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Mol Cell Biol. 2013 May; 33(9): 1819–1829.
PMCID: PMC3624184

BRG1 Is Required for Formation of Senescence-Associated Heterochromatin Foci Induced by Oncogenic RAS or BRCA1 Loss

Abstract

Cellular senescence is an important tumor suppression mechanism. We have previously reported that both oncogene-induced dissociation of BRCA1 from chromatin and BRCA1 knockdown itself drive senescence by promoting formation of senescence-associated heterochromatin foci (SAHF). However, the molecular mechanism by which BRCA1 regulates SAHF formation and senescence is unclear. BRG1 is a chromatin-remodeling factor that interacts with BRCA1 and pRB. Here we show that BRG1 is required for SAHF formation and senescence induced by oncogenic RAS or BRCA1 loss. The interaction between BRG1 and BRCA1 is disrupted during senescence. This correlates with an increased level of chromatin-associated BRG1 in senescent cells. BRG1 knockdown suppresses the formation of SAHF and senescence, while it has no effect on BRCA1 chromatin dissociation induced by oncogenic RAS, indicating that BRG1 functions downstream of BRCA1 chromatin dissociation. Furthermore, BRG1 knockdown inhibits SAHF formation and senescence induced by BRCA1 knockdown. Conversely, BRG1 overexpression drives SAHF formation and senescence in a DNA damage-independent manner. This effect depends upon BRG1's chromatin-remodeling activity as well as the interaction between BRG1 and pRB. Indeed, the interaction between BRG1 and pRB is enhanced during senescence. Chromatin immunoprecipitation analysis revealed that BRG1's association with the human CDKN2A and CDKN1A gene promoters was enhanced during senescence induced by oncogenic RAS or BRCA1 knockdown. Consistently, knockdown of pRB, p21CIP1, and p16INK4a, but not p53, suppressed SAHF formation induced by BRG1. Together, these studies reveal the molecular underpinning by which BRG1 acts downstream of BRCA1 to promote SAHF formation and senescence.

INTRODUCTION

Activation of oncogenes (such as RAS) in primary mammalian cells typically triggers cellular senescence, a state of irreversible cell growth arrest (1, 2). Oncogene-induced senescence is an important tumor suppression mechanism in vivo (1). Senescent cells display several morphological and molecular characteristics. For instance, they are positive for senescence-associated β-galactosidase (SA-β-gal) activity (3). In addition, chromatin in the nuclei of senescent human cells typically reorganizes to form specialized domains of facultative heterochromatin called senescence-associated heterochromatin foci (SAHF) (48). SAHF are enriched in markers of heterochromatin such as histone H2A variant macroH2A (mH2A), di- or trimethylated lysine 9 histone H3 (H3K9Me2/3), and heterochromatin protein 1 (HP1) proteins (5, 7). SAHF formation contributes to the senescence-associated cell cycle exit by directly sequestering and silencing proliferation-promoting genes (4, 7). The p53 and pRB tumor suppressor pathways are the key regulators of senescence (1). Indeed, p16INK4a, an upstream regulator of pRB, and p21CIP1, a downstream target of p53, promote SAHF formation (7, 9). In addition, senescence induced by oncogenic RAS is characterized by a DNA damage response (10) and is accompanied by the accumulation of markers of DNA damage such as upregulation of γH2AX protein expression and increased formation of γH2AX DNA damage foci (10, 11).

BRCA1 plays an important role in DNA damage repair (12, 13). Germ line mutations in the BRCA1 gene predispose women to breast and ovarian cancer (12). We have previously demonstrated that BRCA1 becomes dissociated from chromatin in response to activation of oncogenes such as RAS (14). This promotes senescence by driving SAHF formation (14). In addition, BRCA1 chromatin dissociation contributes to the accumulation of DNA damage by impairing the BRCA1-mediated DNA repair response (14). Similarly, we showed that BRCA1 knockdown drives SAHF formation and senescence and triggers the DNA damage response (14). It has also been shown that cells from the BRCA1 exon 11 knockout mouse exhibit signs of premature senescence (15, 16). However, the molecular mechanism by which BRCA1 regulates SAHF formation and senescence remains to be determined. In addition, it is unclear whether SAHF formation induced by BRCA1 chromatin dissociation or BRCA1 knockdown is independent of the DNA damage response.

