Gata is ubiquitously required for the earliest zygotic gene transcription in the ascidian embryo

In ascidian embryos, the earliest transcription from the zygotic genome begins between the 8-cell and 16-cell stages. Gata.a, a maternally expressed Gata transcription factor, activates target genes specifically in the animal hemisphere, whereas the complex of β-catenin and Tcf7 antagonizes the activity of Gata.a and activates target genes specifically in the vegetal hemisphere. Here, we show that genes zygotically expressed at the 16-cell stage have significantly more Gata motifs in their upstream regions. These genes included not only genes with animal hemisphere-specific expression but also genes with vegetal hemisphere-specific expression. On the basis of this finding, we performed knockdown experiments for Gata.a and reporter assays, and found that Gata.a is required for the expression of not only genes with animal hemisphere-specific expression, but also genes with vegetal hemisphere-specific expression. Our data indicated that weak Gata.a activity that cannot induce animal hemisphere-specific expression can allow β-catenin/Tcf7 targets to be expressed in the vegetal cells. Because genes zygotically expressed at the 32-cell stage also had significantly more Gata motifs in their upstream regions, Gata.a function may not be limited to the genes expressed specifically in the animal or vegetal hemispheres at the 16-cell stage, and Gata.a may play an important role in the earliest transcription of the zygotic genome

Opposite transcriptional switching through TCF/-catenin for binary cell fate decisions in the nematode "C. elegans" and the chordate "Ciona intestinalis"

