (Figure 2B–C). This asymmetry is more pronounced at stage 8, but balances somewhat as genome activation progresses, suggesting timing differences for homeolog activation that could result from subtle regulatory divergence (Figure 2A, Figure 2—figure supplement 1A–C); and slightly less pronounced for strictly zygotic genes compared to maternal-zygotic genes (maternal contribution ≥1 TPM; Figure 2D, Figure 2—figure supplement 1D), which are often reactivated with different homeolog expression patterns compared to their maternal contribution (Figure 2E). Figure 2 with 2 supplements see all Download asset Open asset Homeologous genes are differentially activated in the early embryo. (A) Proportion of genes encoded as homeologs on both subgenomes versus only one subgenome (singleton) (left), as compared to expression patterns in the early embryo. p Values are from χ-squared tests, 10 d.o.f., comparing genomic to expressed proportions, 16 d.o.f., comparing proportions between activated genes and the maternal contribution, 12 d.o.f., comparing proportions at subsequent stages of activation. (B) Browser tracks showing log2 reads-per-million RNA-seq coverage of equivalently activated homeologs (top) and differentially activated homeologs (L-specific, middle; S-specific, bottom). Trip = triptolide, CHX = cycloheximide. (C) Biplot comparing log2 fold primary activation over triptolide treated embryos for the S homeolog (x axis) versus the L homeolog. (D) Left, proportion of genes activated symmetrically or asymmetrically from the L or S subgenomes, stratified into whether there is a maternal contribution for either homeolog (MZ) or not (Z) (p=0.02, χ-squared test, 4 d.o.f.); and whether a gene is activated only in the stage 9 blastula or is additionally increased in only one or more than one differentiated lineage from stages 10–13 (p=1.3 × 10–22, χ-squared test, 8 d.o.f.). Right, homeolog proportions of later gene activation in epidermal (Epi), neural progenitor (Neur), ventral mesodermal (Meso), and endodermal (Endo) lineages from stages 10–13 (p=3.3 × 10–64, χ-squared test comparing stage 9 and the four lineages, 16 d.o.f.). Lineage-specific gene expression data are from Johnson et al., 2022. (E) Homeolog-specific stage 9 activation proportions, versus maternal contribution homeolog expression patterns, for maternal-zygotic genes. (F) Concordance of homeolog activation patterns across the differentiated lineages at stages 10–13, for genes initially activated at stage 9 and also increased in at least two differentiated lineages. (G) Browser track showing strand-separated reads-per-million RNA-seq coverage over the mir-427 encoding locus on the distal end of Chr1L (v10.1). After genome activation, a heightened imbalance in favor of the L homeolog emerges, as measured by activation patterns in four differentiated cell lineages (Johnson et al., 2022; Figure 2D, Figure 2—figure supplement 1E–I), that appears to indicate a shift toward more divergent homeolog regulation as development proceeds, as was observed previously (Session et al., 2016). However, it is likely that some of the shared homeolog activation as measured in the whole blastula is actually composed of homeolog-specific regional activation (Chen and Good, 2022). Indeed, for one-third of genes activated in more than one lineage, different homeologs are activated in different lineages (Figure 2F, Figure 2—figure supplement 1F and G), and for those genes that are already activated at stage 9, this seems to result in a higher proportion of both-homeolog activation, as compared to genes with single-lineage or blastula-specific activation (p=1.3 × 10–22, χ-squared test, 8 d.o.f.). Overall, this indicates a high degree of divergent cis-regulatory architecture between gene homeologs throughout early development. Genes activated from both subgenomes are enriched in transcriptional regulators (p<0.01, Fisher’s exact test, two-sided) (Figure 2—figure supplement 1J), suggesting that gene function may have influenced homeolog expression patterns. However, there is no evidence for stronger functional divergence between homeologs expressed asymmetrically between the subgenomes, as estimated by non-synonymous versus synonymous mutation rate in coding regions (dN/dS ratio; Figure 2—figure supplement 1K and L). The microRNA mir-427 is encoded on only one subgenome Among the first-wave genes is the microRNA mir-427, which plays a major role in clearance of maternally contributed mRNA (Lund et al., 2009). Similar to X. tropicalis mir-427 (Owens et al., 2016) and the related zebrafish mir-430 (Lee et al., 2013), mir-427 is one of the most strongly activated genes in the X. laevis embryonic genome (Figure 2D, Figure 2—figure supplements 1C and 2A). In version 9.2 of the X. laevis genome assembly, the miR-427 precursor hairpin sequence is found in only five copies overlapping a Xenbase-annotated long non-coding RNA on chr1L (Figure 2—figure supplement 2B). To better capture the genomic configuration of the