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Cell type-dependent requirement for PIP box-regulated Cdt1 destruction during S phase.

Lee HO, Zacharek SJ, Xiong Y, Duronio RJ - Mol. Biol. Cell (2010)

Bottom Line: In this tissue the combination of Dup(ΔPIP) expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration.We could not detect Dup hyperaccumulation using mutations in the CRL4(Cdt2) components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation.These data indicate that PIP box-mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain Dup(ΔPIP) activity in some endocycling cell types.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Genetics and Molecular Biology, Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
DNA synthesis-coupled proteolysis of the prereplicative complex component Cdt1 by the CRL4(Cdt2) E3 ubiquitin ligase is thought to help prevent rereplication of the genome during S phase. To directly test whether CRL4(Cdt2)-triggered destruction of Cdt1 is required for normal cell cycle progression in vivo, we expressed a mutant version of Drosophila Cdt1 (Dup), which lacks the PCNA-binding PIP box (Dup(ΔPIP)) and which cannot be regulated by CRL4(Cdt2). Dup(ΔPIP) is inappropriately stabilized during S phase and causes developmental defects when ectopically expressed. Dup(ΔPIP) restores DNA synthesis to dup mutant embryonic epidermal cells, but S phase is abnormal, and these cells do not progress into mitosis. In contrast, Dup(ΔPIP) accumulation during S phase did not adversely affect progression through follicle cell endocycles in the ovary. In this tissue the combination of Dup(ΔPIP) expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration. We could not detect Dup hyperaccumulation using mutations in the CRL4(Cdt2) components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation. These data indicate that PIP box-mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain Dup(ΔPIP) activity in some endocycling cell types.

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DupΔPIP can support DNA replication. All panels show BrdU-labeled embryos. (A) dupa1/CyO control. (B) dupa1 homozygous mutant embryo. (C and D) prd-Gal4–driven expression of DupFL-GFP (C) or DupΔPIP-GFP (D) in dupa1 homozygous mutant embryos. Note the restoration of BrdU incorporation in the prd-Gal4 pattern in the dupa1 mutant embryos. (E and F) Higher magnification images of the BrdU incorporation pattern after prd-Gal4 expression of DupFL-GFP (E) and DupΔPIP-GFP (F) in dupa1 homozygous mutant embryos.
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Figure 5: DupΔPIP can support DNA replication. All panels show BrdU-labeled embryos. (A) dupa1/CyO control. (B) dupa1 homozygous mutant embryo. (C and D) prd-Gal4–driven expression of DupFL-GFP (C) or DupΔPIP-GFP (D) in dupa1 homozygous mutant embryos. Note the restoration of BrdU incorporation in the prd-Gal4 pattern in the dupa1 mutant embryos. (E and F) Higher magnification images of the BrdU incorporation pattern after prd-Gal4 expression of DupFL-GFP (E) and DupΔPIP-GFP (F) in dupa1 homozygous mutant embryos.

Mentions: Many studies have reported that overexpression of Cdt1 leads to rereplication (Zhong et al., 2003; Arias and Walter, 2005; May et al., 2005; Arias and Walter, 2006; Sansam et al., 2006). However, these studies did not directly test whether PIP-dependent destruction of Cdt1 is required for normal cell cycle progression in vivo. Moreover, the redundancy between CRL1 and CRL4 for S phase destruction of human Cdt1 and the inhibition of Cdt1 by Geminin raise the possibility that CRL4-mediated destruction of Cdt1 may not be essential for cell cycle progression. We therefore determined if DupFL-GFP and DupΔPIP-GFP could rescue the lack of S phase and consequent cell cycle arrest in dup mutant embryos. Dupa1 mutant embryos develop normally through the first 15 cell cycles, presumably because of maternal stores of Dup protein, but fail to incorporate BrdU in S phase of the 16th cell cycle (Figure 5, A and B; Whittaker et al., 2000). Both DupFL and DupΔPIP expression driven by prd-Gal4 restored BrdU incorporation in dup ectodermal cells (Figure 5, C and D), indicating that these transgenic proteins were capable of assembling pre-RC complexes and supporting the initiation of DNA replication. However, close inspection revealed an unusual BrdU incorporation pattern in DupΔPIP-expressing cells (Figure 5F): the staining appeared less uniform and more punctate than when DupFL was expressed (Figure 5E).


