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Embryonic cell fate specification and axis patterning requires integration of several signaling pathways that orchestrate region-specific gene expression. The transcription factor signal transducer and activator of transcription 3 (Stat3) plays important roles during early development, but it is unclear how Stat3 is activated. Here, using Xenopus as a model, we analyzed the post-translational regulation and functional consequences of Stat3 activation in dorsoventral axis patterning. We show that Stat3 phosphorylation, lysine methylation, and transcriptional activity increase before gastrulation and induce ventralmesoderm formation. Down syndrome critical region gene 6 (DSCR6), a RIPPLY family member that induces dorsal mesoderm by releasing repressive polycomb group proteins from chromatin, bound to the Stat3 C-terminal region and antagonized its transcriptional and ventralizing activities by interfering with its lysine methylation. Enhancer of zeste 2 polycomb-repressive complex 2 subunit (Ezh2) also bound to this region; however, its methyltransferase activity was required for Stat3 methylation and activation. Loss of Ezh2 resulted in dorsalization of ventralmesoderm and formation of a secondary axis. Furthermore, interference with Ezh2 phosphorylation also prevented Stat3 lysine methylation and transcriptional activity. Thus, inhibition of either Ezh2 phosphorylation or Stat3 lysine methylation compensated for the absence of DSCR6 function. These results reveal that DSCR6 and Ezh2 critically and post-translationally regulate Stat3 transcriptional activity. Ezh2 promotes Stat3 activation in ventralmesoderm formation independently of epigenetic regulation, whereas DSCR6 specifies dorsal fate by counteracting this ventralizing activity. This antagonism helps pattern the mesoderm along the dorsoventral axis, representing a critical facet of cell identity regulation during development.
FIGURE 1. DSCR6 binds to the C-terminal region of Stat3. (A) Schema shows the full-length and truncated Stat3 proteins used for analysis of interaction with DCSR6. Myc, 6 myc epitopes at the N-terminus; Tetra, domain involved in Stat3 tetramerization; Coiled-coil, protein interaction domain; DBD, DNA-binding domain; Linker, linker region; TAD, transactivation domain. (B) Temporal interaction between endogenous DSCR6 and Stat3 in blastula (stage 7), early gastrula (stage 10) and late gastrula (stage 12), analyzed by co immunoprecipitation using anti-DSCR6 antibody. (C) GST pull-down of in vitro translated Stat3 by GST-DSCR6. (D) GST pull-down of myc-tagged full-length and truncated Stat3 proteins expressed in the embryos by GST-DSCR6. (E-H) Immunofluorescence staining using anti-DSCR6 antibody shows the expression of endogenous DSCR6 protein in the cytoplasm and nuclei of all three germ layers in blastula (stages 8 and 9), early gastrula (stage 10) and mid-gastrula (stage 11). The sections are oriented with animal pole region up and dorsal region on the right. Scale bar: (E-H) 200 µm.
FIGURE 2. DSCR6 functionally interacts with Stat3 in DV patterning. (A-Dâ€) In situ
hybridization analysis shows the expression patterns of dorsal (chordin and otx2) and ventral (wnt8 and Xvent1) mesoderm genes in early gastrula stage embryos expressing Stat3C or Stat3C and DSCR6 in the dorsal region. Arrowheads indicate the dorsal limit of wnt8 expression in uninjected embryos. All embryos are vegetal view with dorsal region up. (E-G) Live images at larval stage (stage 36) show that DSCR6 rescues anterior deficiency produced by dorsal activation of Stat3C. (H-Kâ€) In situ hybridization analysis shows the expression patterns of
dorsal and ventral markers in early gastrula stage embryos expressing DSCR6 or DSCR6 and Stat3C in the ventral region. Arrows indicate ectopic expression of chordin and otx2; arrowheads show the repression of wnt8 and Xvent1 expression. All embryos are vegetal view with dorsal region up. (L-N) Live images at stage 36 show that DSCR6-induced formation of secondary axis is blocked by activation of Stat3. Scale bars: (A-Dâ€) 500 µm, (E-G) 500 µm, (HKâ€) 500 µm, (L-N) 500 µm.
FIGURE 3. DSCR6 inhibits Stat3 lysine methylation and transcriptional activity. (A)
Western blot analysis shows the temporal expression of endogenous Stat3 and Stat3-Y705P at cleavage (stage 2), blastula (stages 7 to 9), gastrula (stages 10.5 to 13) and tail-bud (stage 26) stages. Tubulin is a loading control. (B-E) Apotome microscopic analysis shows the nuclear localization of total Stat3 and Stat3-Y705P in the large-cell blastula (stage 7) and early gastrula (stage 10.5). Arrowheads indicate the nuclear localization of total Stat3 at stage 7. Stat3-Y705P is not present at this stage. (F) Luciferase reporter assay shows temporal Stat3 transcriptional activity (*, P<0.05; **, P<0.01; ****, P<0.0001). (G) Luciferase reporter assay shows the repression of Stat3 transcriptional activity by DSCR6 at early gastrula stage (***, P<0.001). (H) Immunoprecipitation followed by western blot analysis of lysine-methylated Stat3 protein in the large-cell blastula (stage 7) and early gastrula (stage 10.5). (I) DSCR6 represses Stat3 methylation at early gastrula stage. Scale bar: (B-E’) 200 µm.
