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Science 336 (6082): 744-747

Copyright © 2012 by the American Association for the Advancement of Science

Transcription-Independent Function of Polycomb Group Protein PSC in Cell Cycle Control

Adone Mohd-Sarip1, Anna Lagarou1,*,{dagger}, Cecile M. Doyen1,{dagger}, Jan A. van der Knaap1,{dagger}, Ülkü Aslan1, Karel Bezstarosti2, Yasmin Yassin3, Hugh W. Brock3, Jeroen A. A. Demmers2, and C. Peter Verrijzer1,{ddagger}

1 Department of Biochemistry and Centre for Biomedical Genetics, Erasmus University Medical Centre, Post Office Box 1738, 3000 DR, Rotterdam, Netherlands.
2 Proteomics Centre, Erasmus University Medical Centre, Post Office Box 1738, 3000 DR, Rotterdam, Netherlands.
3 Department of Zoology, University of British Columbia, Vancouver, BC, Canada.


Figure 1
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Fig. 1. PSC function in mitosis. (A) S2 cell accumulation after RNAi-mediated KD of PC, PH, PSC, or dRING. (B) Cell cycle distribution of S2 cells after KD of the indicated proteins, determined by FACS. (C) Micrograph of cell cycle 11 embryos from a cross of Psch27/CyO females mated to wild-type (wt) fathers displaying chromatin bridges (yellow arrowheads). A comparable wt embryo is shown for comparison. Scale bars, 10 μm. (D) Indirect immunofluorescence of mitotic S2 cells after KD of PC, PH, PSC, or dRING. Cells were stained with antibodies against α-tubulin (red) and centromere identifier (CID) (green); DNA was visualized by 4',6-diamidino-2-phenylindole (blue). Arrowheads indicate misalignment and missegregation. (E) Number of cells with normal mitosis was plotted as a percentage of the total number of mitotic cells. 95% confidence intervals are indicated (error bars). The asterisk marks the significant difference between mock- and PSC KD cells (P < 0.0001), as determined by a {chi}2 test. See also table S1.

 

Figure 2
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Fig. 2. PSC interacts biochemically and genetically with CYC-B. (A) Interaction heatmap, based on mascot scores, depicting associated factors identified by mass spectrometry after immunopurification of PSC, PC, dRING, and PH. See table S2 for details. (B) Co-IPs of PcG proteins. Associated proteins were detected by immunoblotting with antibodies against the indicated proteins. Input represents 10% of the binding reactions. (C) CDC2 and CYC-B co-IPs. (D) Cartoon summarizing the proteomics results. MR, Morula. (E) Scanning electron micrographs of fly eyes in which ectopic CYC-B expression was driven by the GMR enhancer. CYC-B overexpression was combined with RNAi-mediated depletion of PSC or PC. Genotypes are indicated.

 

Figure 3
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Fig. 3. Regulation of CYC-B ubiquitylation and degradation by PSC. Micrographs of wing imaginal discs from either wt or ptc-Gal4; tub-Gal80ts; UAS-PSCRNAi (PTC>PSCRNAi) third instar larvae. The ptc driver directs expression of dsRNA directed against Psc. (A) Immunostaining of wt disc with antibodies against PSC (green) or CYC-B (red). (B) Staining of disc from PTC>PSCRNAi larvae after heatshock. Arrowheads indicate areas where PSC is knocked down. (C) In situ hybridization to detect Psc and cyc-B mRNAs in discs from either PTC>PSCRNAi. See fig. S4 for wt larvae. (D) Immunoblotting of CYC-B immunoprecipitated from extracts of S2 cells depleted for PSC, LMG, or both proteins in the presence of proteasome inhibitors (fig. S6), using antibodies against ubiquitin (α-Ub) or CYC-B(α-CYC-B). (E) Quantification of mitotic phenotypes of S2 cells in which PSC, LMG, or PC were overexpressed (OE) as indicated. Asterisks mark a significant difference with mock; error bars denote 95% confidence intervals. See legend to Fig. 1E, fig. S7, and table S3.

 

Figure 4
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Fig. 4. Cooperation between PSC and LMG during CYC-B ubiquitylation. (A) Reconstituted CYC-B ubiquitylation by PSC and LMG, detected by immunoblotting with α-Ub or α-CYC-B. CYC-B was incubated with a buffer control or in the presence of increasing amounts (~20 or 80 nM) of PSC alone or in addition to ~20 nM LMG (+), ~20 nM or 80 nM of LMG alone, or in addition to ~20 nM PSC (+). For purified proteins, see fig. S8. (B) Quantification of CYC-B ubiquitylation by PSC, LMG, or PSC and LMG (~20 nM each) by lumiimager fluorometry of gels (fig. S9). Ub–CYC-B was plotted as a function of the reaction time. a.u., arbitrary units. (C) Effect of PSC-C287A on CYC-B ubiquitylation. Analysis as described above. (D) Quantification of mitotic phenotypes of S2 cells in which PSC, PSC-C287A, and LMG were overexpressed as indicated. See legend to Fig. 1E, fig. S11, and table S3.

 


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