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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.

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SON, DONSON and CD3EAP are required for the DNA damage response. (A) SON, DONSON and CD3EAP depletion leads to a decreased phosphorylation of CHEK1 on γ irradiation, similar to ATR; 48 h after siRNA transfection, cells were γ irradiated and collected 1 or 2 h later for immunoblot analysis, probing with indicated antibodies. (B) SON and DONSON depletion leads to attenuated phosphorylation of CHEK1 on UV exposure, similar to ATR depletion. U2OS cells were UVC irradiated (20 J/m2) 48 h after siRNA transfection. Cell lysates were collected for immunoblotting 2 h later and probed with indicated antibodies. (C) Knock down of DONSON and SON impairs RPA2 recruitment onto chromatin and the phosphorylation of ATR substrates. U2OS cells were transfected with siRNAs and UVC irradiated (20 J/m2). Subsequently, the chromatin-associated insoluble fraction was extracted 2 h after UV exposure, and the fractions were analysed for the indicated proteins by immunoblot.
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f6: SON, DONSON and CD3EAP are required for the DNA damage response. (A) SON, DONSON and CD3EAP depletion leads to a decreased phosphorylation of CHEK1 on γ irradiation, similar to ATR; 48 h after siRNA transfection, cells were γ irradiated and collected 1 or 2 h later for immunoblot analysis, probing with indicated antibodies. (B) SON and DONSON depletion leads to attenuated phosphorylation of CHEK1 on UV exposure, similar to ATR depletion. U2OS cells were UVC irradiated (20 J/m2) 48 h after siRNA transfection. Cell lysates were collected for immunoblotting 2 h later and probed with indicated antibodies. (C) Knock down of DONSON and SON impairs RPA2 recruitment onto chromatin and the phosphorylation of ATR substrates. U2OS cells were transfected with siRNAs and UVC irradiated (20 J/m2). Subsequently, the chromatin-associated insoluble fraction was extracted 2 h after UV exposure, and the fractions were analysed for the indicated proteins by immunoblot.

Mentions: The maintenance of genomic integrity is dependent on a functional DDR, a process comprising multiple signal transduction pathways that coordinate cell-cycle transitions, DNA replication, DNA repair and apoptosis (Cimprich and Cortez, 2008). The phosphorylation of CHEK1 is an early event in the DDR. To analyse the function of DONSON in DDR, we used immunoblot analysis to assess the phosphorylation of CHEK1 on γ irradiation in U2OS cells 48 h after transfection with DONSON. We observed that the phosphorylation of CHEK1 was strongly reduced in DONSON-depleted cells compared to negative control (Figure 6A). Moreover, DONSON-depleted cells showed an impaired phosphorylation of CHEK1 (Figure 6B and C) after UV exposure, similar to the effect of γ irradiation. Although ATM phosphorylation was not affected in DONSON-depleted cells, we observed an attenuated phosphorylation of RPA2, H2AX and NBS1 (Figure 6C). These results indicate that DONSON acts downstream of ATM and upstream of CHEK1 in the DDR signalling cascade.


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)

SON, DONSON and CD3EAP are required for the DNA damage response. (A) SON, DONSON and CD3EAP depletion leads to a decreased phosphorylation of CHEK1 on γ irradiation, similar to ATR; 48 h after siRNA transfection, cells were γ irradiated and collected 1 or 2 h later for immunoblot analysis, probing with indicated antibodies. (B) SON and DONSON depletion leads to attenuated phosphorylation of CHEK1 on UV exposure, similar to ATR depletion. U2OS cells were UVC irradiated (20 J/m2) 48 h after siRNA transfection. Cell lysates were collected for immunoblotting 2 h later and probed with indicated antibodies. (C) Knock down of DONSON and SON impairs RPA2 recruitment onto chromatin and the phosphorylation of ATR substrates. U2OS cells were transfected with siRNAs and UVC irradiated (20 J/m2). Subsequently, the chromatin-associated insoluble fraction was extracted 2 h after UV exposure, and the fractions were analysed for the indicated proteins by immunoblot.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: SON, DONSON and CD3EAP are required for the DNA damage response. (A) SON, DONSON and CD3EAP depletion leads to a decreased phosphorylation of CHEK1 on γ irradiation, similar to ATR; 48 h after siRNA transfection, cells were γ irradiated and collected 1 or 2 h later for immunoblot analysis, probing with indicated antibodies. (B) SON and DONSON depletion leads to attenuated phosphorylation of CHEK1 on UV exposure, similar to ATR depletion. U2OS cells were UVC irradiated (20 J/m2) 48 h after siRNA transfection. Cell lysates were collected for immunoblotting 2 h later and probed with indicated antibodies. (C) Knock down of DONSON and SON impairs RPA2 recruitment onto chromatin and the phosphorylation of ATR substrates. U2OS cells were transfected with siRNAs and UVC irradiated (20 J/m2). Subsequently, the chromatin-associated insoluble fraction was extracted 2 h after UV exposure, and the fractions were analysed for the indicated proteins by immunoblot.
Mentions: The maintenance of genomic integrity is dependent on a functional DDR, a process comprising multiple signal transduction pathways that coordinate cell-cycle transitions, DNA replication, DNA repair and apoptosis (Cimprich and Cortez, 2008). The phosphorylation of CHEK1 is an early event in the DDR. To analyse the function of DONSON in DDR, we used immunoblot analysis to assess the phosphorylation of CHEK1 on γ irradiation in U2OS cells 48 h after transfection with DONSON. We observed that the phosphorylation of CHEK1 was strongly reduced in DONSON-depleted cells compared to negative control (Figure 6A). Moreover, DONSON-depleted cells showed an impaired phosphorylation of CHEK1 (Figure 6B and C) after UV exposure, similar to the effect of γ irradiation. Although ATM phosphorylation was not affected in DONSON-depleted cells, we observed an attenuated phosphorylation of RPA2, H2AX and NBS1 (Figure 6C). These results indicate that DONSON acts downstream of ATM and upstream of CHEK1 in the DDR signalling cascade.

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