Linder B et al. (2007), CHD4/Mi-2beta activity is required for the posi...

XB-ART-35807

Genes Dev 2007 Apr 15;218:973-83. doi: 10.1101/gad.409507.

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

Linder B , Mentele E , Mansperger K , Straub T , Kremmer E , Rupp RA .

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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.

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Species referenced: Xenopus laevis
Genes referenced: cdh4 chd4 fgf4 gemin2 gsc nherf2 nodal sia1 tbxt
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	Figure 1. Perturbation of CHD4 activity leads to specific alterations in gastrula gene expression. Embryos were unilaterally injected with a dose of 1.0 ng of CHD4 mRNA or 40 ng of CHD4-Mo into one blastomere at the two-cell stage. At gastrula stages, they were fixed, presorted into left-side- or right-side-injected populations by fluorescence of coinjected eGFP (not shown), and used for RNA in situ hybridizations for the marker genes indicated to the left. Displayed are representative embryos from three to five independent experimental repeats, in which the left side serves as an internal control for normal marker gene expression, while the right side shows the expression under the experimental condition (either vegetal or dorsovegetal views). The injected side is to the right.
	Figure 2. CHD4 regulates Sip1 and Xbra mRNA expression specifically. CHD4 overexpression suppresses Sip1 mRNA levels in animal cap explants. (A) Rescue of Xbra and Sip1 mRNA expression at mid-gastrula (injections and embryo presentation as in Fig. 1). CHD4-Mo (40 ng) or dnCHD4 mRNA (1.0 ng) causes similar inhibition of Xbra mRNA on the injected side, which is rescued by coinjection of wtCHD4 mRNA (1.0 ng). Unilateral reduction of Sip1 mRNA in the prospective neural plate area is rescued by CHD4-Mo, but not Control-Mo, coinjection. The injected side is to the right. (B, left panel) Semiquantitative RTâ€“PCR; 1.0 ng of wtCHD4 mRNA was injected. (Right panel) Real-time RTâ€“PCR quantification of Sip1 mRNA levels after normalization to histone H4; 0.25 ng of wtCHD4 mRNA was injected.
	Figure 3. CHD4 levels define the position of the mesoderm/neuroectoderm boundary. Embryos were unilaterally injected with the reagents indicated on the left. In each panel, the right side was injected, and the left side serves as internal control. In A and C, embryos were subjected to in situ hybridization for the indicated markers prior to sectioning. (A) Gastrula embryos, stained for endogenous Xbra and Sip1 mRNAs, were bisected as indicated in the cartoon. Black bars delimit the normal expression domains, and white bars mark the domains in the injected halfs. (d) Dorsal; (v) ventral; (l) left; (r) right. Note reduced Sip1 staining in wtCHD4-injected embryos (white asterisks). (B) Transverse, hematoxylin-and-eosin-stained sections of tailbud-stage embryos at the head (left column) or anterior trunk (right column) level. WtCHD4-injected embryos showed absent or reduced eyes but enlarged somites on the injected side. CHD4-Mo-injected embryos showed hyperproliferation of the retina and disorganized, loosely packed somites on the injected side. (C) Hindbrain and trunk somites of either wtCHD4 mRNA or CHD4-Mo-injected embryos express cognate differentiation markers in neural (nrp1) and muscle (m-actin) tissues.
	Figure 4. CHD4 binds to the Sip1 gene. (A) The cartoon depicts the organization of frog and mouse Sip1 gene loci around the first translated exon E1 (AUG indicated by arrow). While exons U5 and E1 are highly conserved in sequence (connected by dashed lines), mouse exons U6â€“U9 apparently are not conserved in Xenopus. Black bars indicate the relative positions of the ChIP amplicons xU5, xE1, and xE2 for Xenopus. Not drawn to scale; however, absolute distances between ChIP probes are given in brackets. (B) ChIPs were performed on mid-gastrula Xenopus embryos (NF11), using a rat monoclonal antibody mix against xCHD4 protein followed by real-time PCR analysis. They revealed preferential binding of endogenous xCHD4 protein to E1 (n = 3 independent experiments). The relative xCHD4 occupancy was normalized to the xGAPDH amplicon; xTH/bZIP is a silent gene, which becomes activated during metamorphosis. Error bars are the mean standard deviation.
	Figure 5. CHD4 defines the activation threshold for Activin-dependent Xbra induction. Animals cap explants, preloaded with various mRNAs and Morpholinos as indicated, were lysed at mid-gastrula and analyzed for Xbra mRNA by RTâ€“PCR. Histone H4 served as loading control; RTâˆ’ represents mock RTâ€“PCR reactions without reverse transcriptase. (A) Overexpression of CHD4 protein sensitized the Xbra promoter for Activin induction in a Sip1-dependent manner. Note that Xbra was hardly induced by 5 pg of Activin mRNA alone (lane 4), but already by 1 pg of Activin mRNA, when CHD4 mRNA was coinjected (lane 7). The CHD4-dependent sensitization of the Xbra gene was reverted by coinjection of Sip1 mRNA. (B) Partial ablation of CHD4 protein by Morpholino knockdown desensitized Xbra for Activin induction. Note that CHD4-Mo-injected caps still expressed only basal Xbra mRNA levels at an inducer dose of 24 ng of Activin mRNA (cf. lanes 4 and 8), while at 100 pg induction was partially restored (lane 9). The observed desensitization was reverted by coinjection of Sip1-Mo (cf. lanes 12 and 13). These key observations were confirmed in two additional, independent experiments by real-time RTâ€“PCR quantification: (C) The wtCHD4-dependent sensitization of the Xbra promoter response is inhibited by either dnCHD4 or Sip1 mRNA coinjection. (D) Both dnCHD4 and CHD4-Mo desensitize the Xbra promoter; Sip1-Mo coinjection partially rescues the CHD4-Mo down-regulation of Xbra induction. Xbra mRNA levels were normalized to histone H4 and uninjected control explants.
	Figure 6. Specificity of CHD4-dependent threshold control. The specificity of the Xbra promoter response was further investigated in animal caps. (A) The CHD4-Mo-dependent desensitization of the Xbra promoter can be rescued by wtCHD4 mRNA coinjection. (B) The Activin threshold of the Gsc promoter is not altered by CHD4 overexpression. (C) Elevated or reduced CHD4 protein levels had no significant effect on the eFGF-dependent induction of Xbra transcription (cf. lanes 4, 8, and 12). (D) Neither wtCHD4 nor dnCHD4 mRNA levels affect the induction of Siamois or Xnodal related 3 by Wnt8/canonical Wnt signaling.

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Becker, ATP-dependent nucleosome remodeling. 2002, Pubmed