BRCA1 has also been implicated in regulating high-order chromatin structure. For example, targeting BRCA1 to an amplified lac operator-containing chromosome region in the mammalian genome results in large-scale chromatin unfolding (17). This suggests that BRCA1 antagonizes heterochromatin formation. Notably, BRCA1 also interacts with the BRG1 subunit of the ATP-dependent SWI/SNF chromatin-remodeling complex (18). BRG1 acts as an activator or repressor of gene expression in a context-dependent manner (19). Loss of BRG1 function is associated with malignant transformation (19), and BRG1 heterozygous deletion results in spontaneous tumor development in mouse models, indicating its role as a tumor suppressor (20, 21). Notably, BRG1 interacts with pRB (22), a key regulator of SAHF formation and senescence (4, 7, 23). BRG1 also plays a role in promoting cell growth arrest and senescence phenotypes (22, 2427). However, whether the interaction between BRG1 and BRCA1 or pRB is regulated during senescence is unknown. In addition, whether BRG1 contributes to SAHF formation induced by oncogenic RAS or BRCA1 knockdown has never been investigated.

Here we show that the interaction between BRCA1 and BRG1 is disrupted in cells undergoing senescence. This correlates with an increased level of chromatin-associated BRG1 in senescent cells. BRG1 is required for SAHF formation and senescence induced by BRCA1 chromatin dissociation or BRCA1 knockdown. Conversely, ectopic BRG1 drives SAHF formation and senescence, which requires its chromatin-remodeling activity to upregulate p16INK4a and p21CIP1. Indeed, the association of BRG1 with the promoters of the SAHF-regulating genes CDKN2A (encoding p16INK4a) and CDKN1A (encoding p21CIP1) was enhanced during senescence induced by oncogene activation or BRCA1 knockdown. Interestingly, BRG1 promotes SAHF and senescence in a DNA damage response-independent manner. Further, we show that the interaction between BRG1 and pRB is enhanced during senescence and that SAHF formation induced by ectopic BRG1 requires its interaction with pRB. Finally, we found that knockdown of pRB, p16INK4a, and p21CIP1, but not p53, impairs SAHF formation induced by BRG1.

MATERIALS AND METHODS

Cell culture.

IMR90 cells were cultured according to the ATCC and as previously described (5, 23). Experiments were performed on IMR90 cells between population doublings 25 and 35.

Plasmids, antibodies, and immunoblotting.

pBABE-puro, pBABE-puro-H-RASG12V, pBABE-puro-BRG1, and pBABE-puro-BRG1 (K798R) were obtained from Addgene. The BRCA1 short hairpin RNAs (shRNAs) were described previously (14). pBABE-puro-BRG1 delRB with a deletion of amino acids (aa) 1357 to 1361 (BRG1 ΔRB) was generated using standard molecular cloning techniques. Lentivirus-encoded shRNAs were purchased from Open Biosystems. The sense strand sequences for the BRG1 shRNAs are 5′-CCCGTGGACTTCAAGAAGATA-3′ and 5′-CGGCAGACACTGTGATCATTT-3′. The sense strand sequences for the p16INK4a shRNAs are 5′-CATGGAGCCTTCGGCTGACT-3′ and 5′-GCGCTGCCCAACGCACCGAAT-3′. The sense strand sequences for the p21CIP1 shRNAs are 5′-CGCTCTACATCTTCTGCCTTA-3′ and 5′-GACAGATTTCTACCACTCCAA-3′. The sense strand sequences for the pRB shRNAs are 5′-CCACATTATTTCTAGTCCAAA-3′ and 5′-CAGAGATCGTGTATTGAGATT-3′. The sense strand sequences for the p53 shRNAs are 5′-GAGGGATGTTTGGGAGATGTA-3′ and 5′-GTCCAGATGAAGCTCCCAGAA-3′. The following antibodies were obtained from the indicated suppliers: rabbit anti-BRCA1 (Upstate), mouse anti-BRCA1 (Calbiochem) (for immunofluorescence staining), rabbit anti-BRG1 (Santa Cruz), mouse anti-p21CIP1 (Santa Cruz), mouse anti-p16INK4a (Santa Cruz), mouse anti-β-actin (Sigma), mouse anti-RAS (BD Bioscience), mouse anti-p53 (Calbiochem), rabbit anti-serine 15-phosphorylated p53 (Cell Signaling), rabbit anti-FLAG (Cell Signaling), mouse anti-pRB (Cell Signaling), rabbit anti-histone H3 (Millipore), and mouse anti-H3K9me2 (Abcam). Rabbit anti-mH2A1.2 was described previously (4, 28). Immunoblotting was performed using standard protocols with the antibodies indicated above.

