McGarry TJ (2002), Geminin deficiency causes a Chk1-dependent G2 a...

XB-ART-6362

Mol Biol Cell 2002 Oct 01;1310:3662-71. doi: 10.1091/mbc.e02-04-0199.

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Geminin deficiency causes a Chk1-dependent G2 arrest in Xenopus.

McGarry TJ .

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Geminin is an unstable inhibitor of DNA replication that gets destroyed at the metaphase/anaphase transition. The biological function of geminin has been difficult to determine because it is not homologous to a characterized protein and has pleiotropic effects when overexpressed. Geminin is thought to prevent a second round of initiation during S or G2 phase. In some assays, geminin induces uncommitted embryonic cells to differentiate as neurons. In this study, geminin was eliminated from developing Xenopus embryos by using antisense techniques. Geminin-deficient embryos show a novel and unusual phenotype: they complete the early cleavage divisions normally but arrest in G2 phase immediately after the midblastula transition. The arrest requires Chk1, the effector kinase of the DNA replication/DNA damage checkpoint pathway. The results indicate that geminin has an essential function and that loss of this function prevents entry into mitosis by a Chk1-dependent mechanism. Geminin may be required to maintain the structural integrity of the genome or it may directly down-regulate Chk1 activity. The data also show that during the embryonic cell cycles, rereplication is almost entirely prevented by geminin-independent mechanisms.

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Species referenced: Xenopus laevis
Genes referenced: cdc25c cdk1 cds1 chek1 gmnn myc pold1
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	Fig. 1. Depletion of geminin by antisense oligonucleotides. (A) Stage VI Xenopus oocytes were injected with a phosphorothioate antigeminin or control oligonucleotide and fertilized by the host transfer technique. At various points in development, the amount of geminin was determined by immunoblotting (arrowheads). The higher molecular weight bands are unrelated proteins that cross-react with the antibody. Oo, oocyte before progesterone treatment; Egg, oocyte after progesterone treatment; 16, 16-cell embryo; MBT, mid-blastula transition. (B) Same as A, except two-cell embryos were injected with morpholino antigeminin or control oligonucleotide; 8–128, cell number at time of harvest; stage 10, onset of gastrulation.
	Fig. 2. Development of geminin-depleted embryos. (A–D) Geminin depletion causes a cell cycle arrest. Two-cell embryos were injected with morpholino control oligonucleotide (CO) or antisense-geminin oligonucleotide (AS). (A) Early blastula stage. (B) Shortly after the midblastula transition. (C) Gastrula stage 12, animal view. (D) Stage 12, detail of animal view. (E–G) Geminin RNA rescues geminin depletion. Stage VI oocytes were injected with control phosphorothioate oligonucleotide (control), antigeminin oligo (antisense), or antigeminin oligo followed by 240 pg of WT geminin RNA (rescue). Oocytes were fertilized by the host transfer technique and cultured in vitro. The embryos in the middle panel were fixed at stage 12, those in the top and bottom panels were fixed at stage 41–45.
	Fig. 3. Geminin-deficient embryos arrest in G2 phase. (A) Histogram of nuclear DNA content for control (white) and geminin-depleted cells (black). (B) Nuclei of control and geminin-depleted embryos stained with 4,6-diamidino-2-phenylindole at the gastrula stage. Both micrographs are at the same magnification.
	Fig. 4. Cdc2 is hyperphosphorylated on tyrosine-15 in the absence of geminin. (A) Cyclin B1 accumulates in geminin-depleted embryos. Two-cell embryos were injected with antigeminin (AS) or control (CO) oligonucleotide and cultured in vitro. At various times after fertilization, the concentration of cyclin B1 was determined by immunoblotting. U, unfertilized egg. (B) Cdc2 becomes hyperphosphorylated geminin-depleted embryos. Same protocol as in A but a different experiment. The extent of Cdc2 phosphorylation was determined by immunoblotting with a mouse monoclonal Cdc2 antibody. The Y15-phosphorylated form of Cdc2 migrates through a polyacrylamide gel more slowly than the unphosphorylated form (Hartleyet al., 1996).
	Fig. 5. Suppression of the Chk1 pathway rescues the cell cycle arrest caused by geminin depletion. (A) Top, Cdc2 AF rescues geminin deficiency. Two-cell embryos were sequentially injected with morpholino antigeminin oligo and RNA encoding Cdc2 WT or Cdc2 AF. The number of embryos that continued dividing past the MBT was determined by visual inspection (Figure 2, B and D). The chart shows the combined results of two independent experiments. Each data point represents 22–36 embryos. Bottom, after the MBT, the extent of Cdc2 phosphorylation and the cyclin B1 level were determined by immunoblotting. CO, uninjected embryos; AS, embryos injected with antisense oligo; AS + Cdc2 WT, embryos injected with antisense oligo and 1000 pg/side of WT Cdc2 RNA; AS + Cdc2 AF, same except Cdc2 AF RNA was injected. (B) Cdc25 S287A rescues geminin deficiency. Same as described above, except the embryos were injected with RNA encoding WT Cdc25 or Cdc25 S287A. (C) Chk1 D148A rescues geminin deficiency. Same as described above, except the embryos were injected with RNA encoding Chk1 D148A or an inert Myc tag.
	Fig. 6. Geminin depletion causes increased Chk1 phosphorylation. Top, Xenopus embryos were injected with RNA encoding his6-Chk1ΔKD at the two-cell stage. At various times the degree of his6-Chk1ΔKD phosphorylation was determined by immunoblotting with anti-his6-tag antibody. Injected embryos were either left untreated (control), cultured in 20 mM hydroxyurea (+HU), or injected with antigeminin oligo (geminin depleted). Times indicate hours postfertilization (p.f.). In this experiment the MBT occurred just after 9 h p.f. Middle, same experiment, but the blot was probed with anti-Cdc2 antibody. The loss of Cdc2 staining after 10 h in the geminin-depleted embryos is not reproducible (Figures 4 and5). Bottom, same experimental protocol as described above, except the embryos were not injected with his6-Chk1ΔKD RNA and the blot was probed with anti-Cds1 antibody. In this experiment the MBT occurred around 10 h p.f.

References [+] :

Blow, A role for the nuclear envelope in controlling DNA replication within the cell cycle. 1988, Pubmed, Xenbase