Cell type-dependent requirement for PIP box-regulated Cdt1 destruction during S phase.

Lee HO, Zacharek SJ, Xiong Y, Duronio RJ - Mol. Biol. Cell (2010)

DupΔPIP can support DNA replication. All panels show BrdU-labeled embryos. (A) dupa1/CyO control. (B) dupa1 homozygous mutant embryo. (C and D) prd-Gal4–driven expression of DupFL-GFP (C) or DupΔPIP-GFP (D) in dupa1 homozygous mutant embryos. Note the restoration of BrdU incorporation in the prd-Gal4 pattern in the dupa1 mutant embryos. (E and F) Higher magnification images of the BrdU incorporation pattern after prd-Gal4 expression of DupFL-GFP (E) and DupΔPIP-GFP (F) in dupa1 homozygous mutant embryos.
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Related In: Results  -  Collection

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Figure 5: DupΔPIP can support DNA replication. All panels show BrdU-labeled embryos. (A) dupa1/CyO control. (B) dupa1 homozygous mutant embryo. (C and D) prd-Gal4–driven expression of DupFL-GFP (C) or DupΔPIP-GFP (D) in dupa1 homozygous mutant embryos. Note the restoration of BrdU incorporation in the prd-Gal4 pattern in the dupa1 mutant embryos. (E and F) Higher magnification images of the BrdU incorporation pattern after prd-Gal4 expression of DupFL-GFP (E) and DupΔPIP-GFP (F) in dupa1 homozygous mutant embryos.
Mentions: Many studies have reported that overexpression of Cdt1 leads to rereplication (Zhong et al., 2003; Arias and Walter, 2005; May et al., 2005; Arias and Walter, 2006; Sansam et al., 2006). However, these studies did not directly test whether PIP-dependent destruction of Cdt1 is required for normal cell cycle progression in vivo. Moreover, the redundancy between CRL1 and CRL4 for S phase destruction of human Cdt1 and the inhibition of Cdt1 by Geminin raise the possibility that CRL4-mediated destruction of Cdt1 may not be essential for cell cycle progression. We therefore determined if DupFL-GFP and DupΔPIP-GFP could rescue the lack of S phase and consequent cell cycle arrest in dup mutant embryos. Dupa1 mutant embryos develop normally through the first 15 cell cycles, presumably because of maternal stores of Dup protein, but fail to incorporate BrdU in S phase of the 16th cell cycle (Figure 5, A and B; Whittaker et al., 2000). Both DupFL and DupΔPIP expression driven by prd-Gal4 restored BrdU incorporation in dup ectodermal cells (Figure 5, C and D), indicating that these transgenic proteins were capable of assembling pre-RC complexes and supporting the initiation of DNA replication. However, close inspection revealed an unusual BrdU incorporation pattern in DupΔPIP-expressing cells (Figure 5F): the staining appeared less uniform and more punctate than when DupFL was expressed (Figure 5E).

Bottom Line: In this tissue the combination of Dup(ΔPIP) expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration.We could not detect Dup hyperaccumulation using mutations in the CRL4(Cdt2) components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation.These data indicate that PIP box-mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain Dup(ΔPIP) activity in some endocycling cell types.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Genetics and Molecular Biology, Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
DNA synthesis-coupled proteolysis of the prereplicative complex component Cdt1 by the CRL4(Cdt2) E3 ubiquitin ligase is thought to help prevent rereplication of the genome during S phase. To directly test whether CRL4(Cdt2)-triggered destruction of Cdt1 is required for normal cell cycle progression in vivo, we expressed a mutant version of Drosophila Cdt1 (Dup), which lacks the PCNA-binding PIP box (Dup(ΔPIP)) and which cannot be regulated by CRL4(Cdt2). Dup(ΔPIP) is inappropriately stabilized during S phase and causes developmental defects when ectopically expressed. Dup(ΔPIP) restores DNA synthesis to dup mutant embryonic epidermal cells, but S phase is abnormal, and these cells do not progress into mitosis. In contrast, Dup(ΔPIP) accumulation during S phase did not adversely affect progression through follicle cell endocycles in the ovary. In this tissue the combination of Dup(ΔPIP) expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration. We could not detect Dup hyperaccumulation using mutations in the CRL4(Cdt2) components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation. These data indicate that PIP box-mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain Dup(ΔPIP) activity in some endocycling cell types.

Show MeSH
Related in: MedlinePlus