A systemic cell cycle block impacts stage-specific histone modification profiles during Xenopus embryogenesis.

Forming an embryo from a zygote poses an apparent conflict for epigenetic regulation. On the one hand, the de novo induction of cell fate identities requires the establishment and subsequent maintenance of epigenetic information to harness developmental gene expression. On the other hand, the embryo depends on cell proliferation, and every round of DNA replication dilutes preexisting histone modifications by incorporation of new unmodified histones into chromatin. Here, we investigated the possible relationship between the propagation of epigenetic information and the developmental cell proliferation during Xenopus embryogenesis. We systemically inhibited cell proliferation during the G1/S transition in gastrula embryos and followed their development until the tadpole stage. Comparing wild-type and cell cycle-arrested embryos, we show that the inhibition of cell proliferation is principally compatible with embryo survival and cellular differentiation. In parallel, we quantified by mass spectrometry the abundance of a large set of histone modification states, which reflects the developmental maturation of the embryonic epigenome. The arrested embryos developed abnormal stage-specific histone modification profiles (HMPs), in which transcriptionally repressive histone marks were overrepresented. Embryos released from the cell cycle block during neurulation reverted toward normality on morphological, molecular, and epigenetic levels. These results suggest that the cell cycle block by HUA alters stage-specific HMPs. We propose that this influence is strong enough to control developmental decisions, specifically in cell populations that switch between resting and proliferating states such as stem cells.

H4K20 Methylation Is Differently Regulated by Dilution and Demethylation in Proliferating and Cell-Cycle-Arrested Xenopus Embryos.

DNA replication during cell division leads to dilution of histone modifications and can thus affect chromatin-mediated gene regulation, raising the question of how the cell-cycle shapes the histone modification landscape, particularly during embryogenesis. We tackled this problem by manipulating the cell cycle during early Xenopus laevis embryogenesis and analyzing in vivo histone H4K20 methylation kinetics. The global distribution of un-, mono-, di-, and tri-methylated histone H4K20 was measured by mass spectrometry in normal and cell-cycle-arrested embryos over time. Using multi-start maximum likelihood optimization and quantitative model selection, we found that three specific biological methylation rate constants were required to explain the measured H4K20 methylation state kinetics. While demethylation is essential for regulating H4K20 methylation kinetics in non-cycling cells, demethylation is very likely dispensable in rapidly dividing cells of early embryos, suggesting that cell-cycle-mediated dilution of H4K20 methylation is an essential regulatory component for shaping its epigenetic landscape during early development. A record of this paper's transparent peer review process is included in the Supplemental Information.

Quantification of Proteins and Histone Marks in Drosophila Embryos Reveals Stoichiometric Relationships Impacting Chromatin Regulation.

Gene transcription in eukaryotes is regulated through dynamic interactions of a variety of different proteins with DNA in the context of chromatin. Here, we used mass spectrometry for absolute quantification of the nuclear proteome and methyl marks on selected lysine residues in histone H3 during two stages of Drosophila embryogenesis. These analyses provide comprehensive information about the absolute copy number of several thousand proteins and reveal unexpected relationships between the abundance of histone-modifying and -binding proteins and the chromatin landscape that they generate and interact with. For some histone modifications, the levels in Drosophila embryos are substantially different from those previously reported in tissue culture cells. Genome-wide profiling of H3K27 methylation during developmental progression and in animals with reduced PRC2 levels illustrates how mass spectrometry can be used for quantitatively describing and comparing chromatin states. Together, these data provide a foundation toward a quantitative understanding of gene regulation in Drosophila.

DNp73-induced degradation of tyrosinase links depigmentation with EMT-driven melanoma progression.

Melanoma is an aggressive cancer with poor prognosis, requiring personalized management of advanced stages and establishment of molecular markers. Melanomas derive from melanocytes, which specifically express tyrosinase, the rate-limiting enzyme of melanin-synthesis. We demonstrate that melanomas with high levels of DNp73, a cancer-specific variant of the p53 family member p73 and driver of melanoma progression show, in contrast to their less-aggressive low-DNp73 counterparts, hypopigmentation in vivo. Mechanistically, reduced melanin-synthesis is mediated by a DNp73-activated IGF1R/PI3K/AKT axis leading to tyrosinase ER-arrest and proteasomal degradation. Tyrosinase loss triggers reactivation of the EMT signaling cascade, a mesenchymal-like cell phenotype and increased invasiveness. DNp73-induced depigmentation, Slug increase and changes in cell motility are recapitulated in neural crest-derived melanophores of Xenopus embryos, underscoring a previously unnoticed physiological role of tyrosinase as EMT inhibitor. This data provides a mechanism of hypopigmentation accompanying cancer progression, which can be exploited in precision diagnosis of patients with melanoma-associated hypopigmentation (MAH), currently seen as a favorable prognostic factor. The DNp73/IGF1R/Slug signature in colorless lesions might aid to clinically discriminate between patients with MAH-associated metastatic disease and those, where MAH is indeed a sign of regression.

