Sox17 and β-catenin co-occupy Wnt-responsive enhancers to govern the endodermal gene regulatory network

Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN in Xenopus gastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and β-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and β-catenin synergistically activate transcription independent of Tcfs, whereas on other enhancers, Sox17 represses ß-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/ß-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and ß-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts. Key findings Sox17 regulates germ layer segregation by promoting endoderm differentiation while simultaneously repressing mesectoderm fates. Functional interactions between Sox17 and canonical Wnt-signaling is a major feature of the GRN controlling endoderm specification and patterning. Sox17 and β-catenin co-occupy a subset of enhancers to synergistically stimulate transcription in the absence of Tcfs. Sox17 regulates the spatial expression domains of Wnt/β-catenin-responsive transcription in the gastrula.


ABSTRACT
Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN in Xenopus gastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and b-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and b-catenin synergistically activate transcription independent of Tcfs, whereas on other enhancers, Sox17 represses ß-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/ß-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and ß-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.

INTRODUCTION
During embryogenesis, the pluripotent zygote progressively gives rise to specialized cell types expressing distinct sets of genes that, in turn, define the cell's identity and encode proteins necessary for its function. Lineage specific gene expression is controlled by the genomic integration of signaling pathways and transcription factors (TFs) on DNA cis-regulatory modules (CRMs), such as enhancers, that control transcription (Heinz et al., 2015;Stevens et al., 2017).
An important goal of developmental biology is to elucidate how signaling effectors and TFs interact on distinct sets of CRMs within the chromatin landscape to form gene regulatory networks (GRNs) that activate lineage-specific transcriptional programs whilst repressing expression of alternative fates (Charney et al., 2017b). This will provide a deeper understanding of how transcriptional networks are established, dysregulated in disease, and how GRNs might be manipulated for therapeutic purposes.
We have addressed this in the context of endoderm germ layer specification: one of the earliest cell fate decisions in vertebrate development that provides a relatively simple model to elucidate how GRNs control lineage-specific transcriptional programs (Charney et al., 2017b). In embryos and pluripotent stem cells (PSCs), Nodal/Smad2 and Wnt/ß-catenin (Ctnnb1; hereafter Bcat) signaling cooperate to initiate the mesendoderm program and subsequent development of definitive endoderm progenitors, which gives rise to the epithelia of the digestive and respiratory systems (Sumi et al., 2008;Zorn and Wells, 2009). Downstream of Nodal and Wnt, a core set of endoderm TFs: Sox17, Gata4-6, Eomes, Foxa1/2 and Mix family of homeodomain TFs execute the endoderm GRN (Arnold et al., 2008;Charney et al., 2017b;Engert et al., 2013;Sinner et al., 2006). In Xenopus where endoderm specification is well studied, the Nodal and Wnt pathways interact at several levels. First, maternal (m) Wnt/Bcat, active on the dorsal side of the blastula, is required for high levels of nodal expression at the onset of zygotic transcription (Hyde and Old, 2000). Then Nodal and mWnt cooperate to promote the expression of endoderm TFs Sox17, Foxa2 and many dorsal mesendoderm organizer genes (Xanthos et al., 2002). A few hours later zygotic (z) wnt8 (a Nodal target) is expressed on the ventral side of the gastrula where, together with Nodal signaling, it promotes ventral and posterior mesendoderm identities (Charney et al., 2017b;Stevens et al., 2017). In mammals early Wnt/Bcat similarly activates expression of Nodal and core endoderm TFs, with prolonged Wnt promoting posterior hindgut fate (Engert et al., 2013;Zorn and Wells, 2009).
Functional interactions between the core endoderm TFs and the Wnt pathway are thought to 1) segregate the transient mesendoderm into endoderm and mesoderm, 2) pattern the nascent endoderm into spatially distinct subtypes and 3) execute the downstream differentiation program to give rise to endoderm-derived lineages. But mechanistically, how the Wnt signaling machinery interacts with core endoderm TFs to execute this differentiation cascade is unresolved. The transcriptional targets of the endoderm TFs are largely unknown, and it is unclear exactly how Wnt/Bcat regulates distinct spatiotemporal transcription programs in the embryo. Indeed, how the canonical Wnt pathway elicits context-specific transcriptional responses in its multitude of different biological roles from development, homeostasis and cancer is still poorly understood. According to the dogma of canonical Wnt signaling, Wnt-activated Fzd-Lrp5/6 receptor complexes sequester the Bcat degradation complex, resulting in the stabilization and translocation of Bcat to the nucleus. There, it interacts with one of four Tcf HMG-box TFs (Tcf7, Tcf7L1, Tcf7L2 and Lef1) at Wnt-responsive CRMs (Cadigan and Waterman, 2012;Schuijers et al., 2014), ultimately activating a multiprotein "Wnt-enhanceosome" with the scaffold proteins Bcl9, Pygopus and the ChiLS complex (Gammons and Bienz, 2018). In the absence of Bcat, Tcfs are coupled with corepressor proteins Tle/Groucho and histone deacetylase Hdac to inhibit transcription of Wnt target genes. Bcat displaces Tle and recruits a co-activator complex including histone acetyltransferases Ep300 or Cbp to stimulate transcription (Cadigan and Waterman, 2012). How distinct contextspecific Wnt target genes are selected is unclear since all Tcfs have nearly identical DNA-binding specificities (Badis et al., 2009;Ramakrishnan and Cadigan, 2017) and for the most part they are ubiquitously expressed. An emerging idea is that the Wnt-enhanceosome also interacts with other lineage-specific TFs to integrate lineage-specific inputs (Gammons and Bienz, 2018;Trompouki et al., 2011); yet, how these impact genomic specificity in vivo is largely untested and the idea of Tcf-independent Wnt-mediated transcription remains controversial.
In this study we investigated the possibility that Sox17 functionally interacts with Wnt/Bcat to regulate transcription in the Xenopus endodermal GRN. In all vertebrate embryos, Sox17 is specifically expressed in the gastrula endoderm where it is required for early gut development (Clements et al., 2003;Hudson et al., 1997;Kanai-Azuma et al., 2002;Viotti et al., 2014). Despite the critical role of Sox17 in endoderm development, only few of its direct transcriptional target genes have been identified (e.g.: hnf1b, foxa1 and dhh) (Ahmed et al., 2004;Sinner et al., 2004;Yagi et al., 2008). In Xenopus, ectopic Sox17 is sufficient to initiate endoderm development in pluripotent blastula animal cap cells (Clements et al., 2003;Hudson et al., 1997), and co-injection of stabilized Bcat can enhance this activity (Sinner et al., 2004). Sox17 can physically interact with Bcat in vitro and suppress the transcriptional activity of generic Tcf/Bcat reporter constructs (TOPflash) in tissue culture experiments (Sinner et al., 2007;Zorn et al., 1999). However, the biological relevance of these interactions and whether Sox17 and Bcat functionally interact on chromatin to regulate the endoderm GRN remains unknown.
Here, we defined the genomic targets of Sox17 in the Xenopus gastrula. In addition to promoting expression of endoderm genes, Sox17 also represses ectoderm and mesoderm gene transcription, and acts in a negative feedback loop to restrain Nodal signaling. We demonstrate that functional interaction with canonical Wnt signaling is a key feature of the Sox17-regulated GRN. Over a third of all Bcat and Sox17 genomic binding in the gastrula occur at the same CRMs.
In some instances, Sox17 suppressed Bcat-Tcf mediated transcription, while in other cases, Sox17 and Bcat synergistically activated enhancers apparently independently of Tcfs, indicating a novel mode of regulation. These results provide new insight into the GRN controlling endoderm development and have implications for how Sox TFs and Bcat might interact in diverse biological contexts from development to cancer.

