Limits...
Involvement of an SCFSlmb complex in timely elimination of E2F upon initiation of DNA replication in Drosophila.

Hériché JK, Ang D, Bier E, O'Farrell PH - BMC Genet. (2003)

Bottom Line: We found that expression of the mouse Cul1 (mCul1) in the larval wing disc has a dominant negative effect.A screen for mutations that dominantly modify this phenotype showed effective suppression upon reduction of E2F function, suggesting that compromised downregulation of E2F contributes to the phenotype.These results argue that the SCFSlmb ubiquitin ligase directs E2F destruction in S phase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, 513 Parnassus Street, San Francisco, CA 94143-0448, USA. heriche@cgl.ucsf.edu

ABSTRACT

Background: Cul1 is a core component of the evolutionarily conserved SCF-type ubiquitin ligases that target specific proteins for destruction. SCF action contributes to cell cycle progression but few of the key targets of its action have been identified.

Results: We found that expression of the mouse Cul1 (mCul1) in the larval wing disc has a dominant negative effect. It reduces, but does not eliminate, the function of SCF complexes, promotes accumulation of Cubitus interruptus (a target of SCF action), triggers apoptosis, and causes a small wing phenotype. A screen for mutations that dominantly modify this phenotype showed effective suppression upon reduction of E2F function, suggesting that compromised downregulation of E2F contributes to the phenotype. Partial inactivation of Cul1 delayed the abrupt loss of E2F immunofluorescence beyond its normal point of downregulation at the onset of S phase. Additional screens showed that mild reduction in function of the F-box encoding gene slimb enhanced the mCul1 overexpression phenotype. Cell cycle modulation of E2F levels is virtually absent in slimb mutant cells in which slimb function is severely reduced. This implicates Slimb, a known targeting subunit of SCF, in E2F downregulation. In addition, Slimb and E2F interacted in vitro in a phosphorylation-dependent manner.

Conclusion: We have used genetic and physical interactions to identify the G1/S transcription factor E2F as an SCFSlmb target in Drosophila. These results argue that the SCFSlmb ubiquitin ligase directs E2F destruction in S phase.

Show MeSH

Related in: MedlinePlus

slimb mutant cells accumulate E2F in S phase. (A-C) slmb8, hs-slmb disc cells without heat shock, (D-F) slmb8, hs-slmb disc cells 1.5 hour after a heat shock restoring Slimb expression. E2F is in red, BrdU in green. C and F show merged images of A and B and D and E respectively. Asterisks point to cells with high BrdU incorporation that still express E2F. Scale bar is 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC161817&req=5

Figure 7: slimb mutant cells accumulate E2F in S phase. (A-C) slmb8, hs-slmb disc cells without heat shock, (D-F) slmb8, hs-slmb disc cells 1.5 hour after a heat shock restoring Slimb expression. E2F is in red, BrdU in green. C and F show merged images of A and B and D and E respectively. Asterisks point to cells with high BrdU incorporation that still express E2F. Scale bar is 5 μm.

Mentions: We thus wished to test whether loss of Slimb had an effect on E2F protein in imaginal discs. As slimb mutants die as first instar larvae, direct analysis of the mutant discs was not possible. The severity of the slimb mutant phenotype suggested that the mutation reduced crucial SCF functions much more than did expression of mCul1. We used a slimb mutant stock carrying a slimb transgene under the control of the HSP 70 promoter to modify the time of onset of the slimb mutant phenotype. In the absence of heat shock, slimb homozygotes exhibited the early lethality of slimb mutants, but a single 30 minute heat shock at 37°C during first instar was enough to allow some larvae to reach the wandering third instar stage 4 days later. These larvae have small imaginal discs. Labelling of these discs with the anti-E2F antibody revealed that nearly all cells expressed E2F (fig. 7A). Many of these cells also incorporated low levels of BrdU (fig. 7B and 7C), usually in regions of the nucleus with lower E2F staining. A few cells also had high levels of BrdU incorporation and these also expressed E2F, although at lower levels (asterisks on fig. 7A and 7B). An hour and a half after a heat shock that restored Slimb expression, BrdU-labelled cells no longer contained detectable levels of E2F (fig. 7D,7E,7F). No accumulation of E2F mRNA can be seen in slimb mutant discs (fig. 8) suggesting that any effect of Slimb on E2F occurs posttranscriptionnally. These results indicate that elimination of E2F during DNA replication requires Slimb.


