Tet3 CXXC domain and dioxygenase activity cooperatively regulate key genes for Xenopus eye and neural development.

Ten-Eleven Translocation (Tet) family of dioxygenases dynamically regulates DNA methylation and has been implicated in cell lineage differentiation and oncogenesis. Yet their functions and mechanisms of action in gene regulation and embryonic development are largely unknown. Here, we report that Xenopus Tet3 plays an essential role in early eye and neural development by directly regulating a set of key developmental genes. Tet3 is an active 5mC hydroxylase regulating the 5mC/5hmC status at target gene promoters. Biochemical and structural studies further demonstrate that the Tet3 CXXC domain is critical for specific Tet3 targeting. Finally, we show that the enzymatic activity and CXXC domain are both crucial for Tet3's biological function. Together, these findings define Tet3 as a transcription regulator and reveal a molecular mechanism by which the 5mC hydroxylase and DNA binding activities of Tet3 cooperate to control target gene expression and embryonic development.

Combinatorial transcription factor activities on open chromatin induce embryonic heterogeneity in vertebrates.

During vertebrate gastrulation, mesoderm is induced in pluripotent cells, concomitant with dorsal-ventral patterning and establishing of the dorsal axis. We applied single-cell chromatin accessibility and transcriptome analyses to explore the emergence of cellular heterogeneity during gastrulation in Xenopus tropicalis. Transcriptionally inactive lineage-restricted genes exhibit relatively open chromatin in animal caps, whereas chromatin accessibility in dorsal marginal zone cells more closely reflects transcriptional activity. We characterized single-cell trajectories and identified head and trunk organizer cell clusters in early gastrulae. By integrating chromatin accessibility and transcriptome data, we inferred the activity of transcription factors in single-cell clusters and tested the activity of organizer-expressed transcription factors in animal caps, alone or in combination. The expression profile induced by a combination of Foxb1 and Eomes most closely resembles that observed in the head organizer. Genes induced by Eomes, Otx2, or the Irx3-Otx2 combination are enriched for maternally regulated H3K4me3 modifications, whereas Lhx8-induced genes are marked more frequently by zygotically controlled H3K4me3. Taken together, our results show that transcription factors cooperate in a combinatorial fashion in generally open chromatin to orchestrate zygotic gene expression.

ONECUT2 regulates RANKL-dependent enterocyte and microfold cell differentiation in the small intestine; a multi-omics study.

Microfold (M) cells reside in the intestinal epithelium of Peyer's patches (PP). Their unique ability to take up and transport antigens from the intestinal lumen to the underlying lymphoid tissue is key in the regulation of the gut-associated immune response. Here, we applied a multi-omics approach to investigate the molecular mechanisms that drive M cell differentiation in mouse small intestinal organoids. We generated a comprehensive profile of chromatin accessibility changes and transcription factor dynamics during in vitro M cell differentiation, allowing us to uncover numerous cell type-specific regulatory elements and associated transcription factors. By using single-cell RNA sequencing, we identified an enterocyte and M cell precursor population. We used our newly developed computational tool SCEPIA to link precursor cell-specific gene expression to transcription factor motif activity in cis-regulatory elements, uncovering high expression of and motif activity for the transcription factor ONECUT2. Subsequent in vitro and in vivo perturbation experiments revealed that ONECUT2 acts downstream of the RANK/RANKL signalling axis to support enterocyte differentiation, thereby restricting M cell lineage specification. This study sheds new light on the mechanism regulating cell fate balance in the PP, and it provides a powerful blueprint for investigation of cell fate switches in the intestinal epithelium.

Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus.

Hybridization of eggs and sperm from closely related species can give rise to genetic diversity, or can lead to embryo inviability owing to incompatibility. Although central to evolution, the cellular and molecular mechanisms underlying post-zygotic barriers that drive reproductive isolation and speciation remain largely unknown. Species of the African clawed frog Xenopus provide an ideal system to study hybridization and genome evolution. Xenopus laevis is an allotetraploid with 36 chromosomes that arose through interspecific hybridization of diploid progenitors, whereas Xenopus tropicalis is a diploid with 20 chromosomes that diverged from a common ancestor approximately 48 million years ago. Differences in genome size between the two species are accompanied by organism size differences, and size scaling of the egg and subcellular structures such as nuclei and spindles formed in egg extracts. Nevertheless, early development transcriptional programs, gene expression patterns, and protein sequences are generally conserved. Whereas the hybrid produced when X. laevis eggs are fertilized by X. tropicalis sperm is viable, the reverse hybrid dies before gastrulation. Here we apply cell biological tools and high-throughput methods to study the mechanisms underlying hybrid inviability. We reveal that two specific X. laevis chromosomes are incompatible with the X. tropicalis cytoplasm and are mis-segregated during mitosis, leading to unbalanced gene expression at the maternal to zygotic transition, followed by cell-autonomous catastrophic embryo death. These results reveal a cellular mechanism underlying hybrid incompatibility that is driven by genome evolution and contributes to the process by which biological populations become distinct species.