Sox17 regulates a genomic program controlling germ layer segregation and endoderm development
To identify the transcriptional program regulated by Sox17, we performed RNA-sequencing (RNAseq) on control and Sox17-depleted Xenopus tropicalis embryos at multiple time points during blastula and gastrula stages (NF9-12) when the endoderm germ layer is being specified. In Xenopus tropicalis there are three redundant genes: sox17a, sox17b.1 and sox17b.2 (collectively sox17) with indistinguishable activities and identical expression in presumptive vegetal endoderm cells of gastrula embryos ( Fig. 1A and Fig. S1A) (D'Souza et al., 2003;Hellsten et al., 2010).
Microinjection of a combination of antisense morpholino oligos (sox17aMO and sox17bMO) targeting all 3 paralogs (Fig. S1B) resulted in a robust knockdown of Sox17 protein as confirmed by immunostaining (Fig. 1C,B). The Sox17-MO phenotype was consistent with previous reports (Clements et al., 2003;Sinner et al., 2006) and phenocopied mouse mutants with defective gut development (Kanai-Azuma et al., 2002). Injection of mRNA encoding mouse Sox17 rescued both the anatomical and gene expression phenotypes confirming the efficacy and specificity of the MOs (Fig. S1C,D).
We next investigated how Sox17-regulated genes were spatially expressed, leveraging a previously published RNA-seq of different tissues dissected from gastrula embryos (Blitz et al., 2017). As predicted, a majority of Sox17-dependent genes were enriched in the vegetal endoderm and dorsal mesendoderm (organizer) (Fig. 1E) including osr1, hnf1b, dhh and slc5a8 ( Fig. 1F and transcripts were modestly increased in Sox17-MO embryos even though previous work has demonstrated that Sox17 directly maintains its own transcription (Howard et al., 2007) after initial induction by Nodal/Smad2. These data indicate that Sox17 acts as a negative feedback inhibitor that restricts excessive endoderm development by restraining Nodal activity after initial induction.
The observation that over 10% of Sox17-regulated genes are enriched in the organizer mesendoderm while Sox17 is present throughout the endoderm, suggests that Sox17 might also functionally interact with the Wnt and/or Bmp dorso-ventral patterning pathways to control spatial expression. Intriguingly, we found that Sox17 regulates the expression of several Wnt/Bcat pathway components and targets including: dkk1, dkk2, fzd5, nodal3. 1, pygo1, sia1, ror1, rsop2 and wnt11 (Fig. 1D,F and Table S1). Together these data suggest that Sox17 regulates both endoderm specification and patterning while suppressing mesectoderm fate and that Sox17 participates in feedback loops with the Wnt and Nodal pathways.