Brg1 chromatin remodeling ATPase balances germ layer patterning by amplifying the transcriptional burst at midblastula transition.

Zygotic gene expression programs control cell differentiation in vertebrate development. In Xenopus, these programs are initiated by local induction of regulatory genes through maternal signaling activities in the wake of zygotic genome activation (ZGA) at the midblastula transition (MBT). These programs lay down the vertebrate body plan through gastrulation and neurulation, and are accompanied by massive changes in chromatin structure, which increasingly constrain cellular plasticity. Here we report on developmental functions for Brahma related gene 1 (Brg1), a key component of embyronic SWI/SNF chromatin remodeling complexes. Carefully controlled, global Brg1 protein depletion in X. tropicalis and X. laevis causes embryonic lethality or developmental arrest from gastrulation on. Transcriptome analysis at late blastula, before development becomes arrested, indicates predominantly a role for Brg1 in transcriptional activation of a limited set of genes involved in pattern specification processes and nervous system development. Mosaic analysis by targeted microinjection defines Brg1 as an essential amplifier of gene expression in dorsal (BCNE/Nieuwkoop Center) and ventral (BMP/Vent) signaling centers. Moreover, Brg1 is required and sufficient for initiating axial patterning in cooperation with maternal Wnt signaling. In search for a common denominator of Brg1 impact on development, we have quantitatively filtered global mRNA fluctuations at MBT. The results indicate that Brg1 is predominantly required for genes with the highest burst of transcriptional activity. Since this group contains many key developmental regulators, we propose Brg1 to be responsible for raising their expression above threshold levels in preparation for embryonic patterning.

Suv4-20h histone methyltransferases promote neuroectodermal differentiation by silencing the pluripotency-associated Oct-25 gene.

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state.

Cardiac differentiation in Xenopus is initiated by mespa.

AIMS: Future cardiac repair strategies will require a profound understanding of the principles underlying cardiovascular differentiation. Owing to its extracorporal and rapid development, Xenopus laevis provides an ideal experimental system to address these issues in vivo. Whereas mammalian MesP1 is currently regarded as the earliest marker for the cardiovascular system, several MesP1-related factors from Xenopus-mespa, mespb, and mespo-have been assigned only to somitogenesis so far. We, therefore, analysed these genes comparatively for potential contributions to cardiogenesis. METHODS AND RESULTS: RNA in situ hybridizations revealed a novel anterior expression domain exclusively occupied by mespa during gastrulation, which precedes the prospective heart field. Correspondingly, when overexpressed mespa most strongly induced cardiac markers in vivo as well as ex vivo. Transference to murine embryonic stem (ES) cells and subsequent FACS analyses for Flk-1 and Troponin I confirmed the high potential of mespa as a cardiac inducer. In vivo, Morpholino-based knockdown of mespa protein led to a dramatic loss of pro-cardiac and sarcomeric markers, which could be rescued either by mespa itself or human MesP1, but neither by mespb nor mespo. Epistatic analysis positioned mespa upstream of mespo and mespb, and revealed positive autoregulation for mespa at the time of its induction. CONCLUSIONS: Our findings contribute to the understanding of conserved events initiating vertebrate cardiogenesis. We identify mespa as functional amphibian homologue of mammalian MesP1. These results will enable the dissection of cardiac specification from the very beginning in the highly versatile Xenopus system.

Maternal Wnt/ß-catenin signaling coactivates transcription through NF-κB binding sites during Xenopus axis formation.