ANANSE: an enhancer network-based computational approach for predicting key transcription factors in cell fate determination.

Proper cell fate determination is largely orchestrated by complex gene regulatory networks centered around transcription factors. However, experimental elucidation of key transcription factors that drive cellular identity is currently often intractable. Here, we present ANANSE (ANalysis Algorithm for Networks Specified by Enhancers), a network-based method that exploits enhancer-encoded regulatory information to identify the key transcription factors in cell fate determination. As cell type-specific transcription factors predominantly bind to enhancers, we use regulatory networks based on enhancer properties to prioritize transcription factors. First, we predict genome-wide binding profiles of transcription factors in various cell types using enhancer activity and transcription factor binding motifs. Subsequently, applying these inferred binding profiles, we construct cell type-specific gene regulatory networks, and then predict key transcription factors controlling cell fate transitions using differential networks between cell types. This method outperforms existing approaches in correctly predicting major transcription factors previously identified to be sufficient for trans-differentiation. Finally, we apply ANANSE to define an atlas of key transcription factors in 18 normal human tissues. In conclusion, we present a ready-to-implement computational tool for efficient prediction of transcription factors in cell fate determination and to study transcription factor-mediated regulatory mechanisms. ANANSE is freely available at https://github.com/vanheeringen-lab/ANANSE.

Tissue- and stage-specific Wnt target gene expression is controlled subsequent to ß-catenin recruitment to cis-regulatory modules.

Key signalling pathways, such as canonical Wnt/ß-catenin signalling, operate repeatedly to regulate tissue- and stage-specific transcriptional responses during development. Although recruitment of nuclear ß-catenin to target genomic loci serves as the hallmark of canonical Wnt signalling, mechanisms controlling stage- or tissue-specific transcriptional responses remain elusive. Here, a direct comparison of genome-wide occupancy of ß-catenin with a stage-matched Wnt-regulated transcriptome reveals that only a subset of ß-catenin-bound genomic loci are transcriptionally regulated by Wnt signalling. We demonstrate that Wnt signalling regulates ß-catenin binding to Wnt target genes not only when they are transcriptionally regulated, but also in contexts in which their transcription remains unaffected. The transcriptional response to Wnt signalling depends on additional mechanisms, such as BMP or FGF signalling for the particular genes we investigated, which do not influence ß-catenin recruitment. Our findings suggest a more general paradigm for Wnt-regulated transcriptional mechanisms, which is relevant for tissue-specific functions of Wnt/ß-catenin signalling in embryonic development but also for stem cell-mediated homeostasis and cancer. Chromatin association of ß-catenin, even to functional Wnt-response elements, can no longer be considered a proxy for identifying transcriptionally Wnt-regulated genes. Context-dependent mechanisms are crucial for transcriptional activation of Wnt/ß-catenin target genes subsequent to ß-catenin recruitment. Our conclusions therefore also imply that Wnt-regulated ß-catenin binding in one context can mark Wnt-regulated transcriptional target genes for different contexts.

Activation of a T-box-Otx2-Gsc gene network independent of TBP and TBP-related factors.

Embryonic development relies on activating and repressing regulatory influences that are faithfully integrated at the core promoter of individual genes. In vertebrates, the basal machinery recognizing the core promoter includes TATA-binding protein (TBP) and two TBP-related factors. In Xenopus embryos, the three TBP family factors are all essential for development and are required for expression of distinct subsets of genes. Here, we report on a non-canonical TBP family-insensitive (TFI) mechanism of transcription initiation that involves mesoderm and organizer gene expression. Using TBP family single- and triple-knockdown experiments, α-amanitin treatment, transcriptome profiling and chromatin immunoprecipitation, we found that TFI gene expression cannot be explained by functional redundancy, is supported by active transcription and shows normal recruitment of the initiating form of RNA polymerase II to the promoter. Strikingly, recruitment of Gcn5 (also known as Kat2a), a co-activator that has been implicated in transcription initiation, to TFI gene promoters is increased upon depletion of TBP family factors. TFI genes are part of a densely connected TBP family-insensitive T-box-Otx2-Gsc interaction network. The results indicate that this network of genes bound by Vegt, Eomes, Otx2 and Gsc utilizes a novel, flexible and non-canonical mechanism of transcription that does not require TBP or TBP-related factors.