Retrovirus and lentivirus infections.

Retrovirus production and transduction were performed as described previously (5, 29) using Phoenix cells to package the infection viruses (Gary Nolan, Stanford University). Lentivirus was packaged using a Virapower kit from Invitrogen by following the manufacturer's instructions and as described previously (29, 30). Cells infected with viruses carrying a gene conferring resistance to puromycin or neomycin were selected in 1 μg/ml puromycin or 500 μg/ml neomycin (G418), respectively.

Co-IP analysis.

Cells were washed twice with phosphate-buffered saline (PBS) and lysed using a buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% NP-40, and 150 mM NaCl with the proteinase inhibitors. The supernatant was collected by centrifuging the cell lysates at 12,000 × g for 10 min at 4°C. One milligram of total protein supernatant was subjected to immunoprecipitation (IP) using 2 μg anti-FLAG antibody (Sigma), anti-BRG1 antibody (Santa Cruz), or an isotype-matched IgG control by incubating the antibodies with supernatant for 2 h at 4°C. The IP products were separated on an SDS-PAGE gel and immunoblotted with anti-pRB, anti-BRCA1, or anti-BRG1 antibodies to visualize the immunoprecipitated proteins.

Chromatin isolation and chromatin immunoprecipitation (ChIP) analysis.

Chromatin was prepared according to previously published methods (6, 31). Chromatin-bound proteins in the chromatin fraction were detected by immunoblotting using antibodies against BRCA1, BRG1, and histone H3 as indicated above.

ChIP analysis in control, RAS-infected, or BRCA1 knockdown IMR90 cells was performed at day 4 post-drug selection as previously described (14) using a polyclonal anti-BRG1 antibody (Santa Cruz), an anti-BRCA1 antibody (Millipore), or an isotype-matched IgG control. Immunoprecipitated DNA was analyzed by PCR or SYBR green-based quantitative PCR (Qiagen). Primers against the human CDKN2A gene promoter region are 5′-TGATTTCGATTCTCGGTGGG-3′ and 5′-GGGTGTTTGGTGTCATAGGG-3′. Primers against the human CDKN1A gene promoter region are 5′-TTCAGGAGACAGACAACTCACTC-3′ and 5′-GACACCCCAACAAAGCATCTTG-3′.

Immunofluorescence and SA-β-gal staining.

Immunofluorescence staining was performed as described previously using the antibodies described above (4, 5, 28). SA-β-gal staining was performed as previously described (3).

Comet assay.

A comet assay was performed with a CometAssay (Trevigen) kit following the manufacturer's instruction. DNA damage was measured in artificial Olive Moment units using Cometscore software. A t test for significance was performed using Graph Prism software.

RESULTS

The interaction between BRCA1 and BRG1 is disrupted in cells undergoing senescence, which correlates with an increased level of chromatin-associated BRG1 and an enhanced interaction between BRG1 and pRB.