mir-427 primary transcript, we aligned the miRBase-annotated precursor sequence (Kozomara et al., 2019) to the version 10.1 X. laevis genome assembly. This revealed an expanded mir-427 locus at the distal end of Chr1L composed of 33 precursor copies, encoded in both strand orientations over 55 kilobases (Figure 2D, Figure 2—figure supplement 2A and B). The corresponding region on Chr1S is unalignable (Figure 2—figure supplement 2C), suggesting that mir-427 is encoded on only the L subgenome. We additionally found two mir-427 hairpin sequence matches to the distal end of Chr3S, but these loci were not supported by substantial RNA-seq coverage (Figure 2—figure supplement 2D). This is reminiscent of the X. tropicalis mir-427 genomic configuration (Owens et al., 2016), although smaller in scale and on a non-homologous chromosome. In X. tropicalis, 171 tandemly arrayed mir-427 precursors are found on the distal end of Chr03, which is thought to accelerate mature miR-427 accumulation during the MZT to facilitate rapid maternal clearance (Owens et al., 2016). Zebrafish similarly encodes a large array of more than 2000 mir-430 precursors, which begin to target maternal mRNA for clearance shortly after ZGA (Bazzini et al., 2012; Giraldez et al., 2006; Hadzhiev et al., 2023; Lee et al., 2013). These results strongly suggest that the mir-427 locus has undergone genomic remodeling, resulting in absence from the S subgenome, but possibly also translocation between chromosomes in the tropicalis or laevis lineages. Subgenomes differ in their regulatory architecture To discover the maternal regulators of differential homeolog activation, we first profiled embryonic chromatin using Cleavage Under Target & Release Using Nuclease (CUT&RUN) (Hainer and Fazzio, 2019; Skene and Henikoff, 2017), which we adapted for blastulae. We found that cell dissociation was necessary for efficient nuclear isolation to carry out the on-bead CUT&RUN chemistry (Figure 3A, Figure 3—figure supplement 1A–D). At stages 8 and 9, the active marks H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 27 acetylation (H3K27ac) were enriched in the transcription start site (TSS) regions of activated genes, and differential homeolog activation measured by RNA-seq significantly correlates with differential histone modification profiles, with a slight overall bias toward stronger L homeolog chromatin activity (Figure 3B and C, Figure 3—figure supplement 1E–G, Supplementary file 3). Differential promoter engagement by transcriptional machinery likely underlies the differential histone modification levels; however, we found no promoter sequence differences between homeologs that would implicate differential recruitment of specific transcription factors (Supplementary file 4). Figure 3 with 2 supplements see all Download asset Open asset Differential homeolog activation is regulated by subgenome-specific enhancers. (A) CUT&RUN coverage over all annotated transcription-start site (TSS) regions, sorted by descending stage 8 H3K27ac signal. (B) Bee-swarm plots showing the log2 ratio of L versus S homeolog coverage among genes where only one homeolog is activated (L only, S only), or both homeologs are activated. TSS region is 1 kb centered on the TSS; upstream region is 500 bp to 3 kb upstream of the TSS. Horizontal bars show medians. Individual category p values are from two-sided paired t-tests of log2 L homeolog coverage vs log2 S homeolog coverage, p values across the three categories are from a one-way ANOVA on the log2 ratios. (C) Stage 9 H3K4me3 CUT&RUN coverage over paired homeologous gene regions around the TSS (left) and maps comparing high-confidence predicted enhancer density near homeologous TSSs (middle). Differential predicted enhancers are active in only one subgenome, conserved predicted enhancers are active in both. Average densities are plotted to the right of each paired map. Gene pairs are sorted according to L versus S subgenome RNA-seq activation ratio (right). (D) Schematics showing aligned predicted enhancers and their homeologous regions (gray) mapped onto L (red, top lines) and S (blue, bottom lines) chromosomes. Comparable schematics show Xenbase annotated homeologous gene pairs (lavender). (E) Heatmap of stage 9 ATAC-seq and stage 8 H3K27ac CUT&RUN over L & S homeologous regions for equivalently active high-confidence predicted enhancers (top) and subgenome-specific predicted enhancers. (F) Top enriched transcription factor motif families in L-specific and S-specific active high-confidence predicted enhancers compared to inactive homeologous regions. FDR-corrected p-values from Homer are shown. RPM = reads per million. Instead, we searched for differences in gene-distal regulatory elements – that is enhancers – between the two subgenomes. To identify regions of open chromatin characteristic of enhancers, we performed Assays for Transposase-Accessible Chromatin with sequencing (ATAC-seq) on dissected animal cap explants; the high concentration of yolk in vegetal