Involvement of an SCFSlmb complex in timely elimination of E2F upon initiation of DNA replication in Drosophila.

Hériché JK, Ang D, Bier E, O'Farrell PH - BMC Genet. (2003)

slimb mutant cells accumulate E2F in S phase. (A-C) slmb8, hs-slmb disc cells without heat shock, (D-F) slmb8, hs-slmb disc cells 1.5 hour after a heat shock restoring Slimb expression. E2F is in red, BrdU in green. C and F show merged images of A and B and D and E respectively. Asterisks point to cells with high BrdU incorporation that still express E2F. Scale bar is 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC161817&req=5

Figure 7: slimb mutant cells accumulate E2F in S phase. (A-C) slmb8, hs-slmb disc cells without heat shock, (D-F) slmb8, hs-slmb disc cells 1.5 hour after a heat shock restoring Slimb expression. E2F is in red, BrdU in green. C and F show merged images of A and B and D and E respectively. Asterisks point to cells with high BrdU incorporation that still express E2F. Scale bar is 5 μm.
Mentions: We thus wished to test whether loss of Slimb had an effect on E2F protein in imaginal discs. As slimb mutants die as first instar larvae, direct analysis of the mutant discs was not possible. The severity of the slimb mutant phenotype suggested that the mutation reduced crucial SCF functions much more than did expression of mCul1. We used a slimb mutant stock carrying a slimb transgene under the control of the HSP 70 promoter to modify the time of onset of the slimb mutant phenotype. In the absence of heat shock, slimb homozygotes exhibited the early lethality of slimb mutants, but a single 30 minute heat shock at 37°C during first instar was enough to allow some larvae to reach the wandering third instar stage 4 days later. These larvae have small imaginal discs. Labelling of these discs with the anti-E2F antibody revealed that nearly all cells expressed E2F (fig. 7A). Many of these cells also incorporated low levels of BrdU (fig. 7B and 7C), usually in regions of the nucleus with lower E2F staining. A few cells also had high levels of BrdU incorporation and these also expressed E2F, although at lower levels (asterisks on fig. 7A and 7B). An hour and a half after a heat shock that restored Slimb expression, BrdU-labelled cells no longer contained detectable levels of E2F (fig. 7D,7E,7F). No accumulation of E2F mRNA can be seen in slimb mutant discs (fig. 8) suggesting that any effect of Slimb on E2F occurs posttranscriptionnally. These results indicate that elimination of E2F during DNA replication requires Slimb.

Bottom Line: We found that expression of the mouse Cul1 (mCul1) in the larval wing disc has a dominant negative effect.A screen for mutations that dominantly modify this phenotype showed effective suppression upon reduction of E2F function, suggesting that compromised downregulation of E2F contributes to the phenotype.These results argue that the SCFSlmb ubiquitin ligase directs E2F destruction in S phase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, 513 Parnassus Street, San Francisco, CA 94143-0448, USA. heriche@cgl.ucsf.edu

ABSTRACT

Background: Cul1 is a core component of the evolutionarily conserved SCF-type ubiquitin ligases that target specific proteins for destruction. SCF action contributes to cell cycle progression but few of the key targets of its action have been identified.

Results: We found that expression of the mouse Cul1 (mCul1) in the larval wing disc has a dominant negative effect. It reduces, but does not eliminate, the function of SCF complexes, promotes accumulation of Cubitus interruptus (a target of SCF action), triggers apoptosis, and causes a small wing phenotype. A screen for mutations that dominantly modify this phenotype showed effective suppression upon reduction of E2F function, suggesting that compromised downregulation of E2F contributes to the phenotype. Partial inactivation of Cul1 delayed the abrupt loss of E2F immunofluorescence beyond its normal point of downregulation at the onset of S phase. Additional screens showed that mild reduction in function of the F-box encoding gene slimb enhanced the mCul1 overexpression phenotype. Cell cycle modulation of E2F levels is virtually absent in slimb mutant cells in which slimb function is severely reduced. This implicates Slimb, a known targeting subunit of SCF, in E2F downregulation. In addition, Slimb and E2F interacted in vitro in a phosphorylation-dependent manner.

Conclusion: We have used genetic and physical interactions to identify the G1/S transcription factor E2F as an SCFSlmb target in Drosophila. These results argue that the SCFSlmb ubiquitin ligase directs E2F destruction in S phase.

Show MeSH
Related in: MedlinePlus