We have previously shown that oncogenic RAS dissociates BRCA1 from chromatin, which contributes to formation of SAHF and the senescence phenotype (14). Notably, BRCA1 chromatin dissociation is not a consequence of G1 phase accumulation in the fully established senescence cells (14), because BRCA1 becomes dissociated from chromatin prior to G1 phase accumulation in RAS-infected cells compared with controls (see Fig. S1 in the supplemental material). Likewise, knockdown of BRCA1 in primary human cells induces formation of SAHF and senescence (14). Given the role of p16INK4a and p21CIP1 in promoting SAHF formation and senescence (2), we first examined changes in the expression of p16INK4a and p21CIP1 in IMR90 normal human fibroblasts expressing shRNAs corresponding to the human BRCA1 gene (shBRCA1) that efficiently knock down BRCA1 protein expression (Fig. 1A). Compared with controls, the expression of both p16INK4a and p21CIP1 was upregulated in BRCA1 knockdown cells (Fig. 1B). Notably, similar results were obtained by multiple shBRCA1s (Fig. 1A and andB),B), suggesting that the observed phenotype was not due to off-target effects.

Fig 1
The interaction between BRG1 and BRCA1 is disrupted during senescence, which correlates with an increased level of chromatin-associated BRG1. (A) IMR90 normal human fibroblasts were infected with lentivirus encoding the indicated shBRCA1 or the control. ...

It has been demonstrated that BRCA1 interacts with BRG1, an ATP-dependent chromatin-remodeling factor, in transformed cancer cells (18). In addition, the CDKN2A and CDKN1A genes, which respectively encode p16INK4a and p21CIP1 proteins, are known BRG1 target genes (24, 32). Thus, we sought to determine whether the interaction between BRCA1 and BRG1 is regulated during senescence of IMR90 fibroblasts induced by oncogenic RAS. Toward this goal, IMR90 cells expressing oncogenic RAS to induce senescence or control were subjected to co-immunoprecipitation (co-IP) using an anti-BRG1 antibody. An isotype-matched IgG was used a negative control. Consistent with the previous observations in cancer cells (18), we found that BRG1 is associated with BRCA1 in control proliferating cells by co-IP analysis (Fig. 1C). Interestingly, we found that this association was lost in RAS-infected cells (Fig. 1C). This was not due to a decrease of either BRCA1 or BRG1 expression level, as the input levels of BRCA1 and BRG1 are comparable between control and RAS-infected cells at this time point (Fig. 1C). Notably, the co-IP demonstrated a high stoichiometry of BRG1 and BRCA1 complex formation, as pulling down BRG1 brought down a similar fraction of BRCA1 (Fig. 1C). Since BRCA1 became dissociated from chromatin (14) and the interaction between BRG1 and BRCA1 was disrupted during senescence (Fig. 1C), we examined the changes in the levels of BRG1 in chromatin fractions during senescence. In contrast to BRCA1's chromatin dissociation (14), BRG1 levels in the chromatin fraction were increased in RAS-infected cells compared with controls (Fig. 1D). This was not due to an increase in total BRG1 levels in RAS-infected cells compared to controls (Fig. 1D). Consistently, immunofluorescence staining of BRCA1 and BRG1 showed that the colocalization between BRCA1 and BRG1 was decreased in the nuclei of RAS-induced senescent cells compared with controls (Fig. 1E). Based on this result, we conclude that the interaction between BRCA1 and BRG1 is disrupted and chromatin-associated BRG1 levels are increased in cells undergoing senescence.

BRG1 interacts with pRB, a known regulator of SAHF formation and senescence (7, 22, 23). Thus, we sought to determine whether the interaction between BRG1 and pRB is regulated during senescence. To do so, we performed co-IP using an anti-BRG1 antibody in control and RAS-infected cells. An isotype-matched IgG was used as a control. Interestingly, the interaction between BRG1 and pRB was enhanced in cells undergoing senescence (Fig. 1F).

Ectopic BRG1 drives SAHF formation and senescence.

Knockout of BRG1 results in the dissolution of pericentromeric heterochromatin (33), suggesting that BRG1 plays a role in regulating heterochromatin structure. Therefore, we next sought to determine whether ectopic BRG1 is sufficient to drive SAHF formation. Toward this goal, we ectopically expressed wild-type BRG1 in IMR90 normal human fibroblasts (Fig. 2A). Indeed, there was a significant increase in SAHF formation in cells ectopically expressing BRG1 compared with controls (Fig. 2B and andC).C). Consistently, other markers of SAHF such as formation of H3K9Me2 and mH2A1.2 foci were also induced by the ectopically expressed wild-type BRG1 (Fig. 2B and andC).C). Expression of SA-β-gal activity, a hallmark of cellular senescence (3), was also induced by wild-type BRG1 expression (Fig. 2D and andE).E). Thus, we conclude that ectopic expression of wild-type BRG1 drives SAHF formation and senescence.