FIGURE 4. Ezh2 binds to the C-terminal region of Stat3. (A) Western blot analysis shows the temporal expression of Ezh2 from cleavage to tail-bud stages. Tubulin is a loading control. (B-E) Apotome microscopic analysis shows the nuclear localization of Ezh2 in late blastula (stage 9) and mid-gastrula (stage 11). (F) Temporal interaction between endogenous Ezh2 and Stat3 in blastula (stage 7), early gastrula (stage 10) and late gastrula (stage 12), analyzed by coimmunoprecipitation using anti-Ezh2 antibody. (G) GST pull-down of in vitro translated myctagged Stat3 by GST-Ezh2. (H) GST pull-down of full-length and truncated Stat3 proteins expressed in the embryos by GST-Ezh2. Scale bar: (B-E) 200 µm.
FIGURE 5. Functional interaction between Ezh2 and Stat3 in DV patterning. (A-Dâ€) In situ hybridization analysis shows the expression of dorsal (chordin and otx2) and ventral (wnt8 and Xvent1) mesoderm markers in early gastrula expressing Ezh2-H694L or Ezh2-H694L and Stat3C in the ventral region. Arrows indicate ectopic expression of chordin and otx2; arrowheads show the region where wnt8 and Xvent1 expression is repressed. All embryos are vegetal view with dorsal region up. (E-G) Live images of tail-bud stage embryos. Activation of
Stat3 inhibits the formation of secondary axis (II) induced by ventral expression of Ezh2-H694L. (H) Western blot analysis following immunoprecipitation shows the inhibition of Stat3 methylation by Ezh2-H694L at early gastrula stage. (I) Quantification of lysine-methylated Stat3 level (***, P<0.001). (J) Luciferase reporter assay for Stat3 transcriptional activity in early gastrula stage embryos expressing Ezh2 or Ezh2-H694L (NS, not significant; ***, P<0.001). Scale bars: (A-Dâ€) 500 µm, (E-G) 500 µm.
FIGURE 6. Ezh2 phosphorylation is required for Stat3 methylation and transcriptional activity. (A, B) Analysis of the nuclear localization of Ezh2-S21P in the large-cell blastula (stage 7) and early gastrula (stage 10). (C) Luciferase reporter assay of Stat3 transcriptional activity in early gastrula expressing a constitutively active form of Ezh2, Ezh2-S21D, or a nonphosphorylable form of Ezh2, Ezh2-S21A (*P<0.05; **P<0.01). (D) A control embryo at tail-bud stage. (E) Ventral expression of Ezh2-S21D does not affect embryonic development. (F, G) Ventral expression of Ezh2-S21A induces a secondary axis with somitic tissue. (H, I) Ventral expression of an inhibitory form of Stat3 methylation, Stat3-K49R, induces a secondary axis with somitic tissue. Scale bars: (A, B) 200 µm, (D-F, H) 500 µm, (G, I) 500 µm.
FIGURE 7. Regulation of Stat3 transcriptional activity by DSCR6 and Ezh2 in embryonic patterning. (A-D) Functional interaction between DSCR6, Ezh2 and Stat3. (A) A normal larval stage embryo (stage 40) previously injected with CoMO. (B) Knockdown of Dscr6 produces anterior deficiency. (C) Inhibition of Ezh2 phosphorylation by Ezh2-S21A rescues anterior defects in Dscr6 morphants. (D) Interference with Stat3 lysine methylation by Stat3-K49R rescues anterior defects in Dscr6 morphants. (E) A model of functional interaction between DSCR6, Ezh2 and Stat3. Prior to gastrulation, both Ezh2 and Stat3 are phosphorylated and become active. Ezh2 binds to Stat3 and regulates its transcriptional activity through lysine methylation. DSCR6 binds to Ezh2 and Stat3. It may directly interfere with Stat3 transcriptional activity by binding to the C-terminal region that contains protein interaction cassettes and transactivation domain. It may also competitively binds to Ezh2 and interferes with its methyltransferase activity, thus indirectly preventing Stat3 lysine methylation and activation. Scale bar: (A-D) 500 µm.
Immunofluorescence staining using ripply3 Ab1 [anti-DSCR6] antibody shows the expression of endogenous Ripply3/DSCR6 protein in the cytoplasm and nuclei of all three germ layers in blastula (stages 8 and 9), early gastrula (stage 10) and mid-gastrula (stage 11). The sections are oriented with animal pole region up and dorsal region on the right. Scale bar: 200 µm.
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