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SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair.

Kanu N, Grönroos E, Martinez P, Burrell RA, Yi Goh X, Bartkova J, Maya-Mendoza A, Mistrík M, Rowan AJ, Patel H, Rabinowitz A, East P, Wilson G, Santos CR, McGranahan N, Gulati S, Gerlinger M, Birkbak NJ, Joshi T, Alexandrov LB, Stratton MR, Powles T, Matthews N, Bates PA, Stewart A, Szallasi Z, Larkin J, Bartek J, Swanton C - Oncogene (2015)

Bottom Line: Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity.In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach.Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo.

View Article: PubMed Central - PubMed

Affiliation: UCL Cancer Institute, Paul O'Gorman Building, London, UK.

ABSTRACT
Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.

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Loss of SETD2 leads to impaired RAD51 foci formation and LEDGF association to chromatin. (a) A representative experiment of RAD51 foci formation following IR (8 Gy) in siRNA-transfected RCC4 cells. siRNA for Nijmegen breakage syndrome 1 was used as a positive control for impaired RAD51 foci formation. 45 An arbitrary threshold of 80 RAD51 foci was used. (b) Proportion of cells with >80 RAD51 foci (mean±s.e.m of three independent experiments Statistical test: two-tailed t-test). **P⩽0.01. (c) RCC4 cells were exposed to 0 (non-irradiated) or 3 Gy IR, allowed to form colonies over 16 days, before quantification of colony numbers (mean±s.e.m. of three independent experiments. Statistical test: two-tailed t-test). (d) Cellular fractionation was performed to examine chromatin-bound LEDGF/p75 in RCC4 and FG2 cells. Total levels of LEDGF/p75 in whole-cell lysates (WCL) are shown as control. * denotes a non-specific band.
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fig4: Loss of SETD2 leads to impaired RAD51 foci formation and LEDGF association to chromatin. (a) A representative experiment of RAD51 foci formation following IR (8 Gy) in siRNA-transfected RCC4 cells. siRNA for Nijmegen breakage syndrome 1 was used as a positive control for impaired RAD51 foci formation. 45 An arbitrary threshold of 80 RAD51 foci was used. (b) Proportion of cells with >80 RAD51 foci (mean±s.e.m of three independent experiments Statistical test: two-tailed t-test). **P⩽0.01. (c) RCC4 cells were exposed to 0 (non-irradiated) or 3 Gy IR, allowed to form colonies over 16 days, before quantification of colony numbers (mean±s.e.m. of three independent experiments. Statistical test: two-tailed t-test). (d) Cellular fractionation was performed to examine chromatin-bound LEDGF/p75 in RCC4 and FG2 cells. Total levels of LEDGF/p75 in whole-cell lysates (WCL) are shown as control. * denotes a non-specific band.

Mentions: The transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) contains a proline-tryptophan-tryptophan-proline domain, which has been shown to interact with H3K36me3.20 LEDGF is required for CtIP recruitment leading to RAD51 loading and DNA repair.21 We first investigated the proficiency of homologous recombination (HR) following SETD2 depletion in RCC cell lines by small-interfering RNA (siRNA). We used two alternative siRNA pools targeting SETD2, having excluded some sequences because of off-target effects (Supplementary Figures S4a and b). After 48 h both the pools and individual siRNA sequences efficiently silenced SETD2 and depleted H3K36me3 levels (Supplementary Figures sS4c and e). Consistent with a defect in HR, SETD2-depleted cells displayed reduced RAD51 foci formation, used as surrogate for detecting active HR, following induction of DNA damage with ionizing irradiation (IR) (Figure 4a and Supplementary Figure S5a). Using our newly developed quantitative method to assess RAD51 foci formation in large numbers of cells, we found that the proportion of RAD51-positive cells was reduced from 15.1% in control to 2.2% in SETD2-depleted cells and 2.9% in cells depleted of Nijmegen breakage syndrome 1, which was used as a positive control (both P=0.003, average from n= 3 independent experiments, Figure 4b). Similar results were obtained in U2OS cells (SETD2 wild-type) (Supplementary Figures S5b and c), and RCC-JW cells (SETD2 missense mutation, H3K36me3 competent) (Supplementary Figure S5d). In order to support the specificity of the RNA interference phenotype, we repeated these experiments in the RCC-FG2 line, which harbours bi-allelic aberrations in SETD2 (no detectable SETD2 protein and barely detectable H3K36me3, Supplementary Figures S4c and S5e). No decrease in RAD51 foci formation after IR was observed following SETD2 silencing in RCC-FG2. Consistent with a defect in HR, SETD2-depleted cells were also more sensitive to IR (P=0.035, two-tailed t-test, Figure 4c). As Rad51 foci formation and HR repair require CtIP-mediated double-strand break end resection, and CtIP recruitment to active-chromatin-associated double-strand breaks relies on the proline-tryptophan-tryptophan-proline domain-containing LEDGF,21, 22 we speculated that chromatin loading of LEDGF may depend on SETD2 function. Indeed, we observed reduced chromatin-bound LEDGF/p75 following SETD2 depletion in RCC cell lines, while there was no decrease in total cellular LEDGF levels (Figure 4d). Our data therefore support the hypothesis that the defective DNA repair in the absence of SETD2 could exacerbate the DNA damage observed in vivo. This conclusion is further supported by two recent studies, submitted while the manuscript was under preparation, reporting the link between SETD2, H3K36me3 and HR.20, 22


SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair.