Fig 2
Ectopic expression of BRG1 drives SAHF formation and senescence, which is dependent upon its chromatin-remodeling activity. (A) IMR90 cells were infected with retrovirus encoding a wild-type BRG (BRG WT), a K798R mutant BRG1 that is defective in chromatin-remodeling ...

We next sought to determine whether BRG1's ability to drive SAHF formation is dependent upon its chromatin-remodeling activity. To do so, we obtained a mutant BRG1 with a single amino acid substitution at amino acid residue 798 (BRG1 K798R), which has previously been demonstrated to be deficient in chromatin-remodeling activity (34). Compared with wild-type controls, the mutant BRG1 that is deficient in chromatin-remodeling activity was also severely impaired in its ability to drive SAHF formation and the expression of SA-β-gal activity (Fig. 2A to toE).E). Since p16INK4a and p21CIP1 are key regulators of SAHF formation and are known BRG1 target genes (7, 9, 24, 32), we next examined the expression of p16INK4a and p21CIP1 in cells expressing the control, wild-type BRG1, and the mutant BRG1. Compared with controls, the expression of both p16INK4a and p21CIP1 was upregulated by wild-type BRG1 but not mutant BRG1 that is deficient in chromatin-remodeling activity (Fig. 2F). Together, we conclude that the chromatin-remodeling activity of BRG1 is required for SAHF formation and senescence, which correlates with the upregulation of p16INK4a and p21CIP1 in these cells.

Since formation of SAHF and senescence induced by BRCA1 knockdown or oncogenic RAS is accompanied by the accumulation of DNA damage (see Fig. S1C in the supplemental material) (14), we next examined the expression of γH2AX, a marker of DNA damage, in cells expressing wild-type BRG1 or mutant BRG1 (BRG1 K798R). Interestingly, the expression of γH2AX was not upregulated by the expression of wild-type or mutant BRG1 compared with controls (Fig. 2F). Likewise, the formation of γH2AX foci was not significantly induced by wild-type BRG1 (see Fig. S2 in the supplemental material). Further, the expression of p53 or serine 15 phosphorylated p53 was not upregulated by the expression of either wild-type or mutant BRG1 (Fig. 2F). We next directly measured the extent of DNA damage in these cells using the comet assay. Compared with controls, the extent of DNA damage was not significantly affected by either wild-type or mutant BRG1 (Fig. 2G and andH).H). Interestingly, both wild-type BRG1 and the BRG1 K798R mutant were able to interact with BRCA1 (Fig. 2I), suggesting that the inability of the BRG1 K798R mutant to induce SAHF formation and senescence was not due to an impaired interaction with BRCA1. In addition, the interaction between BRG1 and pRB was not disrupted by the BRG1 K798R mutant, suggesting that the observed effects were not due to a complete misfolding of the mutant protein (Fig. 2J). Notably, ectopically expressed BRG1 did not dissociate BRCA1 from chromatin (Fig. 2K), suggesting that BRG1 acts downstream of BRCA1 chromatin dissociation. We previously showed that oncogene-induced BRCA1 chromatin dissociation contributes to both SAHF formation and DNA damage accumulation (14). Likewise, BRCA1 knockdown triggers SAHF formation and DNA damage response (14). Here we showed that ectopic BRG1 drove SAHF formation but not the DNA damage response and that BRG1 did not dissociate BRCA1 from chromatin (Fig. 2B, ,C,C, ,FF to toH,H, and andK).K). Together, these data support the premise that BRG1 acts downstream of BRCA1 chromatin dissociation to promote SAHF formation during senescence.