Genomic organization and modulation of gene expression of the TGF-ß and FGF pathways in the allotetraploid frog Xenopus laevis.

Inductive interactions mediated by the TGF-ß and FGF-MAPK pathways are essential for specification of the germ layers and embryonic body axes during early vertebrate embryogenesis. TGF-ß and FGF ligands signal through receptor Ser/Thr and Tyr kinases, respectively, and these signaling pathways cross-talk to regulate transcription and cell behavior. The allotetraploid Xenopus laevis and its ancestral diploid Xenopus tropicalis are versatile model organisms with which to study the inductive interactions and mechanisms of these signal transduction pathways. Here we have analyzed the draft genome of X. laevis with respect to the genomic organization and differential expression of genes in the TGF-ß and FGF pathways. Genomic structure and gene expression analyses of pathway components in X. laevis revealed that genetic modulations, including deletions resulting in singletons and differential expression of homeologs, have occurred frequently among extracellular regulatory factors of the TGF-ß pathway after allotetraploidization. Moreover, differential gene expression was found for factors regulating various cellular responses including co-receptors, decoy receptors, and intracellular negative regulators in both the TGF-ß and FGF-MAPK pathways. We summarize the patterns of genetic alterations in the allotetraploid frog X. laevis and discuss the importance of these changes with regard to developmental processes.

Heterochromatic histone modifications at transposons in Xenopus tropicalis embryos.

Transposable elements are parasitic genomic elements that can be deleterious for host gene function and genome integrity. Heterochromatic histone modifications are involved in the repression of transposons. However, it remains unknown how these histone modifications mark different types of transposons during embryonic development. Here we document the variety of heterochromatic epigenetic signatures at parasitic elements during development in Xenopus tropicalis, using genome-wide ChIP-sequencing data and ChIP-qPCR analysis. We show that specific subsets of transposons in various families and subfamilies are marked by different combinations of the heterochromatic histone modifications H4K20me3, H3K9me2/3 and H3K27me3. Many DNA transposons are marked at the blastula stage already, whereas at retrotransposons the histone modifications generally accumulate at the gastrula stage or later. Furthermore, transposons marked by H3K9me3 and H4K20me3 are more prominent in gene deserts. Using intra-subfamily divergence as a proxy for age, we show that relatively young DNA transposons are preferentially marked by early embryonic H4K20me3 and H3K27me3. In contrast, relatively young retrotransposons are marked by increasing H3K9me3 and H4K20me3 during development, and are also linked to piRNA-sized small non-coding RNAs. Our results implicate distinct repression mechanisms that operate in a transposon-selective and developmental stage-specific fashion.

The positive transcriptional elongation factor (P-TEFb) is required for neural crest specification.

Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a 'gate-keeper' for the correct temporal and spatial development of the neural crest.

Principles of nucleation of H3K27 methylation during embryonic development.

During embryonic development, maintenance of cell identity and lineage commitment requires the Polycomb-group PRC2 complex, which catalyzes histone H3 lysine 27 trimethylation (H3K27me3). However, the developmental origins of this regulation are unknown. Here we show that H3K27me3 enrichment increases from blastula stages onward in embryos of the Western clawed frog (Xenopus tropicalis) within constrained domains strictly defined by sequence. Strikingly, although PRC2 also binds widely to active enhancers, H3K27me3 is only deposited at a small subset of these sites. Using a Support Vector Machine algorithm, these sequences can be predicted accurately on the basis of DNA sequence alone, with a sequence signature conserved between humans, frogs, and fish. These regions correspond to the subset of blastula-stage DNA methylation-free domains that are depleted for activating promoter motifs, and enriched for motifs of developmental factors. These results imply a genetic-default model in which a preexisting absence of DNA methylation is the major determinant of H3K27 methylation when not opposed by transcriptional activation. The sequence and motif signatures reveal the hierarchical and genetically inheritable features of epigenetic cross-talk that impose constraints on Polycomb regulation and guide H3K27 methylation during the exit of pluripotency.

Establishing pluripotency in early development.