cells inhibits the Tn5 transposase (Esmaeili et al., 2020). Accessible chromatin is already evident at stage 8 in putative enhancer regions, though the overall signal is weak, and by stage 9, these regions exhibit robust accessibility (Figure 3—figure supplement 2A). We called peaks of elevated sub-nucleosome sized fragment coverage at stage 9, then intersected the open regions with our H3K27ac CUT&RUN. This yielded 15,654 putative open and acetylated gene-distal regulatory regions at genome activation, of which we classified 5047 as high confidence predicted enhancers that had ≥2 fold signal enrichment in each of at least three H3K27ac replicates and three ATAC-seq replicates individually (Figure 3—figure supplement 2A, Supplementary file 5). To identify homeologous L and S enhancer regions, we constructed a subgenome chromosome-chromosome alignment using LASTZ (Harris, 2007). This yielded a syntenic structure consistent with genetic maps (Figure 3D; Session et al., 2016), recapitulating the large inversions between chr3L/chr3S and chr8L/chr8S. Seventy-nine percent of predicted enhancer regions successfully lifted over to homeologous chromosomes, and of these, >92% of these are flanked by the same homeologous genes (Figure 3—figure supplement 2B), confirming local synteny. Among the paired regions involving high-confidence predicted enhancers, only 21% had conserved activity in both homeologs, with the remaining pairs exhibiting differential H3K27ac and chromatin accessibility (Figure 3E, Figure 3—figure supplement 2C). Differential predicted enhancer density around genes significantly correlated with differential activation (p=1.3 × 10–16, Pearson’s correlation test; Figure 3C, middle, Figure 3—figure supplement 2D), with greater L enhancer density around differentially activated L genes, and similarly for S enhancers and S genes. In contrast, conserved enhancers had equivalent density near both homeologs regardless of activation status (p=0.67, Pearson’s correlation test; Figure 3C, right). Thus, differences in enhancer activity likely underlie divergent gene homeolog transcription at genome activation. Maternal pluripotency factors differentially engage the subgenomes Given that these paired enhancer regions are differentially active despite having similar base sequences, we searched for transcription factor binding motifs that distinguished active enhancers from their inactive homeolog. Two motifs were strongly enriched in both active L enhancers and active S enhancers, corresponding to the binding sequences of the pluripotency factors OCT4 and SOX2/3 (SOXB1 family; Figure 3F, Supplementary file 4). Since mammalian OCT4 and SOX2 are master regulators of pluripotent stem cell induction (Li and Belmonte, 2017; Takahashi and Yamanaka, 2016), and zebrafish homologs of these factors are maternally provided and required for embryonic genome activation (Lee et al., 2013; Leichsenring et al., 2013; Miao et al., 2022), we hypothesized that differential enhancer binding by maternal X. laevis OCT4 and SOXB1 homologs underlies asymmetric activation of the L and S subgenomes. RNA-seq confirms high maternal levels of sox3 and pou5f3.3 (OCT4 homolog) mRNA, as well as lower levels of paralog pou5f3.2, each deriving from both subgenomes (Figure 3—figure supplement 2E). To assess their roles in genome activation, we inhibited their translation using previously validated antisense morpholinos (Morrison and Brickman, 2006; Takebayashi-Suzuki et al., 2007; Zhang et al., 2003) injected into stage 1 embryos. Combinations of pou5f3.3+sox3 morpholinos and pou5f3.2+pou5 f3.3 morpholinos led to mild and severe gastrulation defects, respectively, while combining all three morpholinos led to developmental arrest with a complete failure to close the blastopore (Figure 4—figure supplement 1A), consistent with what has been reported in X. tropicalis (Gentsch et al., 2019). RNA-seq of morpholino-treated embryos at stage 9 revealed extensive misregulation of genome activation, though only 15% of genes exhibited deficient activation, while 43% of genes actually exhibited slightly higher levels in the morphants (Figure 4A, Figure 4—figure supplement 1B and G–K), which could be due to direct or indirect transcriptional repression mediated by Pou5f3 and Sox3. Increases were predominantly detected from intron signal (Figure 4—figure supplement 1J), which would largely rule out post-transcriptional effects. A larger proportion of strictly zygotic genes were down-regulated in the morphants compared to maternal-to-zygotic genes (p=6.6 × 10-18, χ-squared test, 3 d.o.f.), perhaps reflecting a more complex regulatory network that regulates maternal gene reactivation (Figure 4—figure supplement 1L). Figure 4 with 2 supplements see all Download asset Open asset Pou5f3.3 and Sox3 binding drives genome activation. (A) Heatmap showing log2 fold activation differences for exonic and intronic regions of primary-activated genes for combinations of pou5f3.2, pou5f3.3, and sox3 