Next, we sought to determine the importance of the interaction between BRG1 and pRB in formation of SAHF induced by BRG1. Toward this goal, we generated a mutant BRG1 that no longer binds to pRB (deletion of aa 1357 to 1361; BRG1 ΔRB) (22, 35, 36) and confirmed the disruption of the interaction between BRG1 ΔRB and pRB by co-IP analysis (Fig. 2L). Notably, the BRG1 ΔRB mutant retained its ability to interact with BRCA1 (Fig. 2L). This result suggests that the effects observed in the BRG1 ΔRB mutant were not due to a complete misfolding of the protein. Indeed, compared with wild-type BRG1, the BRG1 ΔRB mutant was significantly impaired in its ability to drive SAHF formation (Fig. 2M and andN).N). Interestingly, compared with wild-type BRG1, there was no overt difference in induction of p16INK4a and p21CIP1 by the BRG1 ΔRB mutant (Fig. 2O). Given that the BRG1 chromatin-remodeling-activity-deficient mutant (BRG1 K798R) failed to upregulate p16INK4a and p21CIP1 (Fig. 2F), this result suggests that upregulation of p16INK4a and p21CIP1 expression induced by BRG1 functions upstream of pRB, which is independent of the interaction between BRG1 and pRB and dependent on BRG1's chromatin-remodeling activity. Consistently, compared with wild-type BRG1, levels of other markers of senescence such as SA-β-gal activity were decreased in BRG1 ΔRB mutant-expressing cells, albeit to a much lesser extent than that seen SAHF formation in wild-type BRG1-expressing cells (Fig. 2P and andQ).Q). Based on these results, we conclude that the interaction between BRG1 and pRB is enhanced in cells undergoing senescence and that SAHF formation induced by BRG1 requires its interaction with pRB.

Knockdown of BRG1 suppresses SAHF formation and senescence induced by oncogenic RAS.

We next sought to determine whether BRG1 is required for the SAHF formation and senescence that is associated with BRCA1 chromatin dissociation induced by oncogenic RAS. To do so, we obtained two individual shRNAs corresponding to the human BRG1 gene (shBRG1) that can efficiently knock down BRG1 expression (e.g., see Fig. 3D). We infected IMR90 cells with oncogenic RAS to induce BRCA1 chromatin dissociation, SAHF formation, and senescence together with shBRG1 or the control. Compared with RAS-alone cells, knockdown of BRG1 significantly suppressed the RAS-induced SA-β-gal activity and SAHF formation (Fig. 3A to toC).C). However, BRG1 knockdown itself had no effects on expression of markers of senescence such as SA-β-gal activity or SAHF formation (see Fig. S3 in the supplemental material). Interestingly, BRG1 knockdown in mesenchymal stem cells induces the senescence phenotype (27). The discrepancy may be explained by differences in the experimental systems. Notably, the expression of both p16INK4a and p21CIP1 was significantly downregulated by shBRG1 compared with RAS-alone controls (Fig. 3D). This was not due to a lower level of RAS expression in BRG1 knockdown cells, as the RAS expression was comparable (Fig. 3D). Interestingly, BRG1 knockdown had no effects on BRCA1 chromatin dissociation induced by oncogenic RAS (Fig. 3E). This result further supports the premise that BRG1 functions downstream of RAS-induced BRCA1 chromatin dissociation. Consistently, BRG1 knockdown did not overtly affect RAS-induced DNA damage accumulation. For example, RAS-induced upregulation of γH2AX expression or formation of γH2AX foci was not significantly affected by shBRG1 compared with controls (Fig. 3F; see also Fig. S4 in the supplemental material). Based on these results, we conclude that knockdown of BRG1 suppresses RAS-induced SAHF formation and senescence, which is independent of the DNA damage response.

Fig 3
Knockdown of BRG1 suppresses SAHF formation and senescence induced by oncogenic RAS. (A) IMR90 cells were infected with retrovirus encoding oncogenic H-RASG12V together with lentivirus encoding the indicated shBRG1 or the control. Drug-selected cells ...

