RB localizes to DNA double-strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1

Vélez-Cruz, Renier; Swarnalatha, Manickavinayaham; Biswas, Anup; Clary, Regina Weaks; Premkumar, Tolkappiyan; Cole, Francesca; Johnson, D. Gale

doi:10.1101/gad.288282.116

Cited by 91 publications

(181 citation statements)

References 74 publications

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“…Rb heterozygosity leads to loss of E2F1 and Condensin II binding, accompanied by replication stress labelled by increased γH2AX foci. Recently, it was shown that Rb localises to DSBs dependent on E2F1 and ATM, to promote DSB repair through homologous recombination (Velez‐Cruz et al , ). We show that a similar mechanism might operate in plants, where RBR localisation to DSBs requires ATM and ATR activities.…”

Section: Discussionmentioning

confidence: 99%

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control

et al. 2017

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The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX‐labelled DNA damage foci in an ATM‐ and ATR‐dependent manner. These γH2AX‐labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co‐localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta. Genetic interaction between the RBR‐silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

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Section: Discussionmentioning

confidence: 99%

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control

et al. 2017

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“…Statistical analysis of sequence data was performed using R (version 3.4.2), and the plotting function used was lattice (v0. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35].…”

Section: Methodsmentioning

confidence: 99%

“…These include chromosome condensation through RB-dependent recruitment of condensin II and cohesin (20,21). The RB protein also influences the repair of DNA breaks through both nonhomologous end joining (NHEJ) (22) and homologous recombination (HR) (23) and through induction of mitochondrial biogenesis that impacts cell metabolism (24)(25)(26). Some of these functions, such as repair of DNA breaks by HR, are obligatorily outside RB's role in G 1 -to-S-phase regulation.…”

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confidence: 99%

RB1 Deletion in Retinoblastoma Protein Pathway-Disrupted Cells Results in DNA Damage and Cancer Progression

Marshall

Roes

Passos

et al. 2019

Molecular and Cellular Biology

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Proliferative control in cancer cells is frequently disrupted by mutations in the retinoblastoma protein (RB) pathway. Intriguingly, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage and instability in cancer cells with RB pathway defects when RB1 mutations are induced. We generated isogenic RB1 mutant genotypes with CRISPR/Cas9 in a number of cell lines. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immunocompromised mice, RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late-arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the preexisting proliferative control deficit. FIG 1 CRISPR/Cas9-induced mutations in RB1 cause DNA damage. (A) Ethidium bromide-stained agarose gel showing examples of wild-type, heterozygous, and homozygous mutant RB1 genotypes that are detected by PCR amplification of exon 22 sequences. MW, molecular weight. (B, top) Representative Western blot showing RB expression in control, heterozygous, and homozygous mutant cells. (Bottom) Sp1 loading control.(C) Immunofluorescence microscopy was used to detect RB expression (green) in cultures of control, heterozygous, or homozygous mutants. Cells were counterstained with DAPI to visualize nuclei (blue). (D) Representative confocal microscopy images of ␥H2AX foci (red) in control, heterozygous, and homozygous RB1 mutant cells. Cells were counterstained with DAPI to visualize nuclei (blue). (E) Counts of ␥H2AX foci for each of the U2OS RB1 genotypes. The average proportions of cells with discrete numbers of foci are shown as histograms, while the cumulative frequency of foci for each genotype is shown in the inset. The average distributions of foci for RB1 wild-type (4 different clones), heterozygous (3 different clones), and knockout (4 different clones) cells were compared using the Kolmogorov-Smirnov test (*, P Ͻ 0.05). (F) U2OS cells were transfected with CRISPR/Cas9 constructs targeting either a safe-harbor site in the genome or exon 2 of the RB1 gene. Three clones were selected under both control and knockout conditions, and ␥H2AX foci were quantified by fluorescence microscopy. The average proportions of ␥H2AX foci for both RB1 wild-type and knockout genotypes are shown as histograms, while the cumulative relative frequency of foci is shown in the inset. Focus distributions were again compared by a Kolmogorov-Smirnov test. (G) H460 lung cancer cells were stained for ␥H2AX foci and analyzed as described above for panel F. (H) H1792 non-small cell lung cancer cells were analyzed as described above for panel F. All error bars are ϩ1 standard error of the mean (SEM). *, P Ͻ 0.05. RB1Deletion Causes ...

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“…12). BRG1, on the other hand, was shown to be recruited to DSBs by the retinoblastoma (RB) tumor suppressor to stimulate DNA end resection and HRR (13). Thus, targeting chromatin regulating proteins that are involved in DDR after IR may be a new strategy for individual radiosensitization of cancer cells.…”

Section: Introductionmentioning

confidence: 99%

Targeting of BRM Sensitizes BRG1-Mutant Lung Cancer Cell Lines to Radiotherapy

Zernickel

Sak

Riaz

et al. 2019

Molecular Cancer Therapeutics

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Targeting of epigenetic regulators as the chromatin remodeler SWI/SNF is proving to be a promising therapeutic strategy for individualized treatment of cancer patients. Here, we tested whether targeting one of the two mutually exclusive subdomains of the SWI/SNF complex BRM/SMARCA2 can sensitize specifically non-small cell lung carcinoma (NSCLC) cells with mutations in the other subunit BRG1/SMARCA4 toward ionizing radiation (IR). Knockdown of BRM with siRNA or shRNA and its consequences for radiation sensitivity as measured by clonogenic survival and plaque-monolayer control was studied in different NSCLC lines with or without BRG1 mutations and in primary fibroblasts. Furthermore, the effect on double-strand break (DSB) repair markers measured by immunofluorescence staining of 53BP1-, g-H2AX-, and Rad51-foci was investigated. BRG1mutated cell lines showed an increased surviving fraction compared with BRG1 proficient cells. Depletion of BRM (i) leads to a decreased proliferation rate and plating efficiency specifically in BRG1-mutated cells, (ii) specifically sensitized BRG1-mutant NSCLC cells toward IR as characterized by a survival reducing factor of 0.63 [95% confidence interval (CI), 0.57-0.69] in the dose range between 2 and 6 Gy, and (iii) decreased the tumor control doses after daily fractionation at 4 Gy in BRG1-mutant NSCLC cell lines A549 and H1299 in minimonolayers by 9.9% AE 1.3% and 13.6% AE 1.8%, respectively. In addition, an increase of residual Rad51-foci at 24 hours after irradiation in BRG1-mutant cells was demonstrated. Therefore, targeting of BRM in combination with radiotherapy is supposed to improve the therapeutic outcome of lung cancer patients harboring BRG1 mutations.The present study shows that the moderate radioresponsiveness of NSCLC cells with BRG1 mutations can be increased upon BRM depletion that is associated with a prolonged Rad51-foci prevalence at DNA DSBs.

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RB localizes to DNA double-strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1

Cited by 91 publications

References 74 publications

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control

RB1 Deletion in Retinoblastoma Protein Pathway-Disrupted Cells Results in DNA Damage and Cancer Progression

Targeting of BRM Sensitizes BRG1-Mutant Lung Cancer Cell Lines to Radiotherapy

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