Limits...
Clustering phenotype populations by genome-wide RNAi and multiparametric imaging.

Fuchs F, Pau G, Kranz D, Sklyar O, Budjan C, Steinbrink S, Horn T, Pedal A, Huber W, Boutros M - Mol. Syst. Biol. (2010)

Bottom Line: With RNA interference (RNAi), highly parallel phenotyping of loss-of-function effects in cells has become feasible.Experimental evidence supports that DONSON is a novel centrosomal protein required for DDR signalling and genomic integrity.Multiparametric phenotyping by automated imaging and computational annotation is a powerful method for functional discovery and mapping the landscape of phenotypic responses to cellular perturbations.

View Article: PubMed Central - PubMed

Affiliation: German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany.

ABSTRACT
Genetic screens for phenotypic similarity have made key contributions to associating genes with biological processes. With RNA interference (RNAi), highly parallel phenotyping of loss-of-function effects in cells has become feasible. One of the current challenges however is the computational categorization of visual phenotypes and the prediction of biological function and processes. In this study, we describe a combined computational and experimental approach to discover novel gene functions and explore functional relationships. We performed a genome-wide RNAi screen in human cells and used quantitative descriptors derived from high-throughput imaging to generate multiparametric phenotypic profiles. We show that profiles predicted functions of genes by phenotypic similarity. Specifically, we examined several candidates including the largely uncharacterized gene DONSON, which shared phenotype similarity with known factors of DNA damage response (DDR) and genomic integrity. Experimental evidence supports that DONSON is a novel centrosomal protein required for DDR signalling and genomic integrity. Multiparametric phenotyping by automated imaging and computational annotation is a powerful method for functional discovery and mapping the landscape of phenotypic responses to cellular perturbations.

Show MeSH

Related in: MedlinePlus

Functional analysis of candidate genes for roles in cell-cycle progression and spindle organization. (A) Time-resolved cell-cycle analysis in HeLa cells transfected with indicated siRNAs at different time points. Box colours represent fractions of cells with DNA content corresponding to sub-G1, G1/G0, S and G2/M. HeLa cells transfected with siRNAs against DONSON showed a delay in S-phase progression. (B) Assessment of S-phase progression by BrdU incorporation; 48 h after siRNA transfection, U2OS cells were synchronized for 16 h with 1 mM hydroxyurea (HU) and released for 6 h in BrdU-containing medium. BrdU-positive cells were stained with anti-BrdU primary antibody (Calbiochem) and Alexa 488 secondary antibody. DNA was counterstained with propidium iodide. Imaging and quantification were performed with Acumen Explorer microplate reader. Values are shown as mean±s.d. of three biological replicates. (C) Cell-cycle-dependent protein expression of DONSON. U2OS cells were synchronized either in G1/S with HU or in G2/M with nocodazole (Noc). Cells were collected at different time points after release into cell cycle for western blot analysis. (D) DONSON depletion is associated with a 10-fold increase in multipolar spindles compared to control treatments. U2OS cells were transfected with a DONSON siRNA pool and immunostained for α- and γ-tubulin at indicated time points. U2OS cells transfected with Rluc siRNAs serving as negative control. Data represent mean±s.d. of three biological replicates. At least 200 metaphase spindles were counted in each experiment. Scale bar indicates 2.5 μm. (E) DONSON protein co-localizes with centrosomes. HeLa cells were transfected with HA-tagged DONSON for 48 h and immunostained with primary HA antibody and Alexa 488-conjugated secondary antibody. Arrows indicate the centrosomal staining of DONSON (green). DNA was counterstained with DAPI (blue). Scale bar indicates 2.5 μm. (F) DONSON co-localizes with centrin. U2OS cells were transfected with HA-tagged DONSON; 48 h after transfection, cells were immunostained with anti-centrin and anti-HA-tagged primary antibodies and Alexa 488-, Alexa 594-conjugated secondary antibodies, respectively. Scale bar indicates 2.5 μm. Source data is available for this figure at www.nature.com/msb.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2913390&req=5

