ARID1A promotes genomic stability through protecting telomere cohesion

Abstract

ARID1A inactivation causes mitotic defects. Paradoxically, cancers with high ARID1A mutation rates typically lack copy number alterations (CNAs). Here, we show that ARID1A inactivation causes defects in telomere cohesion, which selectively eliminates gross chromosome aberrations during mitosis. ARID1A promotes the expression of cohesin subunit STAG1 that is specifically required for telomere cohesion. ARID1A inactivation causes telomere damage that can be rescued by STAG1 expression. Colony formation capability of single cells in G₂/M, but not G₁ phase, is significantly reduced by ARID1A inactivation. This correlates with an increase in apoptosis and a reduction in tumor growth. Compared with ARID1A wild-type tumors, ARID1A-mutated tumors display significantly less CNAs across multiple cancer types. Together, these results show that ARID1A inactivation is selective against gross chromosome aberrations through causing defects in telomere cohesion, which reconciles the long-standing paradox between the role of ARID1A in maintaining mitotic integrity and the lack of genomic instability in ARID1A-mutated cancers.

Fig. 1

ARID1A inactivation causes defective telomere cohesion. a, b Representative images of prometaphase chromosome spreads (a) and quantification of distance between distal ends of sister chromatids (b) enriched by mitotic shake-off from parental and ARID1A knockout RMG1 cells. c–e Representative images of chromosome spreads (c) and quantification of distance between distal ends of sister chromatids (d) enriched by colcemid treatment from parental and ARID1A knockout RMG1 cells, and ARID1A mutated TOV21G cells. And quantification of distance between distal ends of sister chromatids enriched by colcemid treatment from the indicated clear cell ovarian cancer cell lines or primary cultures highlighted in red (e). f, g Representative images of telomere fluorescent in situ hybridization (f) and quantification of mitotic telomere signal loss (g) in parental and ARID1A knockout RMG1 cells. h Quantification of mitotic telomere signal loss in the indicated clear cell ovarian cancer cell lines. i, j Representative images of telomere fluorescent in situ hybridization (i) and quantification of mitotic telomere signal loss (j) in cells isolated from normal mouse ovary and Arid1a^−/−/Pik3ca^H1047R genetic clear cell ovarian tumors respectively. n = 3 independent experiments unless otherwise stated. Data represent mean ± s.e.m. P values were calculated using a two-tailed t test except in 1e and 1 h by multilevel mixed-effects models

Fig. 2

ARID1A inactivation causes DNA damage at telomeres. a, b Schematic of synchronization and release (a) and immunoblot of DNA damage marker γH2AX (b) in parental and ARID1A knockout RMG1 cells. Phosphorylated Histone H3 at serine 10 (pH3S10) was used as a marker of mitosis. Relative intensities of immunoblot bands were quantified underneath. c, d Co-staining of telomere by FISH and γH2AX (c) and quantification of telomeric DNA damage (d) in mitotic parental and ARID1A knockout RMG1 cells after cytospin. e, f Co-staining of telomere by FISH and γH2AX in ARID1A-mutated TOV21G mitotic cells (e) and quantification of mitotic telomeric DNA damage in a panel of clear cell ovarian cancer cell lines or primary cultures highlighted in red (f). g, h Representative images (g) and quantification (h) of telomere DNA damage in parental, P53^−/−/Pten^−/− and P53^−/−/Pten^−/−/Arid1a^−/− mouse bladder organoid cultures. n = 3 independent experiments unless otherwise stated. Data represent mean ± s.e.m. Scale bar = 10 μm. P values were calculated using a two-tailed t test except in 2f and 2h by multilevel mixed-effects models

Fig. 3

ARID1A inactivation causes chromosomal defects during mitosis. a–c Representative images (a) and quantification of anaphase bridge (b) and lagging chromosomes (c) in parental and ARID1A knockout RMG1 cells. d Telomere-binding TRF1 protein staining in anaphase bridges and lagging chromosomes observed in ARID1A knockout RMG1 cells. e, f Quantification of percentage of anaphase bridge (e) and lagging chromosome (f) positive cells in a panel of clear cell ovarian cancer cell lines or primary cultures highlighted in red. g, h Representative images of metaphase with anaphase bridge (g) and quantification (h) in ARID1A proficient and deficient patient-derived xenografts of clear cell ovarian cancer. i Representative images of mitotic chromosomal fusion in RMG1 ARID1A knockout and ARID1A-mutated TOV21G cell. j Quantification of mitotic chromosomal fusion in parental and ARID1A knockout RMG1 cells. k Quantification of mitotic chromosomal fusion in the indicated clear cell ovarian cancer cell lines or primary cultures highlighted in red. l Parental and ARID1A knockout RMG1 cells, and ARID1A-mutated TOV21G cells were subjected to time-lapse video microscopic analysis for mitosis. Cell nuclei were visualized by staining for DNA using siR-DNA. Time is expressed as hours: minutes. Arrows points to chromosomal bridges or lagging chromosomes. m Quantification of mitosis duration in the indicated cells. n = 3 independent experiments unless otherwise stated. Data represent mean ± s.e.m. P values were calculated using a two-tailed t test except for 3e, 3f, and 3k by multilevel mixed-effects models