Maternal Wnt/ß-Catenin signaling establishes a program of dorsal-specific gene expression required for axial patterning in Xenopus. We previously reported that a subset of dorsally expressed genes depends not only on Wnt/ß-Catenin stimulation, but also on a MyD88-dependent Toll-like receptor/IL1-receptor (TLR/IL1-R) signaling pathway. Here we show that these two signal transduction cascades converge in the nucleus to coactivate gene transcription in blastulae through a direct interaction between ß-Catenin and NF-κB proteins. A transdominant inhibitor of NF-κB, ΔNIκBα, phenocopies loss of MyD88 protein function, implicating Rel/NF-κB proteins as selective activators of dorsal-specific gene expression. Sensitive axis formation assays in the embryo demonstrate that dorsalization by Wnt/ß-Catenin requires NF-κB protein activity, and vice versa. Xenopus nodal-related 3 (Xnr3) is one of the genes with dual ß-Catenin/NF-κB input, and a proximal NF-κB consensus site contributes to the regional activity of its promoter. We demonstrate in vitro binding of Xenopus ß-Catenin to several XRel proteins. This interaction is observed in vivo upon Wnt-stimulation. Finally, we show that a synthetic luciferase reporter gene responds to both endogenous and exogenous ß-Catenin levels in an NF-κB motif dependent manner. These results suggest that ß-Catenin acts as a transcriptional co-activator of NF-κB-dependent transcription in frog primary embryonic cells.

Stage-specific histone modification profiles reveal global transitions in the Xenopus embryonic epigenome.

Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs) for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development.

Autoregulation of XTcf-4 depends on a Lef/Tcf site on the XTcf-4 promoter.

The restricted expression of XTcf-4 in the anterior midbrain is regulated via an active wnt/beta-catenin pathway (Kunz et al.,2004, Dev Biol 273:390-401). The molecular mechanism of this autoregulatory loop, however, remained elusive. Here we show that the activity of a 1,775 bp promoter fragment containing a consensus Lef/Tcf binding site at position -1,437 to -1,428 is upregulated by activating transcription factors of the Lef/Tcf family. Furthermore, chromatin immunoprecipitation revealed that endogenous beta-catenin is bound to the Lef/Tcf site on the promoter. Thus, regulation of XTcf-4 by canonical wnt-signaling is directly controlled by binding to and activating a consensus Lef/Tcf binding site within its own promoter.

CHD4/Mi-2beta activity is required for the positioning of the mesoderm/neuroectoderm boundary in Xenopus.

Experiments in Xenopus have illustrated the importance of extracellular morphogens for embryonic gene regulation in vertebrates. Much less is known about how induction leads to the correct positioning of boundaries; for example, between germ layers. Here we report that the neuroectoderm/mesoderm boundary is controlled by the chromatin remodeling ATPase CHD4/Mi-2beta. Gain and loss of CHD4 function experiments shifted this boundary along the animal-vegetal axis at gastrulation, leading to excess mesoderm formation at the expense of neuroectoderm, or vice versa. This phenotype results from specific alterations in gene transcription, notably of the neural-promoting gene Sip1 and the mesodermal regulatory gene Xbra. We show that CHD4 suppresses Sip1 transcription by direct binding to the 5' end of the Sip1 gene body. Furthermore, we demonstrate that CHD4 and Sip1 expression levels determine the "ON" threshold for Nodal-dependent but not for eFGF-dependent induction of Xbra transcription. The CHD4/Sip1 epistasis thus constitutes a regulatory module, which balances mesoderm and neuroectoderm formation.

Vertebrate Rel proteins exhibit Dorsal-like activities in early Drosophila embryogenesis.

In Drosophila, the Toll/Dorsal pathway triggers the nuclear entry of the Rel protein Dorsal, which controls dorsoventral patterning in early embryogenesis and plays an important role in innate immunity of the adult fly. In vertebrates, the homologous Toll/IL-1 receptor signaling pathway directs the nuclear localization of Rel/NF-kappaB complexes, which activate genes involved in proliferation, apoptosis, and immune response. Recently, first evidence has been reported for the activity of vertebrate Rel proteins and a Toll-like signaling pathway in the dorsoventral patterning process of Xenopus laevis embryos. Given the evolutionary divergence of the fly and frog model organisms, these findings raise the question, to what extent the effector functions of this pathway have been conserved? Here, we report the ability of two Xenopus Rel proteins to partially substitute for several, but not all, functions of the Dorsal protein in Drosophila embryos. Our results suggest the interaction between Rel proteins and their cytoplasmic inhibitors as an important interface of evolutionary adaptation.

Gene regulation by histone H1: new links to DNA methylation.

Linker histones of the H1 family are among the most abundant components of chromatin. In this issue of Cell, the effect of H1 downregulation on gene expression is examined. Although a 50% reduction of histone H1 levels in embryonic stem cells affects chromatin structure globally, the expression of very few genes is altered. Intriguingly, this study reveals a new link between H1 and DNA methylation.

The SNF2 domain protein family in higher vertebrates displays dynamic expression patterns in Xenopus laevis embryos.

