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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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Replication fork progression is impaired following SETD2 depletion. RCC4 cells were treated with control or SETD2 siRNAs for 48 h, sequentially incubated for 20 min with CldU and IdU, followed by DNA fibre preparation. Representative DNA fibres are shown in: (a) (scale bar: 10 μm). (b) Distribution of replication fork progression rates in control versus SETD2-depleted cells.
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fig7: Replication fork progression is impaired following SETD2 depletion. RCC4 cells were treated with control or SETD2 siRNAs for 48 h, sequentially incubated for 20 min with CldU and IdU, followed by DNA fibre preparation. Representative DNA fibres are shown in: (a) (scale bar: 10 μm). (b) Distribution of replication fork progression rates in control versus SETD2-depleted cells.

Mentions: Histone dynamics have an important role in DNA replication, where the stability of the replication complex is dependent on the reassembly of nucleosomes behind the advancing fork.30, 31 In line with this, the FACT complex has been demonstrated to recruit the MCM complex to chromatin during replication.32, 33 In order to specifically study the role of SETD2 during S phase, SETD2- and control siRNA-treated cells were studied after release from a double thymidine block. SETD2-depleted cells exited the block and entered into S phase with similar dynamics as control-treated cells (Figure 6d). We observed reduced chromatin association of MCM7, DNA polymerase δ and histone H3, whereas overall protein levels of the replisome components were unaffected in RCC4, RCC-FG2, UOK121 and U2OS cell lines by SETD2 depletion, indicating that impaired nucleosome assembly may affect replication dynamics (Figure 6e and Supplementary Figure 8d). In support of this, RCC-FG2 cells, possessing SETD2 loss-of-function and absent H3K36me3, also exhibited reduced loading of replisome components onto chromatin (Supplementary Figure 8e). Nucleosome integrity has been demonstrated to have a key role in replisome stability during replication fork progression.30 Given the aberrant nucleosome occupancy and reduced chromatin association of several DNA replication factors (Figures 6c and e and Supplementary Figures 8d and e), we wished to directly assess any impact of SETD2 status on replication fork progression using DNA fibre assays. Replication fork progression was significantly slower following SETD2 depletion (Figures 7a and b) decreasing from 1.0 kb/min in control to 0.75 kb/min in SETD2-depleted cells, P=8.3e–104, two-tailed unpaired t-test with Welch's correction (Supplementary Figure S9a). In agreement with this finding, we observed significantly slower replication fork progression in A498 cells (0.73 kb/min), which have a homozygous truncating SETD2 mutation2 and reduced H3K36me3 levels (Supplementary Figure S4f), compared with RCC4 cells that have one SETD2 allele intact (1.1 kb/min, P=2.35e–75, two-tailed unpaired t-test with Welch's correction, Supplementary Figure S9b). RCC-FG2 cells, which are SETD2 deficient, also had a slower replication fork speed than RCC4 cells (0.85 kb/min), and no further reduction in replication speed was observed following depletion of SETD2 (Supplementary Figure S9c). Consistent with slower replication rates, we noted an accumulation of cells in S phase after SETD2 depletion (50% cells versus 36% in control cells, P=0.048, two-tailed t-test,) and an extended S phase duration (Supplementary Figures S10a and c). These results suggest that in the absence of SETD2, defective nucleosome assembly during S phase contributes to DNA replication fork instability leading to genomic damage.


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)

Replication fork progression is impaired following SETD2 depletion. RCC4 cells were treated with control or SETD2 siRNAs for 48 h, sequentially incubated for 20 min with CldU and IdU, followed by DNA fibre preparation. Representative DNA fibres are shown in: (a) (scale bar: 10 μm). (b) Distribution of replication fork progression rates in control versus SETD2-depleted cells.
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fig7: Replication fork progression is impaired following SETD2 depletion. RCC4 cells were treated with control or SETD2 siRNAs for 48 h, sequentially incubated for 20 min with CldU and IdU, followed by DNA fibre preparation. Representative DNA fibres are shown in: (a) (scale bar: 10 μm). (b) Distribution of replication fork progression rates in control versus SETD2-depleted cells.
Mentions: Histone dynamics have an important role in DNA replication, where the stability of the replication complex is dependent on the reassembly of nucleosomes behind the advancing fork.30, 31 In line with this, the FACT complex has been demonstrated to recruit the MCM complex to chromatin during replication.32, 33 In order to specifically study the role of SETD2 during S phase, SETD2- and control siRNA-treated cells were studied after release from a double thymidine block. SETD2-depleted cells exited the block and entered into S phase with similar dynamics as control-treated cells (Figure 6d). We observed reduced chromatin association of MCM7, DNA polymerase δ and histone H3, whereas overall protein levels of the replisome components were unaffected in RCC4, RCC-FG2, UOK121 and U2OS cell lines by SETD2 depletion, indicating that impaired nucleosome assembly may affect replication dynamics (Figure 6e and Supplementary Figure 8d). In support of this, RCC-FG2 cells, possessing SETD2 loss-of-function and absent H3K36me3, also exhibited reduced loading of replisome components onto chromatin (Supplementary Figure 8e). Nucleosome integrity has been demonstrated to have a key role in replisome stability during replication fork progression.30 Given the aberrant nucleosome occupancy and reduced chromatin association of several DNA replication factors (Figures 6c and e and Supplementary Figures 8d and e), we wished to directly assess any impact of SETD2 status on replication fork progression using DNA fibre assays. Replication fork progression was significantly slower following SETD2 depletion (Figures 7a and b) decreasing from 1.0 kb/min in control to 0.75 kb/min in SETD2-depleted cells, P=8.3e–104, two-tailed unpaired t-test with Welch's correction (Supplementary Figure S9a). In agreement with this finding, we observed significantly slower replication fork progression in A498 cells (0.73 kb/min), which have a homozygous truncating SETD2 mutation2 and reduced H3K36me3 levels (Supplementary Figure S4f), compared with RCC4 cells that have one SETD2 allele intact (1.1 kb/min, P=2.35e–75, two-tailed unpaired t-test with Welch's correction, Supplementary Figure S9b). RCC-FG2 cells, which are SETD2 deficient, also had a slower replication fork speed than RCC4 cells (0.85 kb/min), and no further reduction in replication speed was observed following depletion of SETD2 (Supplementary Figure S9c). Consistent with slower replication rates, we noted an accumulation of cells in S phase after SETD2 depletion (50% cells versus 36% in control cells, P=0.048, two-tailed t-test,) and an extended S phase duration (Supplementary Figures S10a and c). These results suggest that in the absence of SETD2, defective nucleosome assembly during S phase contributes to DNA replication fork instability leading to genomic damage.

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