Wnt / -Catenin / TCF-Weg ist ein wichtiger molekularer Signalweg, der eine entscheidende Rolle in der Embryonalentwicklung, Stammzellregulation und bei der Entstehung von Krebs spielt (zusammengefasst in MacDonald et al, 2009 ;. Cadigan und Peifer, 2009). Während die meisten der direkten Zielgene dieses Signalweges in der klassischen Art reguliert werden, «Klassische direkte Zielgene»: Repression in Abwesenheit von Wnt über TCF und seine kanonische DNA-Bindungsstellen und Aktivierung in Gegenwart von Wnt über TCF/-Catenin, werden einige andere direkte Zielgene gegenläufig reguliert, «Gegenläufige direkte Zielgene»: Aktivierung in Abwesenheit von Wnt über TCF und Repression in Gegenwart von Wnt über TCF/-Catenin. Der genaue Mechanismus (auf der Ebene der DNA), durch den TCF/-Catenin Zielgene gegenläufig regulieren war unbekannt und ist eine faszinierende Frage im Forschungsfeld des Wnt-Signals, vor allem, weil cis-regulatorische Elemente von "gegenläufigen direkten Zielgenen“ keine kanonischen TCF-DNA-Bindestellen haben. Der gegenläufige Mechanismus wurde im Kontext des sich entwickelnde C. elegans-Nervensystems und des frühe Embryos von Ciona intestinalis untersucht. Die Embryonen des Nematoden C. elegans und des Chordaten Ciona intestinalis sind prädestiniert für Studien des gegenläufigen Wnt/-Catenin/TCF-Signalwegs, sowohl auf molekularer als auch zellulärer Ebene. Erstens, ein invariantes Zellteilungsmuster, große und transparente Zellen und ein kompaktes Genom mit kurzen und einfachen cis-regulatorische Regionen ermöglichen gen-regulatorische Studien mit guter zellulärer Auflösung. Zweitens, alle beteiligten Faktoren sind vorhanden und experimentell zugänglich in den untersuchten embryonalen Geweben. Schließlich, sowohl in C. elegans als auch in Ciona intestinalis wurden Daten generiert, die die Hypothese des gegenläufigen Mechanismus untermauern. Das zentrale Ergebnis der Dissertation ist ein neuartiger repressiver Gen-Switch, der durch -Catenin vermittelt wird und wichtig für binären Zellschicksalsentscheidungen ist. Der neuartige Mechanismus ist in C. elegans und Ciona intestinalis konzeptionell sehr ähnlich. Im Zusammenspiel mit anderen Transkriptionsfaktoren fungieren dabei -Catenin und sein Interaktionspartner TCF als Co-Repressoren von neuen Wnt/-Catenin/TCF-Zielgenen. Die Repression wird durch DNA-Bindungsstellen für einen ZIC- und GATA-Faktor, die sich in den cis-regulatorische Regionen der regulierten Zielgene befinden, vermittelt. (1) In C. elegans blockiert -Catenin die Transkription von ttx-3 durch eine ZIC-Bindungsstelle, während TCF in Abwesenheit von -catenin die Transkription von ttx-3 aktiviert. Dafür gehen TCF und ZIC eine biochemische Bindung ein. (2) In Ciona intestinalis blockiert -Catenin eine ganze Gruppe von GATA-Zielgenen, zfpm, efna.d, gdf1 / 3-r, tfap2-rb und fzd4. Die Blockade erfolgt an GATA-Bindungsstellen, bei der TCF/-Catenin einen ternären Komplex mit dem aktivierenden GATA-Faktor bilden. Der tri-molekulare Komplex behindert GATA an der Bindung seiner Bindungsstelle und transkriptionelle Aktivierung der Zielgene findet nicht statt. In Anbetracht der Bedeutung des Wnt/-Catenin-Signalwegs bei der Krebsentstehung und in der Stammzellforschung wird die vorliegende Arbeit hilfreich für die Entwicklung von neuartigen Anti-Krebs-Medikamenten oder in der regenerativen Medizin sein.The Wnt/-catenin/TCF pathway is a key signaling pathway that plays crucial roles in development, stem cell regulation and cancer formation (reviewed in MacDonald et al., 2009; Cadigan and Peifer, 2009). While most of the direct target genes of this pathway display the classical type of regulation, « Classic direct target genes »: Repression in the absence of Wnt via TCF and its canonical binding sites, and Activation in the presence of Wnt via TCF/-catenin and its binding sites, some other direct target genes have been observed to display the opposite regulation, « Opposite direct target genes »: Activation in the absence of Wnt via TCF, and Repression in the presence of Wnt via TCF/-catenin. The precise mechanism (at the level of DNA) by which TCF/-catenin mediate this opposite regulation was unknown and is an intriguing question in the Wnt signaling field, especially because cis-regulatory elements of “opposite direct target” genes bear binding sites other than the ones usually bound by the TCF transcription factor. In the presented study the developing C. elegans nervous system and the early embryo of Ciona intestinalis were used to study the mechanism of opposite gene switching by TCF/-catenin. The embryos of the nematode C. elegans and the chordate Ciona intestinalis are predestined to study Wnt/TCF/-catenin-mediated opposite transcriptional gene switching at both the molecular and cellular level. Firstly, an invariant cell cleavage pattern, large and transparent cells and a well-annotated compact genome with short and simple cis-regulatory regions facilitate gene regulation studies at a good cellular resolution. Secondly, involved factors are present and experimentally accessible in the embryonic tissues at the according developmental stages. Finally, in both C. elegans and Ciona intestinalis evidences have been generated that support the hypotheses for the opposite TCF/-catenin transcriptional switching. The central finding of the Ph.D. thesis is a novel repressive function of -catenin in transcriptional regulation that is important for binary cell fate decisions and conceptually very similar in C. elegans and Ciona intestinalis and. Thereby, -catenin and its partner TCF act as co-repressors of novel Wnt/-catenin/TCF target genes at non-canonical TCF binding sites in their cis-regulatory regions, notably ZIC and GATA binding sites. (1) In C. elegans, -catenin represses the transcription of ttx-3 through a ZIC binding site, while TCF alone activates ttx-3 for which a biochemical interaction of TCF and ZIC is required. (2) In Ciona intestinalis, -catenin suppresses a whole panel of GATA target genes, zfpm, efna.d, gdf1/3-r, tfap2-r.b and fzd4 at GATA sites for which TCF/-catenin form a ternary complex with the activating GATA-factor that prevents GATA from binding its sites for transcriptional activation. Given the crucial importance of the Wnt/-catenin pathway in cancer and stem cell biology the presented work will be helpful for anti-cancer drug development or regenerative medicine treatment.by Willi KariAbweichender Titel laut Übersetzung des VerfassersKumulative Dissertation aus fünf ArtikelnUniversität Innsbruck, Dissertation, 2016OeBB(VLID)149399