Sox17 ChIP-Seq reveals direct endodermal targets and a Nodal and Wnt feedback
To identify direct Sox17-targets we generated and validated anti-Sox17 antibodies (Fig. S2) and performed ChIP-seq of gastrula (NF10.5) embryos, identifying 8436 statistically significant Sox17bound CRMs (IDR; p<0.05). These were associated with 4801 genes ( Fig. 2A), based on annotation to the nearest transcription start-site (TSS) by HOMER. 88% of Sox17-bound loci were in introns or intergenic regions more than 1kb away from the TSS, consistent with distal CRMs (Fig. S3A). A comparison to published ChIP-seq data of Xenopus embryos at the same stage (Hontelez et al., 2015) showed that most of the Sox17-bound genomic loci were also bound by Ep300, (Fig. 2B, E) indicative of active enhancers. Motif analysis of the ChIP-seq peaks confirmed that Sox17 motifs were the most enriched, as expected ( Fig. 2C and Fig. S3B).
A comparison to published human ChIP-Seq data revealed that 20% of the Xenopus Sox17-bound genes were also SOX17-bound in PSC-induced definitive endoderm ( Fig. S3) (Tsankov et al., 2015). GO analysis of the conserved SOX17-bound genes show an enrichment for 'Tgfb receptor activity' and 'Bcat binding' (Fig. S3) reinforcing the notion that functional interaction with the Nodal and Wnt pathways is a conserved feature of the Sox17-regulated endoderm GRN.
Intersecting the RNA-seq and ChIP-seq data identified 315 genes associated with 609 Sox17-bound enhancers, which are likely to be direct transcriptional targets ( Fig. 2A). These putative direct targets had significantly enriched (44%, 139/315 transcripts) expression in the endoderm or dorsal mesendoderm (Fig. 2D). Of the Sox17-bound and regulated genes, 197 genes (383 peaks) were positively regulated by Sox17 (down in Sox17MOs) including slc5a8, hnf1b, foxa1 and dhh (Fig. 2E), all of which were previously suggested to be direct Sox17-targets.
Sox17 negatively regulated 118 putative direct targets (up in Sox17MOs) including ectodermal genes (lhx5, foxi2 and tfap2a), endoderm-enriched Nodal pathway genes (nodal1, gdf3, gdf6 and mix1) and Wnt-regulated organizer genes (dkk and fst), suggesting direct transcriptional repression (Fig. 2E). Interestingly ~45% of peaks associated with Sox17-activated genes were also enriched for LIM-homeodomain binding sites, in contrast to peaks from Sox17-repressed genes which were enriched for Tbx or Pou motifs (Fig. S3B). This suggests that Sox17 may coordinately engage enhancers with other core endoderm GRN TFs and mediate activation or repression of target genes depending on the interacting TFs.
These analyses provide new insight into the endoderm GRN and reveal previously unappreciated roles for Sox17 in germ layer segregation and endoderm patterning involving functional interactions with the Nodal and Wnt pathways. These findings, together with our previous work demonstrating that Sox17-Bcat can physically interact in vitro (Sinner et al., 2007;Zorn et al., 1999) prompted us to investigate their genomic interactions.

b-catenin directly regulates the endodermal transcriptome
To test the hypothesis that Sox17 and Bcat functionally interact in vivo, we set out to identify Bcatregulated genes and compare these to the Sox17-regulated transcriptome. Injection of a well-  Table S2).
Remarkably the Bcat-dependent genes encoded 251 TFs, (~20% of all the TFs in the genome), reinforcing the notion that Wnt/Bcat initiates a transcriptional cascade in the early embryo.
Intersecting the Bcat-MO RNA-seq data with previously published Bcat ChIP-seq data As expected, many of the positively regulated Bcat targets had enriched expression in the dorsal mesoderm ( Fig. 3D) including most known organizer genes such as cer1, chrd, dkk1, frzb, fst and gsc. In contrast ~30% of Bcat-bound genes with increased expression in the early Bcat-MO embryos (NF7-9) were enriched for ectoderm specific transcripts, consistent with the expansion of ectoderm and loss of neural tissue in ventralized embryos (Fig. 3D). We also identify many direct Bcat targets that are likely to be regulated by zygotic Wnt8, (Kjolby and Harland, 2017;Nakamura et al., 2016) including sp5, axin2, cdx1/2, fzd10, msgrn1 and eight hox genes, all of which were downregulated in Bcat-MOs (Fig. 3A, E). A number of zygotic Wnt-targets with enriched expression in the ventral mesoderm were components of BMP pathway including; bambi, bmp7, id2, msx1, smad7, szl, ventx1, and ventx3 (collectively known as the BMPsynexpression group) (von Bubnoff et al., 2005). Most of these genes are known to be directly activated by both Bmp4/Smad1 and zWnt8/Bcat (Itasaki and Hoppler, 2010;Stevens et al., 2017) but were upregulated, rather than downregulated, in Bcat-depleted embryos, likely due to the increase in BMP/zWnt8 signaling in ventralized embryos.
Most importantly, almost 200 Bcat-bound and regulated genes had endoderm enriched expression including most of the core endoderm TFs; sox17, foxa, eomes, gata4, mix1 and mixer ( Fig. 3D and Table S2). Thus, contrary to the prevailing view that Bcat promotes endoderm fate primarily by activating Nodal ligand expression (Blythe et al., 2010;Hyde and Old, 2000), we find that Wnt/Bcat also directly regulates the transcription of many endodermal genes. Surprisingly, de novo motif analysis showed that the Bcat bound peaks were more enriched for Sox than canonical Tcf motifs ( Fig. 3C and Fig. S5A), supporting the hypothesis that Sox17 and Bcat coregulate endodermal transcription.