morpholino-treatments, or Triptolide treatment, compared to controls. Right panel is in the presence of cycloheximide (CHX). (B) Biplot comparing exonic expression levels in cycloheximide-treated control embryos versus embryos also injected with pou5f3.2, pou5f3.3, and sox3 morpholinos. Primary-activated genes with maternal contribution <1 TPM (strictly zygotic) are purple circles, maternal-zygotic genes detected by exonic increases are orange triangles. TPM = transcripts per million. (C) Barplot summarizing the proportion of genes affected by morpholino treatment with cycloheximide on primary-activated genes (left bar), without cycloheximide (middle bar), and all stage 9 activated genes without cycloheximide (right bar). Down = significantly decreased in one of the morpholino treatments, up = significantly increased. (D, F) Biplots showing genes with >2 fold L or S biased activation (upper red and lower blue points, respectively) in control embryos (left panel) versus their activation in pou5f3.2, pou5f3.3, and sox3 morpholino-treated embryos (right panel, maintaining the same color per gene). (E, G) Quantification of the biplots in (D, F) in before-and-after plots. Y-axis is the absolute value of the log2 L vs S activation difference. p Values are from Wilcoxon signed-rank tests (paired). Overlaid boxplots show median, upper and lower quartiles, and 1.5 x interquartile range. (H) Regulatory networks consistent with direct regulation of embryonic gene activation by Pou5f3 and Sox3 (1) versus additional regulation by zygotic factors (2), which likely accounts for genes up-regulated in MO treatments. (I) Stage 8 Pou5f3.3 (left) and Sox3 (right) CUT&RUN coverage near TSSs for genes down-regulated in morpholino-treated embryos with or without cycloheximide (top), genes up-regulated (middle), and genes not significantly affected in any morpholino treatment (bottom). Top enriched motifs for each factor are shown below with p-values from Homer de novo discovery. (J) Aggregate plots of the binding signal in (I), with down-regulated genes further separated into genes down-regulated with morpholino treatment and cycloheximide (1°) or only down-regulated without cycloheximide (2°). p Values are from Kruskal-Wallis tests on summed signal per TSS. (K) Cumulative distributions of distance from a Pou5f3/Sox3-bound regulatory element for genes strongly (≥8 fold) and less strongly (<8 fold) down-regulated in morpholino-treated embryos with or without cycloheximide, compared to up-regulated, unaffected and unactivated genes. p Value is from a Kruskal-Wallis test. (L) Maps showing density of Pou5f3/Sox3-bound regulatory elements around paired homeologous TSSs, divided into elements with differential homeologous L & S binding (left panels) versus both bound (right panels). TSSs are grouped according to L versus S homeolog sensitivity to morpholino treatment. (M) Browser tracks showing CUT&RUN enrichment and ATAC-seq coverage near active homeolog hes3.L and inactive homeolog hes3.S. Seven L-specific high-confidence regulatory regions are highlighted with their homeologous S regions (bold ‘L’), as well as two lower-confidence enhancers, one of which also has weak activity in S, but minimal Pou5f3 or Sox3 binding (labeled ‘LS’). To further clarify the regulatory network, we also performed morpholino treatments followed by cycloheximide treatment at stage 8, collecting at stage 9 for RNA-seq, to focus the loss of function on primary activation. In these embryos, nearly 70% of first-wave genes were down regulated, including the mir-427 transcript (Figure 4A–C, Figure 4—figure supplement 1B–F, I), suggesting that maternal Pou5f3 and Sox3 directly activate a large proportion of first-wave genome activation, but newly synthesized zygotic factors rapidly mobilize to refine target gene expression levels (Figure 4H). In the absence of wild-type Pou5f3 and Sox3 activity, divergent homeolog activation is reduced for a subset of genes, indicating that these factors at least partially underlie differential subgenome activation (Figure 4D–G). Among primary-activated genes, there does not seem to be a strong bias toward greater regulation of either homeolog; however, a significantly larger proportion of strictly zygotic genes encoded on both subgenomes is activated by Pou5f3 and Sox3 compared to singleton genes (p=0.0020, χ-squared test, 5 d.o.f.; Figure 4—figure supplement 1M, N), which may reflect a reliance on these factors to mediate homeolog-specific expression when two copies exist. To interrogate Pou5f3 and Sox3 chromatin binding across the subgenomes, we performed CUT&RUN on stage 8 embryos injected with mRNA encoding V5 epitope-tagged pou5f3.3.L and sox3.S. Peak calling revealed thousands of binding sites for each factor" @default.
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• W4387585511 title "Decision letter: Hybridization led to a rewired pluripotency network in the allotetraploid Xenopus laevis" @default.
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