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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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Functional assays for candidate genes in maintenance of genomic integrity. (A) Depletion of RRM1, CLSPN, CD3EAP, CADM1, DONSON and SON induced γH2AX foci formation. U2OS cells were transfected with siRNA pools and immunostained with γH2AX antibody 72 h after transfection. Representative images of γH2AX foci formation are shown. Scale bar indicates 2.5 μm. (B) Quantification of γH2AX foci formation on depletion of candidate genes; 72 h after siRNA transfection, U2OS cells were fixed, immunostained for γH2AX and γH2AX-positive cells were quantified. Ratios of γH2AX-positive cells were normalized to the negative control Rluc siRNA treatment. Data are mean±s.d. of three biological replicates. (C) γH2AX accumulation in DONSON and SON-depleted cells is not caused by an accumulation of S-phase cells. U2OS cells were collected 72 h after siRNA transfection and cell lysates were analysed by western blotting using indicated antibodies. Source data is available for this figure at www.nature.com/msb.
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f5: Functional assays for candidate genes in maintenance of genomic integrity. (A) Depletion of RRM1, CLSPN, CD3EAP, CADM1, DONSON and SON induced γH2AX foci formation. U2OS cells were transfected with siRNA pools and immunostained with γH2AX antibody 72 h after transfection. Representative images of γH2AX foci formation are shown. Scale bar indicates 2.5 μm. (B) Quantification of γH2AX foci formation on depletion of candidate genes; 72 h after siRNA transfection, U2OS cells were fixed, immunostained for γH2AX and γH2AX-positive cells were quantified. Ratios of γH2AX-positive cells were normalized to the negative control Rluc siRNA treatment. Data are mean±s.d. of three biological replicates. (C) γH2AX accumulation in DONSON and SON-depleted cells is not caused by an accumulation of S-phase cells. U2OS cells were collected 72 h after siRNA transfection and cell lysates were analysed by western blotting using indicated antibodies. Source data is available for this figure at www.nature.com/msb.

Mentions: The bi-orientation attachment of each chromosome to both poles of the mitotic spindle is essential for genomic integrity (Loncarek et al, 2007). To test the involvement of DONSON in genomic integrity, we used quantitative immunofluorescence to measure the formation of γH2AX foci as a marker of DDR signalling (Figure 5A and B; Supplementary Figures 12 and 13). We observed that DONSON depletion led to the formation of γH2AX foci similar to the depletion of the known DDR effectors RRM1 and CHEK1. In addition, we checked other candidate genes regarding their involvement in genomic integrity. Similar to DONSON knockdown, we observed increased γH2AX formation in cells depleted of SON, CADM1 and CD3EAP (Figure 5A and B). One possible explanation of elevated γH2AX foci formation is the accumulation of cells in S phase (Tanaka et al, 2006). To test this hypothesis, we monitored cyclin levels in DONSON-depleted U2OS cells. As shown in Figure 5C, knock down of DONSON in U2OS cells led to increased cyclin D1 and decreased cyclin A and cyclin B1 levels, indicating that cells were arrested in G1 and that the elevated γH2AX foci formation observed in DONSON-depleted cells is not caused by an accumulation of cells in S phase. In contrast to DONSON, SON-depleted cells showed an enrichment of cyclin B1 protein, whereas cyclin A was decreased compared to the control treatment; this indicated that cells were arrested in G2/M (Figure 5C). In summary, these results suggest a role of DONSON, SON, CADM1 and CD3EAP in the maintenance of genomic integrity.


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 assays for candidate genes in maintenance of genomic integrity. (A) Depletion of RRM1, CLSPN, CD3EAP, CADM1, DONSON and SON induced γH2AX foci formation. U2OS cells were transfected with siRNA pools and immunostained with γH2AX antibody 72 h after transfection. Representative images of γH2AX foci formation are shown. Scale bar indicates 2.5 μm. (B) Quantification of γH2AX foci formation on depletion of candidate genes; 72 h after siRNA transfection, U2OS cells were fixed, immunostained for γH2AX and γH2AX-positive cells were quantified. Ratios of γH2AX-positive cells were normalized to the negative control Rluc siRNA treatment. Data are mean±s.d. of three biological replicates. (C) γH2AX accumulation in DONSON and SON-depleted cells is not caused by an accumulation of S-phase cells. U2OS cells were collected 72 h after siRNA transfection and cell lysates were analysed by western blotting using indicated antibodies. 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

f5: Functional assays for candidate genes in maintenance of genomic integrity. (A) Depletion of RRM1, CLSPN, CD3EAP, CADM1, DONSON and SON induced γH2AX foci formation. U2OS cells were transfected with siRNA pools and immunostained with γH2AX antibody 72 h after transfection. Representative images of γH2AX foci formation are shown. Scale bar indicates 2.5 μm. (B) Quantification of γH2AX foci formation on depletion of candidate genes; 72 h after siRNA transfection, U2OS cells were fixed, immunostained for γH2AX and γH2AX-positive cells were quantified. Ratios of γH2AX-positive cells were normalized to the negative control Rluc siRNA treatment. Data are mean±s.d. of three biological replicates. (C) γH2AX accumulation in DONSON and SON-depleted cells is not caused by an accumulation of S-phase cells. U2OS cells were collected 72 h after siRNA transfection and cell lysates were analysed by western blotting using indicated antibodies. Source data is available for this figure at www.nature.com/msb.
Mentions: The bi-orientation attachment of each chromosome to both poles of the mitotic spindle is essential for genomic integrity (Loncarek et al, 2007). To test the involvement of DONSON in genomic integrity, we used quantitative immunofluorescence to measure the formation of γH2AX foci as a marker of DDR signalling (Figure 5A and B; Supplementary Figures 12 and 13). We observed that DONSON depletion led to the formation of γH2AX foci similar to the depletion of the known DDR effectors RRM1 and CHEK1. In addition, we checked other candidate genes regarding their involvement in genomic integrity. Similar to DONSON knockdown, we observed increased γH2AX formation in cells depleted of SON, CADM1 and CD3EAP (Figure 5A and B). One possible explanation of elevated γH2AX foci formation is the accumulation of cells in S phase (Tanaka et al, 2006). To test this hypothesis, we monitored cyclin levels in DONSON-depleted U2OS cells. As shown in Figure 5C, knock down of DONSON in U2OS cells led to increased cyclin D1 and decreased cyclin A and cyclin B1 levels, indicating that cells were arrested in G1 and that the elevated γH2AX foci formation observed in DONSON-depleted cells is not caused by an accumulation of cells in S phase. In contrast to DONSON, SON-depleted cells showed an enrichment of cyclin B1 protein, whereas cyclin A was decreased compared to the control treatment; this indicated that cells were arrested in G2/M (Figure 5C). In summary, these results suggest a role of DONSON, SON, CADM1 and CD3EAP in the maintenance of genomic integrity.

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