Sox17 and b-catenin co-occupy endoderm enhancers
Intersection of the ChIP-seq datasets identified 3956 genomic loci co-occupied by both Sox17 and Bcat, and these had epigenetic signatures of active enhancers (Fig. 4A,B). This represents a third of all Sox17 genomic binding in the gastrula genome. Comparison of the Sox17-MO and Bcat-MO RNA-seq datasets identified 415 transcripts that were regulated by both Sox17 and Bcat; comprising of ~40% of the Sox17-regulated and ~15% of the Bcat-regulated transcriptome ( Fig. 4A). These data suggest that co-regulation with Wnt/Bcat is a major feature of the endoderm GRN.
In total, 84 genes (191 Peaks) were bound and regulated by both Sox17 and Bcat ( Fig.   4A,E). This is likely to be an underestimation of the number of direct coregulated genes, as these had to pass a stringent thresholding of four independent statistical tests to be included in this list.
A comprehensive motif analysis of the 191 co-bound and coregulated peaks using the CIS-BP database (Lambert et al., 2019) revealed that 78% (149/191) of the peaks contained both Sox17 and Tcf DNA-binding motifs, often > 5 sites in each peak (e.g., dkk1, lhx5 and osr1) (Fig. S6 B,D, Table S5). In contrast 21% (40/191) of the peaks had Sox motifs but no obvious Tcf consensus sites (e.g., six1), suggesting that Bcat might be recruited to these loci independent of Tcfs. Only 1/191 peaks had Tcf but no Sox motifs. In most cases individual genes were associated with multiple Sox17 and Bcat co-occupied peaks, suggesting that combinatorial binding and integration of several CRMs is crucial to control transcription (Fig. 4E).
Since the ChIP-seq experiments were performed on whole embryos, it was possible that Sox17 and Bcat chromatin binding might occur in different cell populations. To test whether Sox17 and Bcat bound the same enhancers in the same cells we performed ChIP-reChIP experiments.
First anti-Bcat ChIP was performed on 500 gastrulae. DNA eluted from the first ChIP was then re-precipitated with either anti-Sox17 or a negative control IgG antibody. All 4 loci tested by ChIP-reChIP QPCR (six1, dkk1, lhx5 and gli1) showed enrichment compared to IgG demonstrating that these genomic loci (~150 bp in length) were simultaneously bound by Sox17 and Bcat in endodermal cells. In contrast a negative control locus gtse1 which is bound by Bcat but not Sox17 was not recovered in the ChIP-reChIP experiment (Fig. 4D).
The 84 co-bound and coregulated genes fell into 4 regulatory categories (Fig. 4C, E); 32/84 were activated by both Sox17 and Bcat (e.g., six1 and osr1) and tended to have endoderm enriched expression, 27/84 were activated by Bcat but repressed by Sox17 (e.g., dkk1) and these tended to be enriched in the organizer mesendoderm, 19/84 were positively regulated by Sox17 and negatively regulated by Bcat (up in the Bcat MO) and finally 6/84 appeared to be negatively regulated by both Sox17 and Bcat. The observation that Bcat positively regulated most of these genes (59/84, 70%) was consistent with its known role in transcriptional activation. Sox17 positively regulated 60% of targets and repressed 40% of targets, suggesting context-dependent activity. For further analysis we focused on the two major regulatory groups; genes/enhancers activated by both Sox17-Bcat and activated by Bcat but repressed by Sox17 (Fig. 4E).

Sox17 and b-catenin coordinate spatial expression domains in the embryo
It was possible that the overlap in Sox17-and Bcat-regulated genes was largely due to the Sox17-Wnt feedback loop where Bcat is required for robust sox17 expression and Sox17, in turn, regulates the expression of Wnt-pathway components. While this is likely to be part of the mechanism, we directly tested the functional necessity for both Sox17 and Bcat in epistasis experiments, asking whether injection of sox17 RNA could rescue coregulated genes in Bcat depleted embryos and vice versa. Co-injection of mSox17 mRNA could not rescue the normal expression patterns of six1, osr1, lhx5 or dkk1 in Bcat-MO embryos, but it did rescue foxa1 expression (Fig. 5). This demonstrates that reduced Sox17 in Bcat depleted embryos cannot account for the disrupted six1, osr1, lhx5 or dkk1 expression, but it can account for the reduced foxa1 expression. Similarly, co-injection of stabilized (ca)Bcat mRNA rescued the normal expression of foxa1 but not six1, osr1, lhx5 or dkk1 in Sox17-morphants indicating that the defects in their expression were not simply due to reduced Wnt signaling (Fig. 5).
These experiments also show that Sox17 and Bcat coordinate spatial gene expression in the embryo in a complex gene-specific manner. Wnt/Bcat stimulates expression of lhx5 in the ectoderm, but Sox17 represses this activity in the presumptive endoderm. Sox17 and Bcat are both required for robust expression of osr1 and six1 in the endoderm and dorsal mesendoderm respectively. In contrast Sox17 normally suppresses Bcat-activated expression of dkk1 in the deep endoderm. Together, with their co-occupancy at CRMs, these data show that Sox17 and Bcat are both required to directly regulate transcription in discrete spatial domains.