All eukaryotes share a common nuclear infrastructure, in which DNA is packaged into nucleosomal chromatin. Its functional states, in particular the accessibility of the chromatin fiber to trans-acting factors, are determined by two classes of evolutionarily conserved enzymes, i.e. histone modifying enzymes and ATP-driven nucleosome remodeling machines. Browsing the annotated human genome database, we establish here a family of SNF2-like nuclear ATPases, which are the core enzymatic subunits of chromatin remodeling protein complexes. Homologues of those human genes are also to a large extent found in the Xenopus laevis genome, indicating a high degree of sequence conservation of this family among vertebrates. Expression analyses of the ATPase family of proteins reveal stage- and tissue-specific domains of peak RNA expression during early frog embryogenesis. These dynamic expression profiles suggest specific functional requirements for individual members of this family throughout early stages of vertebrate development.

The Xenopus noggin promoter drives roof-plate specific transcription.

During vertebrate neural development, inductive signals derived from the roof plate specify dorsal neural cell types of spinal cord as well as adjacent dermomytotome. Noggin has been demonstrated to play a pivotal role in this signaling network but how its expression is regulated remains unclear. To investigate this issue, we identified a 2066 bp Xenopus noggin 5' flanking sequence which recapitulates the roof-plate expression of endogenous gene in transgenic frog tadpoles and we further mapped the roof-plate enhancer to a sequence as short as 79 bp. Our results, therefore, not only shed light on but also provide a powerful tool to the study of neural and somite patterning.

Lef/Tcf-dependent Wnt/beta-catenin signaling during Xenopus axis specification.

Though the Wnt/beta-catenin signaling pathway is known to play key roles during Xenopus axis specification, whether it signals exclusively through Lef/Tcf transcription factors in this process remains unclear. To investigate this issue, we generated transgenic frog embryos expressing green fluorescent protein (GFP) driven by a Lef/Tcf-dependent and Wnt/beta-catenin-responsive promoter. This promoter is highly sensitive and even detects maternal beta-catenin activity prior to the large-scale transcription of zygotic genes. Unexpectedly, GFP expression was observed only in some, but not all, known Wnt/beta-catenin-positive territories in Xenopus early development. Furthermore, ubiquitous expression of dominant Lef-1 protein variants from transgenes revealed that zygotic Lef/Tcf activity is required for the ventroposterior development of Xenopus embryos. In summary, our results suggest that endogenous Wnt/beta-catenin activity does not result in obligatory Lef/Tcf-dependent gene activation, and that the ventroposteriorizing activity of zygotic Wnt-8 signaling is mediated by Lef/Tcf proteins.

Repression through a distal TCF-3 binding site restricts Xenopus myf-5 expression in gastrula mesoderm.

The development of skeletal muscle in the vertebrate embryo is controlled by a transcriptional cascade that includes the four myogenic regulatory factors Myf-5, MyoD, Myogenin, and MRF4. The dynamic expression pattern of myf-5 during myogenesis is thought to be consistent with its role during early determination of the myogenic lineage. To study the factors and mechanisms, which regulate myf-5 transcription in Xenopus, we isolated a genomic DNA clone containing 4858 bp of Xmyf-5 5' flanking region. Using a transgenic reporter assay, we show here that this genomic contig is sufficient to recapitulate the dynamic stage- and tissue-specific expression pattern of Xmyf-5 from the gastrula to tail bud stages. For the primary induction of myf-5 transcription, we identify three main regulatory elements, which are responsible for (i) activation in dorsal mesoderm, (ii) activation in ventral mesoderm, and (iii) repression in midline mesoderm, respectively. Their combined activities define the two-winged expression domain of myf-5 in the preinvoluted mesoderm. Repression in midline mesoderm is mediated by a single TCF binding site located in the 5' end of the -4.8 kbp sequence, which binds XTcf-3 protein in vitro. Endogenous Wnt signaling in the lateral mesoderm is required to overcome the long-range repression through this distal TCF site, and to stimulate myf-5 transcription independently from it. The element for ventral mesoderm activation responds to Activin. Together, these results describe a regulatory mosaic of repression and activation, which defines the myf-5 expression profile in the frog gastrula.

When the embryonic genome flexes its muscles.

During the development of multicellular organisms, both transient and stable gene expression patterns have to be established in a precisely orchestrated sequence. Evidence from diverse model organisms indicates that this epigenetic program involves not only transcription factors, but also the local structure, composition, and modification of chromatin, which define and maintain the accessibility and transcriptional competence of the nucleosomal DNA template. A paradigm for the interdependence of development and chromatin is constituted by the mechanisms controlling the specification and differentiation of the skeletal muscle cell lineage in vertebrates, which is the topic of this review.