XB-ART-41767J Biol Chem September 17, 2010; 285 (38): 29525-34.
Histone XH2AX is required for Xenopus anterior neural development: critical role of threonine 16 phosphorylation.
A role for histone H2AX, one of the variants of the nucleosome core histone H2A, has been demonstrated in DNA repair, tumor suppression, apoptosis, and cell cycle checkpoint function. However, the physiological function and post-translational modification of histone H2AX during vertebrate development have not been elucidated. Here, we provide evidence showing that Xenopus histone H2AX (XH2AX) has a role in the anterior neural plate for eye field formation during Xenopus embryogenesis. A loss-of-function study clearly demonstrated a critical role of XH2AX in anterior neural specification. Through a differentiation assay with Xenopus animal cap embryonic stem cells, we confirmed that XH2AX is required for the activin-induced anterior neural specification of the ectoderm. Furthermore, we found that Chk1 is an essential kinase to phosphorylate histone XH2AX at Thr(16), which is involved in the biological function of this histone. Taken together, our findings reveal that XH2AX has a specific role in anterior neural formation of Xenopus, which is mediated through phosphorylation of XH2AX at Thr(16) by Chk1.
PubMed ID: 20639511
PMC ID: PMC2937984
Article link: J Biol Chem
Genes referenced: actl6a chek1 chek2 h2ac21 h2ax h2axl h4c4 hoxb9 myc ncam1 otx2 pax6 rax sox3 tubb2b
Morpholinos: chek1 MO1 hist2h2ab MO1
Article Images: [+] show captions
|FIGURE 1. Temporal and spatial expression profile of XH2AX during embryogenesis. A, RT-PCR analysis of temporal mRNA expression of XH2AX. Developmental stages are indicated above each lane. Histone H4 served as a loading control. −RT, control reaction without reverse transcriptase. B–I, whole-mount in situ hybridization showing the spatial expression of XH2AX during early Xenopus development. B and C, blastula stage: animal hemisphere view (B) and vegetal hemisphere view (C); D and E, gastrula stage: animal hemisphere view (D) and vegetal hemisphere view (E); F, neurula stage: anterior view with posterior right; G and H, tail bud stage: lateral view with anterior left (G) and dorsal view with anterior left (H); I, stages 33–34. The black lines represent the angle of sectioning for J–L. J–L, transverse section through a stage 33–34 embryo stained by whole-mount in situ hybridization for XH2AX mRNA. ba, branchial arches; br, brain; ey, eye; hg, hatching gland; mb, midbrain; ov, otic vesicle; so, somite.|
|FIGURE 4. XH2AX is required for the induction of anterior neural markers by activin. A–I, animal caps, explanted from embryos injected with Control-MO or XH2AX-MO (30 ng) either alone or in combination with the indicated dose of FLAG-XH2AX-8aa mRNA, were incubated with or without the activin protein (40 ng/ml) until stage 24, anterior neural markers (Otx2, Rx1, and Pax6), pan-neural markers (N-CAM and Sox3), a mesoderm marker (actin), and EF1α as a loading control. W.E., whole embryo as a positive control for PCR; −RT, control reaction without reverse transcriptase. A, RT-PCR analysis of animal cap explants. XH2AX-MO selectively blocked the activin-induced expression of Sox3, N-CAM, Pax6, and Otx2, but not actin (lane 4). Control-MO did not change activin induction of any of the genes (lane 2). B–G, phenotype of animal caps explanted from embryos injected as described for A–I. XH2AX-MO blocked activin-induced elongation of animal caps, and this phenotype was rescued by XH2AX-8aa mRNA in a dose-dependent manner. H, RT-PCR analysis of samples shown in B–G. XH2AX-8aa mRNA injection rescued activin induction of Sox3, Pax6, N-CAM in XH2AX-MO-injected animal caps. I, Western blot of embryonic extracts probed with anti-FLAG antibody, showing that all injected constructs were translated equally when injected into Xenopus embryos. Actin served as a loading control.|