Sox17 and Bcat synergistically regulate transcription of endodermal enhancers.
To examine how Sox17 and Bcat directly regulate endodermal transcription, we selected four exemplar co-occupied enhancers: a) a proximal six1 enhancer located -1kb from the TSS, b) a proximal dkk1 enhancer located -1.6kb from its TSS (Fig. 6B), c) a distal -7.5kb dkk1 enhancer and d) a -1kb lhx5 enhancer (Fig. S7C, E). We focused most of our analysis on six1 and dkk1 which are both expressed in the dorsal mesendoderm but have opposite regulation. Sox17-Bcat promotes six1, whilst Sox17 represses Bcat activation of dkk1 (Fig. 5).
The -1kb six1 enhancer is evolutionarily conserved from Xenopus to human containing three Sox-binding sites but no predicted Tcf binding sites (Fig. 6B). In mouse, this enhancer can drive transgenic expression in the embryonic gut tube, although the TFs that modulate it were previously unknown (Sato et al., 2012). Analysis of published ChIP-seq data indicate that, like in Xenopus, the human SIX1 CRM is also bound by SOX17 in human PSC-derived endoderm (Fig.   S7A) (Tsankov et al., 2015). Mammalian Dkk1 is also known to be a direct Bcat-Tcf target (Niida et al., 2004), but the enhancers that regulate its expression in the gastrula had been previously uncharacterized. Unlike the -1kb six1 enhancer, the -1.6kb and -7.5 dkk1 enhancers as well as the -1kb lhx5 enhancer harbors both Tcf and Sox DNA-binding sites (Fig. 6B, S7 and file S1).
We cloned each of these CRMs into luciferase (luc) reporter constructs and tested whether they require Sox17 and Bcat for enhancer activity in Xenopus embryos (Fig. 6A). Targeted microinjection into different cells of 32-cell-stage Xenopus embryos showed that all the reporters recapitulated the endogenous spatial expression indicating that they are bona fide enhancers.
The six1 and dkk1 reporters were both active in the dorsal-anterior endoderm, but not the ventral endoderm or ectoderm (Fig. 6C,E, S7F), whereas the lhx5 reporter was only active in the dorsal ectoderm (Fig. S7D). Depletion of endogenous Sox17 or Bcat abolished expression of the -1six1:luc (Fig.6C), and this was rescued by adding back mSox17 to Sox17-MO or by adding back caBcat mRNA to Bcat-MO embryos. However, co-injection of mSox17 could not rescue expression in Bcat-MO embryos and nor could caBcat RNA rescue expression in Sox17-MOs ( Fig. 5, 6C). These epistasis experiments indicate that both Sox17 and Bcat are required for activation of the -1kb six1 enhancer. The human SIX1:luc construct exhibited identical activity to the Xenopus six1:luc enhancer (Fig. S7B). In contrast, in the case of the -1.6kb dkk1, -7.5kb dkk1 and the -1kb lhx5:luc constructs, depletion of Bcat abrogated reporter activity, whereas Sox17 depletion resulted in significantly elevated reporter activity, indicating that Sox17 restricts Bcatmediated transactivation of the dkk1 and lhx5 enhancers (Fig. 6E, S7D,F).
To test whether Sox17 and Bcat were sufficient to regulate the enhancers, we assayed their activity in ventral ectoderm tissue which does not express endogenous Sox17 and has low Wnt/Bcat activity. Injection of mSox17 but not caBcat mRNA was sufficient to stimulate the -1kb six1:luc and human SIX1:luc reporters in the ectoderm, but co-injection of caBcat along with mSox17 synergistically activate the reporters (Fig. 6C, S7B). On the other hand, caBcat but not mSox17 was able to activate the dkk1 and lhx5 reporters in ventral ectoderm, and co-injection of mSox17 suppressed the ability of caBcat to stimulate these reporters (Fig. 6E, S7D,F), consistent with the endogenous regulation (Fig. 5). The ability of Sox17 to activate the six1 reporters or suppress Bcat-induced activation of the dkk1 and lhx5 reporters required the DNA-binding function of Sox17. A M76A point mutation in mSox17, known to disrupt the DNA-binding HMG domain (Sinner et al., 2007) significantly reduced Sox17's ability to (Fig. 6C, E, S7B,D,F).
The six1 enhancer is predicted to have only Sox17 sites with no evidence of Tcf-binding sites, whereas the dkk1 and lhx5 enhancers have both Sox17 and Tcf sites. To test whether or not Tcfs regulate these enhancers, we injected a Tcf7l1-VP16 fusion construct which constitutively activates Tcf-target gene transcription (Darken and Wilson, 2001). As predicted, this had little to no impact on the six1 reporter, but robustly activated the dkk1 and lhx5 reporters in ventral ectoderm (Fig. 6D,F, S7B,D,G). We next mutated the Sox17 and Tcf binding sites in the six1 and dkk1 enhancers and examined the impact on Sox17, Bcat and Tcf regulation. Loss of the Sox17 sites abolished normal and Sox17/Bcat-induced expression of the six1:luc reporter (Fig. 6D). In contrast mutation of the Sox sites in the enhancers resulted in elevated reporter activity consistent with Sox17-mediated repression ( Fig. 6F and S7G). Deletion of the Tcf sites in the dkk1 enhancers abolished expression in the dorsal mesendoderm as well as Bcat or Tcf7l1-VP16 induced activation in the ventral ectoderm, consistent with Bcat-Tcf complexes activating these enhancers ( Fig. 6F and S7G).
From these collective data, we conclude that Sox17 and Bcat synergistically activate the six1 enhancer, likely in a Tcf-independent manner. In contrast, the dkk1 and lhx5 enhancers are activated through a Bcat/Tcf transcription mechanism, with Sox17 spatially restricting Bcat/Tcf activity to maintain cell-type specific gene expression (Fig. 7).