A Maternal System Initiating the Zygotic Developmental Program through Combinatorial Repression in the Ascidian Embryo

Maternal factors initiate the zygotic developmental program in animal embryos. In embryos of the chordate, Ciona intestinalis, three maternal factors—Gata.a, β-catenin, and Zic-r.a—are required to establish three domains of gene expression at the 16-cell stage; the animal hemisphere, vegetal hemisphere, and posterior vegetal domains. Here, we show how the maternal factors establish these domains. First, only β-catenin and its effector transcription factor, Tcf7, are required to establish the vegetal hemisphere domain. Second, genes specifically expressed in the posterior vegetal domain have additional repressive cis-elements that antagonize the activity of β-catenin/Tcf7. This antagonizing activity is suppressed by Zic-r.a, which is specifically localized in the posterior vegetal domain and binds to DNA indirectly through the interaction with Tcf7. Third, Gata.a directs specific gene expression in the animal hemisphere domain, because β-catenin/Tcf7 weakens the Gata.a-binding activity for target sites through a physical interaction in the vegetal cells. Thus, repressive regulation through protein-protein interactions among the maternal transcription factors is essential to establish the first distinct domains of gene expression in the chordate embryo

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Correction: A Maternal System Initiating the Zygotic Developmental Program through Combinatorial Repression in the Ascidian Embryo

<p>Correction: A Maternal System Initiating the Zygotic Developmental Program through Combinatorial Repression in the Ascidian Embryo</p

Tcf7-binding sites are critical for genes expressed specifically in the posterior vegetal hemisphere domain (PVD).

<p>(A–C) Analysis of a regulatory region of <i>Tbx6</i>.<i>b</i>. (A) Illustrations on the left depict the constructs. The numbers indicate the relative nucleotide positions from the transcription start site of <i>Tbx6</i>.<i>b</i>. Mutated Tcf7-binding sites are indicated by X. Graphs show the percentage of blastomeres expressing the reporter in the anterior vegetal blastomeres, in the posterior vegetal blastomeres, and in the animal blastomeres. Note that not all cells or embryos could express the reporter because of mosaic incorporation of the electroporated plasmid. (B, C) Images showing <i>Gfp</i> expression in embryos electroporated with the fourth and last constructs shown in (A). Scale bar, 100 μm. (D–F) Mapping of the Tcf7 and Zic-r.a ChIP data onto genomic regions consisting of the exons and upstream regions of (D) <i>Tbx6</i>.<i>b</i>, (E) <i>Wnttun5</i>, and (F) <i>Admp</i>. The ChIP-chip data are shown in bars, and the ChIP-seq data are shown as magenta lines. Each graph shows the fold enrichment (y-axis) for the chromosomal regions (x-axis). Green and yellow boxes indicate the regions essential for specific expression, which were revealed by the reporter gene assays shown in (A), and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006045#pgen.1006045.s006" target="_blank">S5 Fig</a>. Regions indicated by green boxes overlap peak regions identified by the peak caller programs for ChIP-seq and ChIP-chip, while the peak caller programs did not identify peaks within regions indicated by yellow boxes. (G) Gel-shift analysis showing that the proximal Tcf7-binding site did not bind GST protein but bound the Tcf7-GST fusion protein. The shifted band disappeared by incubation with a specific competitor, but not a competitor with a mutant Tcf7-binding site.</p

Interactions between Tcf7, Gata.a, and β-catenin.