f4: Functional analysis of candidate genes for roles in cell-cycle progression and spindle organization. (A) Time-resolved cell-cycle analysis in HeLa cells transfected with indicated siRNAs at different time points. Box colours represent fractions of cells with DNA content corresponding to sub-G1, G1/G0, S and G2/M. HeLa cells transfected with siRNAs against DONSON showed a delay in S-phase progression. (B) Assessment of S-phase progression by BrdU incorporation; 48 h after siRNA transfection, U2OS cells were synchronized for 16 h with 1 mM hydroxyurea (HU) and released for 6 h in BrdU-containing medium. BrdU-positive cells were stained with anti-BrdU primary antibody (Calbiochem) and Alexa 488 secondary antibody. DNA was counterstained with propidium iodide. Imaging and quantification were performed with Acumen Explorer microplate reader. Values are shown as mean±s.d. of three biological replicates. (C) Cell-cycle-dependent protein expression of DONSON. U2OS cells were synchronized either in G1/S with HU or in G2/M with nocodazole (Noc). Cells were collected at different time points after release into cell cycle for western blot analysis. (D) DONSON depletion is associated with a 10-fold increase in multipolar spindles compared to control treatments. U2OS cells were transfected with a DONSON siRNA pool and immunostained for α- and γ-tubulin at indicated time points. U2OS cells transfected with Rluc siRNAs serving as negative control. Data represent mean±s.d. of three biological replicates. At least 200 metaphase spindles were counted in each experiment. Scale bar indicates 2.5 μm. (E) DONSON protein co-localizes with centrosomes. HeLa cells were transfected with HA-tagged DONSON for 48 h and immunostained with primary HA antibody and Alexa 488-conjugated secondary antibody. Arrows indicate the centrosomal staining of DONSON (green). DNA was counterstained with DAPI (blue). Scale bar indicates 2.5 μm. (F) DONSON co-localizes with centrin. U2OS cells were transfected with HA-tagged DONSON; 48 h after transfection, cells were immunostained with anti-centrin and anti-HA-tagged primary antibodies and Alexa 488-, Alexa 594-conjugated secondary antibodies, respectively. Scale bar indicates 2.5 μm. Source data is available for this figure at www.nature.com/msb.

Mentions: To further investigate the role of DONSON in cell-cycle progression, we performed time-course experiments with HeLa cells depleted for DONSON and measured the DNA content. As shown in Figure 4A, we observed a slower S-phase progression compared to controls, suggesting a DNA replication defect, followed by accumulation of cells in G2/M at 48 h. We then assessed whether DONSON is required for DNA replication during S-phase progression. U2OS cells were arrested with hydroxyurea (HU) in G1/S, and incorporation of bromodeoxyuridine (BrdU) was measured after release. As shown in Figure 4B, depletion of DONSON resulted in significant decrease in BrdU incorporation, indicating that DONSON is required for S-phase transition. We next asked whether the level of DONSON protein is regulated during the cell cycle. U2OS cells were arrested either in G1/S and G2/M by HU or nocodazole, respectively. After release from HU, we assessed DONSON protein levels for up to 24 h by immunoblot analysis. As shown in Figure 4C, DONSON levels peaked at 6 h after release and preceded the cyclin A expression as a marker for G2. Furthermore, when cells were released from G2/M-phase arrest, peak DONSON levels were observed later than phospho-H3 as a marker for M phase (Figure 4C). Taken together, these experiments indicate that DONSON levels are regulated during cell division, peaking during S phase and that DONSON is required for cell-cycle progression.


Clustering phenotype populations by genome-wide RNAi and multiparametric imaging.

Fuchs F, Pau G, Kranz D, Sklyar O, Budjan C, Steinbrink S, Horn T, Pedal A, Huber W, Boutros M - Mol. Syst. Biol. (2010)

Functional analysis of candidate genes for roles in cell-cycle progression and spindle organization. (A) Time-resolved cell-cycle analysis in HeLa cells transfected with indicated siRNAs at different time points. Box colours represent fractions of cells with DNA content corresponding to sub-G1, G1/G0, S and G2/M. HeLa cells transfected with siRNAs against DONSON showed a delay in S-phase progression. (B) Assessment of S-phase progression by BrdU incorporation; 48 h after siRNA transfection, U2OS cells were synchronized for 16 h with 1 mM hydroxyurea (HU) and released for 6 h in BrdU-containing medium. BrdU-positive cells were stained with anti-BrdU primary antibody (Calbiochem) and Alexa 488 secondary antibody. DNA was counterstained with propidium iodide. Imaging and quantification were performed with Acumen Explorer microplate reader. Values are shown as mean±s.d. of three biological replicates. (C) Cell-cycle-dependent protein expression of DONSON. U2OS cells were synchronized either in G1/S with HU or in G2/M with nocodazole (Noc). Cells were collected at different time points after release into cell cycle for western blot analysis. (D) DONSON depletion is associated with a 10-fold increase in multipolar spindles compared to control treatments. U2OS cells were transfected with a DONSON siRNA pool and immunostained for α- and γ-tubulin at indicated time points. U2OS cells transfected with Rluc siRNAs serving as negative control. Data represent mean±s.d. of three biological replicates. At least 200 metaphase spindles were counted in each experiment. Scale bar indicates 2.5 μm. (E) DONSON protein co-localizes with centrosomes. HeLa cells were transfected with HA-tagged DONSON for 48 h and immunostained with primary HA antibody and Alexa 488-conjugated secondary antibody. Arrows indicate the centrosomal staining of DONSON (green). DNA was counterstained with DAPI (blue). Scale bar indicates 2.5 μm. (F) DONSON co-localizes with centrin. U2OS cells were transfected with HA-tagged DONSON; 48 h after transfection, cells were immunostained with anti-centrin and anti-HA-tagged primary antibodies and Alexa 488-, Alexa 594-conjugated secondary antibodies, respectively. Scale bar indicates 2.5 μm. Source data is available for this figure at www.nature.com/msb.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913390&req=5