Overview of findings
Sox17 is a key regulator of vertebrate endoderm development and morphogenesis, yet surprisingly, the transcriptional program that it regulates has been poorly characterized until now.
Here we defined the Sox17-regulated GRN in Xenopus gastrulae, which includes many conserved SOX17 genomic targets in PSC-derived human definitive endoderm. We show that in addition to acting atop a transcriptional hierarchy promoting endoderm differentiation, Sox17 has previously unappreciated roles in endoderm patterning and germ layer segregation. We demonstrate that functional interaction with the canonical Wnt signaling pathway is a major feature of the Sox17-regulated GRN. Over a third of all Bcat and Sox17 genomic binding events in the gastrula occur at the same enhancers, where they coordinately regulate spatial gene expression in the embryo. Our data suggest that Sox17 and Bcat synergistically activate a subset of endodermal enhancers in the absence of Tcfs, whereas at other enhancers, Sox17 modulates Bcat-Tcf mediated transcriptional activation. Together these results provide novel insights into the establishment of the endoderm GRN and suggest a new paradigm where Sox17 acts as a tissue specific modifier of Wnt-signaling responses.

The Sox17-Bcat regulated endodermal GRN
Our genomic study revealed an extensive overlap between Sox17-and Bcat-regulated genes.
Together with previous work (Charney et al., 2017b;Zorn and Wells, 2009), this suggests that Sox17 and the other core endoderm TFs interact with Wnt and Nodal in a series of positive and negative feedback loops to control germ layer segregation and modulate spatial gene expression.
We showed that in addition to its known role in activating transcription of endoderm-inducing nodal ligands (Blythe et al., 2010;Hyde and Old, 2000), Bcat directly promoted the expression of many endodermal genes including most of the core endoderm TFs; sox17, eomes, foxa, gata4 /5, mix1 and mixer. Sox17 also regulated the expression of other endoderm GRN components; promoting expression of foxa1 and gata4, whilst negatively regulating the expression of some nodal and mix/bix genes. Thus on one hand, Sox17 promotes endodermal fate, while it restrains excessive Nodal signaling on the other. There is precedence for Sox factors influencing nodal ligand transcription. Maternal Sox3, localized in the blastula ectoderm, directly binds the nodal5/6 enhancers to repress Bcat-stimulated transcription, whereas vegetally localized maternal Sox7 enhances nodal5/6 expression in the vegetal cells (Zhang and Klymkowsky, 2007). This suggests a potential handoff model where different Sox TFs might co-occupy the same enhancers with Bcat in different cells to activate or repress transcription in a context-dependent manner.
Our data demonstrate that Sox17-Bcat interactions regulate spatial gene expression in the embryo (Fig. 7). Sox17-Bcat interact at some enhancers like six1 and osr1 to promote transcription in the endoderm and dorsal mesendoderm. In addition, Sox17 also has a key role in repressing alternative lineages in the vegetal cells, similar to the maternal TFs Foxh1, Vegt and Otx1 (Chiu et al., 2014;Paraiso et al., 2019). For example, Sox17 represses Bcat/Tcf-stimulated expression of the ectoderm specifying TFs lhx5 (Houston and Wylie, 2003). Sox17-Bcat interactions also impact spatial expression within the endoderm. Sox17 inhibits the Bcat-Tcf stimulation of the dkk1 enhancer in the deep endoderm, but in the dorsal mesendoderm where Bcat activity is higher, Sox17 appears insufficient to suppress dkk1. The finding that six1 and dkk1 are under opposite regulation by Sox17 and Bcat despite having very similar expression patterns warrants future analysis of the temporal-spatial dynamics in Bcat, Tcf and Sox17 genomic occupancy in dorsal vs ventral cells over time.

DNA Binding Specificity of SOX TFs
An important consideration is how Sox17 selects specific target enhancers. In DNA-binding experiments, all Sox TFs bind to variants of the DNA sequence 5

'-(A/T)(A/T)CAA(A/T)3', which is similar but distinct from Tcf DNA-binding sites 5'-T(A/T)(A/T)CAAG 3' (Hou et al., 2017; She and
Yang, 2015). SOX TFs can act either as monomers or heterodimers with different cell-type specific cofactors, which in turn impact their DNA-binding affinity and ability to activate or repress transcription (Bernard and Harley, 2010;Hou et al., 2017). For example, in PSCs, Oct4-Sox2 heterodimers promote pluripotency and bind to distinct sites than those bound by Oct4-Sox17 complexes that initiate mesendoderm development (Aksoy et al., 2013). It is, therefore, likely, that Sox17 and Bcat cooperate with other TFs to provide lineage specificity to Wnt-responsive transcription during germ layer segregation.
Motif analysis revealed that Sox17 bound loci were also enriched for Tbx and Gata motifs indicating that in addition to regulating each other's expression, the core endoderm TFs act together in combinatorial manner, perhaps to establish endodermal super enhancers crucial for downstream lineage specification (Paraiso et al., 2019). Interestingly, enhancers that were negatively regulated by Sox17 were enriched for Tbx and Oct motifs whereas enhancers activated by Sox17 were enriched for homeodomain motifs. This suggests that Sox17 interactions with distinct lineage-specific TFs could elicit a context-specific transcriptional response. In the future, it will be important to identify and integrate the genomic targets of all the core endoderm-specifying TFs and examine how combinatorial enhancer binding is integrated into a unified endodermal GRN.