The earliest steps of embryonic development involve important changes in chromatin and transcription factor networks, which are orchestrated to establish pluripotent cells that will form the embryo. DNA methylation, histone modifications, the pluripotency regulatory network of transcription factors, maternal factors and newly translated proteins all contribute to these transitions in dynamic ways. Moreover, these dynamics are linked to the onset of zygotic transcription. We will review recent progress in our understanding of chromatin state and regulation of gene expression in the context of embryonic development in vertebrates, in particular mouse, Xenopus and zebrafish. We include work on mouse embryonic stem cells and highlight work that illustrates how early embryonic dynamics establish gene regulatory networks and the state of pluripotency.

Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs.

While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.

Dynamics of enhancer chromatin signatures mark the transition from pluripotency to cell specification during embryogenesis.

The generation of distinctive cell types that form different tissues and organs requires precise, temporal and spatial control of gene expression. This depends on specific cis-regulatory elements distributed in the noncoding DNA surrounding their target genes. Studies performed on mammalian embryonic stem cells and Drosophila embryos suggest that active enhancers form part of a defined chromatin landscape marked by histone H3 lysine 4 mono-methylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac). Nevertheless, little is known about the dynamics and the potential roles of these marks during vertebrate embryogenesis. Here, we provide genomic maps of H3K4me1/me3 and H3K27ac at four developmental time-points of zebrafish embryogenesis and analyze embryonic enhancer activity. We find that (1) changes in H3K27ac enrichment at enhancers accompany the shift from pluripotency to tissue-specific gene expression, (2) in early embryos, the peaks of H3K27ac enrichment are bound by pluripotent factors such as Nanog, and (3) the degree of evolutionary conservation is higher for enhancers that become marked by H3K27ac at the end of gastrulation, suggesting their implication in the establishment of the most conserved (phylotypic) transcriptome that is known to occur later at the pharyngula stage.

Nucleotide composition-linked divergence of vertebrate core promoter architecture.

Transcription initiation involves the recruitment of basal transcription factors to the core promoter. A variety of core promoter elements exists; however for most of these motifs, the distribution across species is unknown. Here we report on the comparison of human and amphibian promoter sequences. We have used oligo-capping in combination with deep sequencing to determine transcription start sites in Xenopus tropicalis. To systematically predict regulatory elements, we have developed a de novo motif finding pipeline using an ensemble of computational tools. A comprehensive comparison of human and amphibian promoter sequences revealed both similarities and differences in core promoter architecture. Some of the differences stem from a highly divergent nucleotide composition of Xenopus and human promoters. Whereas the distribution of some core promoter motifs is conserved independently of species-specific nucleotide bias, the frequency of another class of motifs correlates with the single nucleotide frequencies. This class includes the well-known TATA box and SP1 motifs, which are more abundant in Xenopus and human promoters, respectively. While these motifs are enriched above the local nucleotide background in both organisms, their frequency varies in step with this background. These differences are likely adaptive as these motifs can recruit TFIID to either CpG island or sharply initiating promoters. Our results highlight both the conserved and diverged aspects of vertebrate transcription, most notably showing co-opted motif usage to recruit the transcriptional machinery to promoters with diverging nucleotide composition. This shows how sweeping changes in nucleotide composition are compatible with highly conserved mechanisms of transcription initiation.

Temporal uncoupling of the DNA methylome and transcriptional repression during embryogenesis.

DNA methylation is a tightly regulated epigenetic mark associated with transcriptional repression. Next-generation sequencing of purified methylated DNA obtained from early Xenopus tropicalis embryos demonstrates that this genome is heavily methylated during blastula and gastrula stages. Although DNA methylation is largely absent from transcriptional start sites marked with histone H3 lysine 4 trimethylation (H3K4me3), we find both promoters and gene bodies of active genes robustly methylated. In contrast, DNA methylation is absent in large H3K27me3 domains, indicating that these two repression pathways have different roles. Comparison with chromatin state maps of human ES cells reveals strong conservation of epigenetic makeup and gene regulation between the two systems. Strikingly, genes that are highly expressed in pluripotent cells and in Xenopus embryos but not in differentiated cells exhibit relatively high DNA methylation. Therefore, we tested the repressive potential of DNA methylation using transient and transgenic approaches and show that methylated promoters are robustly transcribed in blastula- and gastrula-stage embryos, but not in oocytes or late embryos. These findings have implications for reprogramming and the epigenetic regulation of pluripotency and differentiation and suggest a relatively open, pliable chromatin state in early embryos followed by reestablished methylation-dependent transcriptional repression during organogenesis and differentiation.

A genome-wide survey of maternal and embryonic transcripts during Xenopus tropicalis development.