f4: Functional analysis of candidate genes for roles in cell-cycle progression and spindle organization. (A) Time-resolved cell-cycle analysis in HeLa cells transfected with indicated siRNAs at different time points. Box colours represent fractions of cells with DNA content corresponding to sub-G1, G1/G0, S and G2/M. HeLa cells transfected with siRNAs against DONSON showed a delay in S-phase progression. (B) Assessment of S-phase progression by BrdU incorporation; 48 h after siRNA transfection, U2OS cells were synchronized for 16 h with 1 mM hydroxyurea (HU) and released for 6 h in BrdU-containing medium. BrdU-positive cells were stained with anti-BrdU primary antibody (Calbiochem) and Alexa 488 secondary antibody. DNA was counterstained with propidium iodide. Imaging and quantification were performed with Acumen Explorer microplate reader. Values are shown as mean±s.d. of three biological replicates. (C) Cell-cycle-dependent protein expression of DONSON. U2OS cells were synchronized either in G1/S with HU or in G2/M with nocodazole (Noc). Cells were collected at different time points after release into cell cycle for western blot analysis. (D) DONSON depletion is associated with a 10-fold increase in multipolar spindles compared to control treatments. U2OS cells were transfected with a DONSON siRNA pool and immunostained for α- and γ-tubulin at indicated time points. U2OS cells transfected with Rluc siRNAs serving as negative control. Data represent mean±s.d. of three biological replicates. At least 200 metaphase spindles were counted in each experiment. Scale bar indicates 2.5 μm. (E) DONSON protein co-localizes with centrosomes. HeLa cells were transfected with HA-tagged DONSON for 48 h and immunostained with primary HA antibody and Alexa 488-conjugated secondary antibody. Arrows indicate the centrosomal staining of DONSON (green). DNA was counterstained with DAPI (blue). Scale bar indicates 2.5 μm. (F) DONSON co-localizes with centrin. U2OS cells were transfected with HA-tagged DONSON; 48 h after transfection, cells were immunostained with anti-centrin and anti-HA-tagged primary antibodies and Alexa 488-, Alexa 594-conjugated secondary antibodies, respectively. Scale bar indicates 2.5 μm. Source data is available for this figure at www.nature.com/msb.
Mentions: To further investigate the role of DONSON in cell-cycle progression, we performed time-course experiments with HeLa cells depleted for DONSON and measured the DNA content. As shown in Figure 4A, we observed a slower S-phase progression compared to controls, suggesting a DNA replication defect, followed by accumulation of cells in G2/M at 48 h. We then assessed whether DONSON is required for DNA replication during S-phase progression. U2OS cells were arrested with hydroxyurea (HU) in G1/S, and incorporation of bromodeoxyuridine (BrdU) was measured after release. As shown in Figure 4B, depletion of DONSON resulted in significant decrease in BrdU incorporation, indicating that DONSON is required for S-phase transition. We next asked whether the level of DONSON protein is regulated during the cell cycle. U2OS cells were arrested either in G1/S and G2/M by HU or nocodazole, respectively. After release from HU, we assessed DONSON protein levels for up to 24 h by immunoblot analysis. As shown in Figure 4C, DONSON levels peaked at 6 h after release and preceded the cyclin A expression as a marker for G2. Furthermore, when cells were released from G2/M-phase arrest, peak DONSON levels were observed later than phospho-H3 as a marker for M phase (Figure 4C). Taken together, these experiments indicate that DONSON levels are regulated during cell division, peaking during S phase and that DONSON is required for cell-cycle progression.

Bottom Line: With RNA interference (RNAi), highly parallel phenotyping of loss-of-function effects in cells has become feasible.Experimental evidence supports that DONSON is a novel centrosomal protein required for DDR signalling and genomic integrity.Multiparametric phenotyping by automated imaging and computational annotation is a powerful method for functional discovery and mapping the landscape of phenotypic responses to cellular perturbations.

View Article: PubMed Central - PubMed

Affiliation: German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany.

ABSTRACT
Genetic screens for phenotypic similarity have made key contributions to associating genes with biological processes. With RNA interference (RNAi), highly parallel phenotyping of loss-of-function effects in cells has become feasible. One of the current challenges however is the computational categorization of visual phenotypes and the prediction of biological function and processes. In this study, we describe a combined computational and experimental approach to discover novel gene functions and explore functional relationships. We performed a genome-wide RNAi screen in human cells and used quantitative descriptors derived from high-throughput imaging to generate multiparametric phenotypic profiles. We show that profiles predicted functions of genes by phenotypic similarity. Specifically, we examined several candidates including the largely uncharacterized gene DONSON, which shared phenotype similarity with known factors of DNA damage response (DDR) and genomic integrity. Experimental evidence supports that DONSON is a novel centrosomal protein required for DDR signalling and genomic integrity. Multiparametric phenotyping by automated imaging and computational annotation is a powerful method for functional discovery and mapping the landscape of phenotypic responses to cellular perturbations.

Show MeSH
Related in: MedlinePlus