Sox-Bcat-Tcf Interactions on chromatin
Many SOX proteins, including Sox17, can bind to recombinant Bcat and Tcfs in vitro and modulate Wnt/Bcat-stimulated transcription of synthetic reporters assays with multimerized Tcf-sites (pTOPFlash) (Sinner et al., 2007;Zorn et al., 1999). While a number of models have been proposed to explain how Sox factors might impact Bcat activity, (Kormish et al., 2010;She and Yang, 2015;Tan et al., 2019), in most cases the in vivo biological relevance of these interactions was unclear. Although the resolution of ChIP-reChIP assays only allow us to demonstrate that Sox17 and Bcat bind chromatin within 150bp of each other, taken together with published in vitro data, our results suggest that Sox-Bcat or Sox-Bcat-Tcf transcriptional complexes can be assembled on enhancers in vivo.
At enhancers like six1 where Sox17 and Bcat synergistically stimulate transcription, we postulate that Sox17 and Bcat recruit Ep300/Cbp to chromatin, analogous to Tcf-Bcat in canonical Wnt-signaling. Consistent with this, structure-function studies have found that a conserved 10 amino-acid domain (VDXXEFEQYL) in the Sox17 C-terminus is required for both Bcat-binding and transcriptional transactivation (Sinner et al., 2004). On the other hand, most Bcat and Sox17 co-occupied enhancers contain multiple Sox and Tcf sites clustered within 50 bp, suggesting that Sox17 and Tcf, together with Bcat and scaffolding proteins like Bcl9, (Gammons and Bienz, 2018; Masuda and Ishitani, 2017) might assemble higher-order multiprotein complexes facilitating Wntmediated transcription. Both Soxs and Tcfs are HMG-domain containing TFs, capable of binding to the minor groove of DNA and inducing DNA bending. This could further indicate that they are brought into close spatial proximity to establish the assembly of functional enhanceosomes, despite the distance between their binding sites on linear DNA (Bernard and Harley, 2010;Hou et al., 2017).
The possibility of Sox-Bcat-Tcf ternary complexes raises the question of competitive vs cooperative DNA binding that, in turn, could influence transcriptional repression versus activation.
For example, Sox17 might stabilize the formation of Bcat-Tcf-Ep300 complexes or destabilize the binding of co-repressors such as Tle and Hdac to promote transcription (e.g., osr1). To negatively regulate Bcat-Tcf activity (e.g.,dkk1) Sox17 might destabilize Bcat-Tcf-Ep300-DNA or stabilize Tcf-Tle complexes. This idea is supported by work in mouse cortical neuron progenitors where Sox2 binds within 4-10bp of Tcf/Lef sites and recruits Tle/Gro corepressors to repress Tcf/Bcatmediated Ccnd1 expression (Hagey and Muhr, 2014). Future biochemical studies will need to determine if and how Sox-Bcat-Tcf complexes assemble on DNA.

Broader Implications
Although a growing number of non-Tcf Bcat-binding TFs have been reported, (Gammons and Bienz, 2018;Notani et al., 2010;Trompouki et al., 2011), how these impact the genomic specificity of Wnt-responsive transcription has not been well characterized and the idea of functional Tcfindependent Wnt transcription remains controversial (Doumpas et al., 2019;Schuijers et al., 2014). Our data suggest that in the gastrula endoderm, Sox17 might influence Bcat recruitment to chromatin to control cell-type specific Wnt-responsive transcription, and in some cases, this might could occur independent of Tcfs. Given that there are 20 Sox TFs encoded in the vertebrate genome with most cell types expressing at least one, it is possible that SOX factors are widespread and previously unappreciated accessory effectors of Bcat that finetune Wnt-regulated transcription across many biological contexts.

Xenopus embryo manipulations
Xenopus laevis and Xenopus tropicalis were purchased from Nasco or the National Xenopus Resource and experiments were performed according to CCHMC IACUC approved protocols.

In situ hybridization
In situ hybridization of Xenopus tropicalis embryos was performed as described (Sive et al., 2000) with the following minor modifications. After overnight fixation at 4°C in MEMFA, embryos were washed (2x10 minutes) in MEMFA buffer without the formaldehyde and stored in 100% ethanol at -20°C. Embryos were serially rehydrated to PBS+0.01% Tween-20 and bisected through the dorsal-ventral axis in on 2% agarose coated dishes using a fine razor blade (Electron Microscopy Services 72003-01), followed by Proteinase K (ThermoFisher AM2548) treatment at 1ug/mL for 10 minutes. TEA/Acetic Anhydride steps were skipped on day1. The RNAse A step was 0.25ug/mL for 5 minutes on day 2; and finally the anti-DIG-alkaline phosphatase antibody (Sigma 11093274910) was used at a 1:6,000 dilution in MAB buffer + 20% heat-inactivated lamb serum + 2% blocking reagent (Sigma 11096176001) on day2/3. Anti-sense DIG labeled in situ probes were generated using linearized cDNA templates with the 10X DIG RNA labeling mix (Sigma 11277073910) according to manufacturer's instructions. Details on plasmids used for probe synthesis are listed in supplementary table S7.

Antibodies and Immunostaining
Affinity purified anti-Xenopus Sox17 rabbit polyclonal antibodies were generated by Bethyl labs to the Sox17a/b N-terminal, Sox17b C-terminal and Sox17a C-terminal with the peptides illustrated in Fig. S2.
Antibodies were validated by immunostaining, Western blot, immunoprecipitation and ChIP. Immunoreactivity was not detected in Sox17 deficient tissue and specifically competed by addition of the target peptide to reactions. In preliminary ChIP-qPCR experiments, the pan-Sox17 and Sox17bC-terminal antibodies bound to the same loci, but the Sox17bC-terminal was more efficient and was used for ChIP-seq.