Kanu N, Grönroos E, Martinez P, Burrell RA, Yi Goh X, Bartkova J, Maya-Mendoza A, Mistrík M, Rowan AJ, Patel H, Rabinowitz A, East P, Wilson G, Santos CR, McGranahan N, Gulati S, Gerlinger M, Birkbak NJ, Joshi T, Alexandrov LB, Stratton MR, Powles T, Matthews N, Bates PA, Stewart A, Szallasi Z, Larkin J, Bartek J, Swanton C - Oncogene (2015)

Loss of SETD2 leads to impaired RAD51 foci formation and LEDGF association to chromatin. (a) A representative experiment of RAD51 foci formation following IR (8 Gy) in siRNA-transfected RCC4 cells. siRNA for Nijmegen breakage syndrome 1 was used as a positive control for impaired RAD51 foci formation. 45 An arbitrary threshold of 80 RAD51 foci was used. (b) Proportion of cells with >80 RAD51 foci (mean±s.e.m of three independent experiments Statistical test: two-tailed t-test). **P⩽0.01. (c) RCC4 cells were exposed to 0 (non-irradiated) or 3 Gy IR, allowed to form colonies over 16 days, before quantification of colony numbers (mean±s.e.m. of three independent experiments. Statistical test: two-tailed t-test). (d) Cellular fractionation was performed to examine chromatin-bound LEDGF/p75 in RCC4 and FG2 cells. Total levels of LEDGF/p75 in whole-cell lysates (WCL) are shown as control. * denotes a non-specific band.
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fig4: Loss of SETD2 leads to impaired RAD51 foci formation and LEDGF association to chromatin. (a) A representative experiment of RAD51 foci formation following IR (8 Gy) in siRNA-transfected RCC4 cells. siRNA for Nijmegen breakage syndrome 1 was used as a positive control for impaired RAD51 foci formation. 45 An arbitrary threshold of 80 RAD51 foci was used. (b) Proportion of cells with >80 RAD51 foci (mean±s.e.m of three independent experiments Statistical test: two-tailed t-test). **P⩽0.01. (c) RCC4 cells were exposed to 0 (non-irradiated) or 3 Gy IR, allowed to form colonies over 16 days, before quantification of colony numbers (mean±s.e.m. of three independent experiments. Statistical test: two-tailed t-test). (d) Cellular fractionation was performed to examine chromatin-bound LEDGF/p75 in RCC4 and FG2 cells. Total levels of LEDGF/p75 in whole-cell lysates (WCL) are shown as control. * denotes a non-specific band.
Mentions: The transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) contains a proline-tryptophan-tryptophan-proline domain, which has been shown to interact with H3K36me3.20 LEDGF is required for CtIP recruitment leading to RAD51 loading and DNA repair.21 We first investigated the proficiency of homologous recombination (HR) following SETD2 depletion in RCC cell lines by small-interfering RNA (siRNA). We used two alternative siRNA pools targeting SETD2, having excluded some sequences because of off-target effects (Supplementary Figures S4a and b). After 48 h both the pools and individual siRNA sequences efficiently silenced SETD2 and depleted H3K36me3 levels (Supplementary Figures sS4c and e). Consistent with a defect in HR, SETD2-depleted cells displayed reduced RAD51 foci formation, used as surrogate for detecting active HR, following induction of DNA damage with ionizing irradiation (IR) (Figure 4a and Supplementary Figure S5a). Using our newly developed quantitative method to assess RAD51 foci formation in large numbers of cells, we found that the proportion of RAD51-positive cells was reduced from 15.1% in control to 2.2% in SETD2-depleted cells and 2.9% in cells depleted of Nijmegen breakage syndrome 1, which was used as a positive control (both P=0.003, average from n= 3 independent experiments, Figure 4b). Similar results were obtained in U2OS cells (SETD2 wild-type) (Supplementary Figures S5b and c), and RCC-JW cells (SETD2 missense mutation, H3K36me3 competent) (Supplementary Figure S5d). In order to support the specificity of the RNA interference phenotype, we repeated these experiments in the RCC-FG2 line, which harbours bi-allelic aberrations in SETD2 (no detectable SETD2 protein and barely detectable H3K36me3, Supplementary Figures S4c and S5e). No decrease in RAD51 foci formation after IR was observed following SETD2 silencing in RCC-FG2. Consistent with a defect in HR, SETD2-depleted cells were also more sensitive to IR (P=0.035, two-tailed t-test, Figure 4c). As Rad51 foci formation and HR repair require CtIP-mediated double-strand break end resection, and CtIP recruitment to active-chromatin-associated double-strand breaks relies on the proline-tryptophan-tryptophan-proline domain-containing LEDGF,21, 22 we speculated that chromatin loading of LEDGF may depend on SETD2 function. Indeed, we observed reduced chromatin-bound LEDGF/p75 following SETD2 depletion in RCC cell lines, while there was no decrease in total cellular LEDGF levels (Figure 4d). Our data therefore support the hypothesis that the defective DNA repair in the absence of SETD2 could exacerbate the DNA damage observed in vivo. This conclusion is further supported by two recent studies, submitted while the manuscript was under preparation, reporting the link between SETD2, H3K36me3 and HR.20, 22

Bottom Line: Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity.In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach.Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo.

View Article: PubMed Central - PubMed

Affiliation: UCL Cancer Institute, Paul O'Gorman Building, London, UK.

ABSTRACT
Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.

Show MeSH
Related in: MedlinePlus