|FIGURE 5. Depletion of Chk1 causes defective anterior neural formation. A, Chk1-MO (20 ng) was injected into the DMZ or VMZ of four-cell stage embryos. B, Chk1-MO specifically knocked down the translation of an overexpressed C-terminal HA-tagged Chk1 protein. Actin served as a control for specificity. C and D, dorsally injected Chk1-MO caused severe head and eye defects with a shortened axis (C) compared with the normal embryo that was not injected (D). E, RT-PCR analysis of whole embryos dorsally or ventrally expressing Chk1-MO. Dorsally (but not ventrally) expressed Chk1-MO repressed anterior neural markers (Pax6, Rx1, and Otx2) and the neural marker (N-CAM), but not the actin and globin mesoderm markers, compared with whole embryos (W.E.) not injected. EF1α served as a loading control. −RT, control reaction without reverse transcriptase. F, whole-mount in situ hybridization showing the spatial expression of Chk1 at the tadpole stage. The upper panel shows a magnified anterior region of an embryo.|
|FIGURE 6. XH2AX is phosphorylated by Chk1 at Thr16 of the N terminus. A, in vivo interaction between XH2AX and Chk1. Lysates from embryos injected with mRNAs for HA-tagged Xenopus Chk1 and Myc-tagged XH2AX expression were used to immunoprecipitate (IP) Myc-XH2AX (left panels) or HA-Xenopus Chk1 (right panels). IB, immunoblot. B, schematic representation of the structure of XH2AX. The histone fold domain (HFD) is a globular domain comprising the nucleosome core. αN, α-helix of the N-terminal tail; αC, α-helix of the C-terminal tail. C, Coomassie Brilliant Blue (CBB) staining of a bacterially expressed GST-XH2AX deletion mutant. D–G, Western blot using an anti-phosphothreonine antibody after an in vitro kinase reaction. D, Chk1 phosphorylated a threonine residue of XH2AX. E, Chk1 phosphorylated a threonine residue in the N terminus (but not the C terminus) of XH2AX. F, Chk1 phosphorylated a threonine residue in the N terminus of XH2AX (*), whereas Chk2 had little effect. G, phosphorylation of a threonine residue in the N terminus of XH2AX (*) was absent when Thr16 was mutated to alanine. H–J, Western blot using anti-phospho-Thr16 XH2AX antibody after an in vitro kinase reaction. H, anti-phospho-Thr16 XH2AX antibody was used for detecting phosphorylation of XH2AX. The upper arrow indicates full-length GST-XH2AX, and the lower arrow indicates the N terminus of GST-XH2AX. Asterisks indicate phosphorylation of full-length XH2AX and its N terminus. I, phosphorylation of XH2AX at Thr16 by Chk1 is shown by an in vitro kinase assay using immunoprecipitated FLAG-tagged Chk1. Extracts from embryos injected or uninjected (control) with FLAG-Chk1 mRNA were immunoprecipitated using an anti-FLAG antibody, and the kinase reaction was performed with the indicated GST-recombinant protein as a substrate. Phosphorylation of the threonine residue was analyzed by Western blotting using anti-phospho-Thr16 XH2AX antibody. Western blotting with anti-FLAG antibody showed that equal amounts of Chk1 immunoprecipitates were loaded. The upper arrow indicates the N terminus of GST-XH2AX, and the lower arrow indicates the IgG light chain. The asterisk indicates phosphorylated XH2AX. J, immunoprecipitated Chk1 from DMZ-expressed FLAG-Chk1 mRNA phosphorylated Thr16 of XH2AX. Extracts from embryos dorsally or ventrally injected with FLAG-Chk1 mRNA were immunoprecipitated using an anti-FLAG antibody and subjected to a kinase reaction with the indicated GST-recombinant protein as a substrate. Western blotting using anti-FLAG antibody showed that equal amounts of Chk1 were immunoprecipitated. The upper arrow indicates the N terminus of GST-XH2AX, and the lower arrow indicates the IgG light chain. The asterisk indicates phosphorylated XH2AX.|
|h2afx.1 (H2A.X variant histone) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.|
|h2afx.1 (H2A.X variant histone) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.|
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