RNA isolation and library preparation:
Approximately 200 X. tropicalis embryos were injected with 8 ng of either control-MO or Sox17-MO, or 2 ng of control-MO and Bcat-MO and 10 sibling embryos were collected at every time point from blastula to late gastrula in biological duplicate.
Total RNA was extracted with the Nucleo-spin RNA kit (Machery-Nagel). RNA-seq libraries were generated using Smart-seq2 cDNA synthesis followed by tagmentation, quality-tested using an Agilent Bioanalyzer 2100, quantified using KAPA qPCR and sequenced using Illumina sequencers at the UC Irvine Genomics High Throughput Facility.

RNA-Seq Analysis:
Raw reads were quality checked using FastQC and adapters/low quality reads were removed with Trimmomatic (Bolger et al., 2014).  (Risso et al., 2014). Gene ontology enrichment analysis was performed using the Gene Ontology Consortium Online Resource (http://geneontology.org).
To assess the spatial expression of transcripts we examined the TPM expression values in dissected stage 10.5 embryos from published data in GEO GSE81458 (Blitz et al., 2017).
Enriched expression in a specific tissue was defined as the normalized expression being >1.5x of the mean expression across dissected tissues. Statistical significance of enriched expression pattern in groups of genes was determined by chi-square test p<0.05 performed in R package MASS.

Chromatin Immunoprecipitation
ChIP-Seq: Large scale Sox17 ChIP was carried out as previously described (Charney et al., 2017a) using 2000 NF10.5 X. tropicalis embryos and 30 µg rabbit anti-Sox17b-Cterminal antibody. ChIP-seq libraries were generated using Nextflex ChIP-seq kit (Bio Scientific), analyzed using an Agilent Bioanalyzer 2100, quantified using KAPA qPCR and sequenced using Illumina instruments at the UC Irvine Genomics High Throughput Facility.
ChIP-qPCR: was carried out largely as described in (Charney et al., 2017a;Hontelez et al., 2015) with ChIP-reChIP: Before the first ChIP, the anti-b-catenin antibody was crosslinked to Protein G Dynabeads. ChIP assay was then carried out on extracts from 500 embryos as described above.
At the end of the first ChIP, DNA was eluted with elution buffer supplemented with 10mM DTT.
The eluate was then diluted in 10 volumes of wash buffer (50mM Tris-HCl pH 8.0, 100mM NaCl, 2mM EDTA, 1% NP-40) supplemented with 1x Protease Inhibitor Cocktail and 1mM DTT. The 2 nd ChIP was then carried out as a normal ChIP as described above.  GSE72657 (Nakamura et al., 2016) with an IDR threshold of 0.01. The location of peaks (intron, intergenic, exon and promoter) were annotated using HOMER v4. 1.0 (Heinz et al., 2010). To visualize the data, aligned bam files generated as above were converted to bigwig format using deeptools v3.4.1 and visualized on IGV v2. 4.1.3 (Thorvaldsdottir et al., 2013). Significance of intersected ChIP-seq and RNA-seq datasets was assessed using hyper geometric test (HGT).

Motif analysis:
De-novo motif analysis was performed with HOMER v4. 1.0 (Heinz et al., 2010). results were merged to generate BED files and Bedtools v2.27.1 (Quinlan and Hall, 2010) intersect function was then used to extract coordinates of 'Sox only' 'Tcf only' and 'Sox and Tcf overlapping' categories.
Peak density heatmaps: From the Sox17 and Bcat peaks called by MACS2, Bedtools intersect function was used to stratify the peaks into 'Sox bound only', 'Bcat bound only' and 'Sox and Bcat overlapping peaks'. For each category of peaks, the peak summit was extracted from the TF summit file generated by MACS2 and 2000 bp were added to both sides of the summit using awk.
Bedtools computematrix was used to generate per genome regions. The generated matrix was then used with bedtools plotHeatmap to generate heatmaps and signal density plots.

Analysis of public datasets:
The following datasets were downloaded from GEO: Embryo dissection RNA-seq; GSE81458 (Blitz et al., 2017), GSE67974 (Hontelez et al., 2015). Wherever necessary, raw data was downloaded from GEO and aligned to Xenopus genome version 9.0, and processing to generate bigwig files and heatmaps was carried out as described above.

Data and software availability:
The RNA-seq and ChIP-seq data generated by this study have been deposited in the NCBI Gene Expression Omnibus (GEO) and can be accessed under GSE148726 (reviewer password: choluuuefpkprsb).

Fig. 5 Sox17 and Bcat epistatsis
In situ hybridization of gastrula embryos (dorsal right) injected with sox17-MO or bcat-MO some of which were co-injected with RNA encoding either mSox17 or a human S37A stabilized Bcat. The genes osr1, six1 and foxa1 are activated by both Sox17 and Bcat, whereas dkk1 and lhx5 are activated by Bcat but repressed by Sox17. Co-injection of mSox17 cannot resuce normal expression in Bcat-MOs, nor can hBcat S37A RNA rescue Sox17-MOs indicating that both Sox17 and Bcat required for normal expression.  Sox17 exerts a repressive indluence on Bcat/Tcf-activation of lhx5 and dkk1. We postulate however that in the dorsal region Sox17-mediated repression is insufficient to overcome Bcat/Tcf activation of dkk1. (C) In deep endoderm cells where Bcat activity is lower, Sox17 mediated repression of Bcat/Tcf is sufficient to repress dkk1 and lhx5 transcription, but Bcat activity is not high enough to activate six1.