BACKGROUND: Dynamics of polyadenylation vs. deadenylation determine the fate of several developmentally regulated genes. Decay of a subset of maternal mRNAs and new transcription define the maternal-to-zygotic transition, but the full complement of polyadenylated and deadenylated coding and non-coding transcripts has not yet been assessed in Xenopus embryos. RESULTS: To analyze the dynamics and diversity of coding and non-coding transcripts during development, both polyadenylated mRNA and ribosomal RNA-depleted total RNA were harvested across six developmental stages and subjected to high throughput sequencing. The maternally loaded transcriptome is highly diverse and consists of both polyadenylated and deadenylated transcripts. Many maternal genes show peak expression in the oocyte and include genes which are known to be the key regulators of events like oocyte maturation and fertilization. Of all the transcripts that increase in abundance between early blastula and larval stages, about 30% of the embryonic genes are induced by fourfold or more by the late blastula stage and another 35% by late gastrulation. Using a gene model validation and discovery pipeline, we identified novel transcripts and putative long non-coding RNAs (lncRNA). These lncRNA transcripts were stringently selected as spliced transcripts generated from independent promoters, with limited coding potential and a codon bias characteristic of noncoding sequences. Many lncRNAs are conserved and expressed in a developmental stage-specific fashion. CONCLUSIONS: These data reveal dynamics of transcriptome polyadenylation and abundance and provides a high-confidence catalogue of novel and long non-coding RNAs.

Mass production of lumenogenic human embryoid bodies and functional cardiospheres using in-air-generated microcapsules.

Organoids are engineered 3D miniature tissues that are defined by their organ-like structures, which drive a fundamental understanding of human development. However, current organoid generation methods are associated with low production throughputs and poor control over size and function including due to organoid merging, which limits their clinical and industrial translation. Here, we present a microfluidic platform for the mass production of lumenogenic embryoid bodies and functional cardiospheres. Specifically, we apply triple-jet in-air microfluidics for the ultra-high-throughput generation of hollow, thin-shelled, hydrogel microcapsules that can act as spheroid-forming bioreactors in a cytocompatible, oil-free, surfactant-free, and size-controlled manner. Uniquely, we show that microcapsules generated by in-air microfluidics provide a lumenogenic microenvironment with near 100% efficient cavitation of spheroids. We demonstrate that upon chemical stimulation, human pluripotent stem cell-derived spheroids undergo cardiomyogenic differentiation, effectively resulting in the mass production of homogeneous and functional cardiospheres that are responsive to external electrical stimulation. These findings drive clinical and industrial adaption of stem cell technology in tissue engineering and drug testing.

The epigenome in early vertebrate development.

Epigenetic regulation defines the commitment and potential of cells, including the limitations in their competence to respond to inducing signals. This review discusses the developmental origins of chromatin state in Xenopus and other vertebrate species and provides an overview of its use in genome annotation. In most metazoans the embryonic genome is transcriptionally quiescent after fertilization. This involves nucleosome-dense chromatin, repressors and a temporal deficiency in the transcription machinery. Active histone modifications such as H3K4me3 appear in pluripotent blastula embryos, whereas repressive marks such as H3K27me3 show a major increase in enrichment during late blastula and gastrula stages. The H3K27me3 modification set by Polycomb restricts ectopic lineage-specific gene expression. Pluripotent chromatin in Xenopus embryos is relatively unconstrained, whereas the pluripotent cell lineage in mammalian embryos harbors a more enforced type of pluripotent chromatin.

GimmeMotifs: a de novo motif prediction pipeline for ChIP-sequencing experiments.

SUMMARY: Accurate prediction of transcription factor binding motifs that are enriched in a collection of sequences remains a computational challenge. Here we report on GimmeMotifs, a pipeline that incorporates an ensemble of computational tools to predict motifs de novo from ChIP-sequencing (ChIP-seq) data. Similar redundant motifs are compared using the weighted information content (WIC) similarity score and clustered using an iterative procedure. A comprehensive output report is generated with several different evaluation metrics to compare and evaluate the results. Benchmarks show that the method performs well on human and mouse ChIP-seq datasets. GimmeMotifs consists of a suite of command-line scripts that can be easily implemented in a ChIP-seq analysis pipeline. AVAILABILITY: GimmeMotifs is implemented in Python and runs on Linux. The source code is freely available for download at http://www.ncmls.eu/bioinfo/gimmemotifs/. CONTACT: s.vanheeringen@ncmls.ru.nl SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.