Fig. 4

ARID1A promotes STAG1 expression. a ARID1A ChIP-seq and input tracks of the STAG1 gene locus in RMG1 cells. b Validation of ARID1A binding to the STAG1 promoter by ChIP-qPCR in parental and ARID1A knockout RMG1 cells. c, d Validation of STAG1 downregulation by ARID1A knockout at the mRNA levels determined by qRT-PCR analysis (c) and at the protein levels determined by immunoblot (d) in RMG1 cells. Expression of other cohesin subunits STAG2 and SMC1 was used negative controls. e, f Expression of STAG1 at the mRNA levels determined by qRT-PCR analysis (e) and at the protein levels by immunoblot (f) in the indicated clear cell ovarian cancer cell lines or primary cultures highlighted in red. g Immunoblot of cohesin subunits Stag1 and Stag2 in wild-type controls, P53^−/−/Pten^−/− and P53^−/−/Pten^−/−/Arid1a^−/− mouse bladder organoid cultures. h Correlation analysis between the expression of ARID1A and STAG1 in tumor microarray (TMA) of clear cell ovarian carcinomas determined by immunohistochemical staining. n = 3 independent experiments unless otherwise stated. Data represent mean ± s.e.m. P values were calculated using a two-tailed t test except for 4e by multilevel mixed-effects models, and for 4 h by Pearson correlation analysis. Relative intensities of immunoblot bands were quantified underneath

Fig. 5

Ectopic STAG1 rescues the telomere damage and mitotic defects in ARID1A-inactivated cells. a Immunoblot validation of STAG1 knockdown in RMG1 cells. b, c Representative images (b) and quantification (c) of telomere DNA damage in RMG1 shRNA vector control and STAG1 knockdown cells determined by telomere FISH and γH2AX co-staining. d Quantification of distance between distal ends of sister chromatids enriched by colcemid treatment from the indicated RMG1 cells. e–g Schematics of STAG1 wild-type and mutant that lacks nuclear localization sequence (e), and validation of ectopic STAG1 expression by immunoblot (f) or immunofluorescence (g) in ARID1A knockout RMG1 cells. h–j Quantification of distance between distal ends of sister chromatids (h), and percentage of anaphase bridge (i) and lagging chromosome (j) positive-mitotic cells in the indicated parental, ARID1A knockout, and ARID1A knockout RMG1 cells rescued with wild-type or mutant STAG1, respectively. k, l Co-staining of telomere FISH and γH2AX (k) and quantification of telomeric DNA damage (l) in mitotic parental, ARID1A knockout, and ARID1A knockout RMG1 cells rescued with wild-type or mutant STAG1. m RMG1 cells expressing shSTAG1 or ARID1A knockout RMG1 cells rescued with wild-type or mutant STAG1 were subjected to time-lapse video microscopic analysis. Cell nuclei were visualized by staining for DNA using siR-DNA. Time is expressed as minutes: seconds. Arrows point to examples of lagging chromosomes. n = 3 independent experiments unless otherwise stated. Data represent mean ± s.e.m. Scale bar = 10 μm. P values were calculated using a two-tailed t test. Relative intensities of immunoblot bands were quantified underneath

Fig. 6

ARID1A inactivation is selective against the survival of cells during mitosis. a, b Representative images (a) and quantification of colony formation efficiency (b) of colonies formed by single parental or ARID1A knockout RMG1 cells at the indicated G₁ or G₂/M phases of the cell cycle sorted by flow cytometry based on Hoechst 33342 staining. c, d Representative images (c) and quantification of colony formation efficiency (d) of colonies formed by single parental or ARID1A knockout RMG1 cells at the indicated synchronized G₁ or G₂/M phases of the cell cycle. e, f Representative images (e) and quantification of colony formation efficiency (f) of colonies formed by single ARID1A knockout RMG1 cells rescued with wild-type or mutant STAG1 at the indicated G₁ or G₂/M phases of the cell cycle sorted by flow cytometry based on Hoechst 33342 staining. g, h Expression of ARID1A and apoptosis markers cleaved caspase 3 or cleaved PAPR p85 in a panel of clear cell ovarian cancer cell lines (g) or endometrial cancer cell lines (h), respectively. i, j Images of orthotopic tumors formed by parental and ARID1A knockout RMG1 cells (i) and the sizes of the tumors formed were quantified (j). k, l Integrated density analysis of colonies formed by single cell G₁ phase RMG1 parental and ARID1A knockout cells (k) or ARID1A knockout RMG1 cells rescued by wild-type or mutant STAG1 (l). m Compared with ARID1A wild-type tumors, ARID1A-mutated tumors exhibit a significant less copy number variations in the indicated cancer types in the TCGA datasets. n = 3 independent experiments unless otherwise stated. Data represent mean ± s.e.m. P values were calculated using a two-tailed t test except in 6 m by multilevel mixed-effects models. Relative intensities of immunoblot bands were quantified underneath