<p>(A–D) Using the upstream sequence of <i>Dlx</i>.<i>b</i>, (A) 3xmyc-tagged Tcf7, (B) 3xmyc-tagged Gfp, (C) 3xmyc-tagged Gata.a, and (D) 3xmyc-tagged Tcf7 and 3xflag-tagged Gata.a were misexpressed in epidermal cells. Note that Gfp protein expressed using the upstream sequence of <i>Dlx</i>.<i>b</i> is present in both the nucleus and cytoplasm (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006045#pgen.1006045.s011" target="_blank">S10 Fig</a>). Lysates of embryos were used for immunoprecipitation assays with an anti-myc antibody. Western blotting was performed with an (A–C) anti-β-catenin antibody and (D) anti-flag antibody. (E) Immunoprecipitation assay to examine interactions among recombinant β-catenin, Tcf7, and Gata.a proteins produced in <i>E</i>. <i>coli</i>.</p

Gata.a binding activity is suppressed in a ternary complex with Tcf7 and β-catenin.

<p>(A) Gata.a protein produced <i>in vitro</i> specifically recognized the Gata site critical for <i>Efna</i>.<i>d</i> expression. As a negative control, we used a rabbit reticulocyte lysate that did not contain template plasmids (NT control). (B) Co-incubation of Gata.a with either β-catenin or Tcf7 did not affect the binding activity of Gata.a for the proximal Gata site (Gata site 1) in the upstream sequence of <i>Efna</i>.<i>d</i>, whereas co-incubation of Gata.a with β-catenin and Tcf7 reduced the binding activity of Gata.a. (C) Co-incubation of Gata.a with β-catenin and Tcf7 reduced the binding activity of Gata.a for Gata sites in the upstream sequences of <i>Efna</i>.<i>d</i> and <i>Gdf1/3-r</i>. (D) Expression of <i>Efna</i>.<i>d</i> was suppressed in embryos injected with β<i>-catenin</i> mRNA. (E) ChIP followed by quantitative PCR revealed that BIO treatment reduced Gata-a binding to the <i>Efna</i>.<i>d</i> upstream region. Error bars indicate standard errors of three independent experiments.</p

Tcf7-binding sites are critical for expression in the vegetal hemisphere domain (VD).

<p>(A) Analysis of a regulatory region of <i>Fgf9/16/20</i>. Illustrations on the left depict the constructs. Green boxes indicate the <i>Gfp</i> gene and SV40 polyadenylation signal. The numbers indicate the relative nucleotide positions from the transcription start site of <i>Fgf9/16/20</i>. Mutant Tcf7-binding sites are indicated by X. The graphs show the percentage of blastomeres expressing the reporter in the anterior vegetal blastomeres, posterior vegetal blastomeres, and animal blastomeres. Note that not all cells or embryos could express the reporter because of mosaic incorporation of the electroporated plasmid. (B, C) Images showing <i>Gfp</i> expression, which was revealed by in situ hybridization, in embryos electroporated with the fourth and eighth constructs shown in (A). Scale bar, 100 μm. (D) Mapping of the Tcf7 ChIP data onto genomic regions consisting of the exons and upstream region of <i>Fgf9/16/20</i>. ChIP-chip data are shown in bars. ChIP-seq data are shown as a magenta line. Each graph shows the fold enrichment (y-axis) for the chromosomal region over <i>Fgf9/16/20</i> (x-axis). A green box indicates the region essential for specific expression, which was revealed by reporter gene assays. This region overlapped peaks identified by the peak caller programs for ChIP-seq and ChIP-chip. (E) Gel-shift analysis showing that Tcf7-binding site b did not bind the GST protein but bound the Tcf7-GST fusion protein. The shifted band was greatly reduced by incubation with a specific competitor, but not a competitor with a mutant Tcf7-binding site b.</p