Our results suggest that BRG1 contributes to SAHF formation and senescence induced by oncogenic RAS by promoting p16INK4a and p21CIP1 expression, which depends upon BRG1's chromatin-remodeling activity (Fig. 2). Thus, we examined the association of BRG1 with the promoters of the CDKN2A (encoding p16INK4a) and CDKN1A (encoding p21CIP1) genes in IMR90 cells infected with oncogenic RAS by ChIP using an anti-BRG1 antibody. An isotype-matched IgG was used a negative control for ChIP analysis. Compared with controls, BRG1's association with the promoters of both CDKN2A and CDKN1A genes was significantly enhanced in RAS-infected cells (Fig. 3G to toJ).J). In contrast, compared with controls, there was no overt change in BRCA1's association with the promoters of either the CDKN2A or CDKN1A gene in RAS-infected cells (see Fig. S5 in the supplemental material).

BRG1 enhances senescence induced by BRCA1 knockdown, and knockdown of BRG1 suppresses SAHF formation and senescence induced by BRCA1 knockdown.

We next sought to determine whether ectopic expression of BRG1 enhances the senescence phenotype induced by BRCA1 knockdown. Toward this goal, we infected IMR90 fibroblasts with a lentivirus encoding shBRCA1 together with a retrovirus encoding the wild type or the chromatin-remodeling-activity-deficient BRG1 mutant (BRG1 K798R). Compared with shBRCA1-alone cells, expression of wild-type BRG1 but not mutant BRG1 induced a more pronounced senescence phenotype, as indicated by a significant increase in SAHF formation and expression of SA-β-gal activity (Fig. 4A to toC).C). Notably, this correlated with an increased induction of p16INK4a and p21CIP1 but not γH2AX expression (Fig. 4D). Based on these results, we conclude that ectopic BRG1 enhances the senescence phenotype induced by BRCA1 knockdown, which correlates with enhanced expression of p16INK4a and p21CIP1.

Fig 4
Knockdown of BRG1 suppresses SAHF formation and senescence induced by BRCA1 knockdown. (A) IMR90 cells were infected with lentivirus encoding shBRCA1 (#3) together with retrovirus encoding wild-type BRG1 (BRG1 WT) or a mutant BRG1 that is defective in ...

Our evidence supports the hypothesis that BRG1 acts downstream of BRCA1 chromatin dissociation to promote SAHF formation during RAS-induced senescence. We next wanted to directly test the effects of BRG1 knockdown on SAHF formation and senescence induced by BRCA1 knockdown. To do this, we infected IMR90 cells with a lentivirus encoding shBRCA1 together with a lentivirus encoding shBRG1 or the control. Compared with shBRCA1-alone cells, BRG1 knockdown significantly suppressed the expression of SA-β-gal activity and SAHF formation induced by BRCA1 knockdown (Fig. 4E to toG).G). This was accompanied by a notable downregulation of p16INK4a and p21CIP1 in shBRCA1/shBRG1-expressing cells compared with shBRCA1-alone cells (Fig. 4H). Taken together, these data indicate that BRG1 contributes to SAHF formation and senescence induced by BRCA knockdown and that this correlates with its effects on the expression of p16INK4a and p21CIP1.

We next sought to determine the effects of BRG1 knockdown on the DNA damage induced by BRCA1 knockdown by examining γH2AX expression and directly measuring the extent of DNA damage in these cells using the comet assay. Notably, BRG1 knockdown did not overtly affect BRCA1 knockdown-induced γH2AX expression (Fig. 4H). Likewise, BRG1 knockdown did not significantly affect the extent of DNA damage induced by BRCA1 knockdown as measured by the comet assay (Fig. 4I and andJ).J). Taking these results together, we conclude that BRG1 knockdown does not suppress the DNA damage response induced by BRCA1 knockdown.

Notably, chromatin-associated BRG1 levels were increased in BRCA1 knockdown cells (Fig. 4K), further supporting the notion that BRCA1 functions upstream of BRG1. This was not due to an increase in the total BRG1 protein level in BRCA1 knockdown cells (Fig. 4K). Since BRCA1 knockdown induces the expression of p21CIP1 and p16INK4a, we next examined the association of BRG1 with the promoters of CDKN2A and CDKN1A genes in BRCA1 knockdown cells. Similar to what we observed in cells expressing oncogenic RAS, which induces dissociation of BRCA1 from chromatin, we observed that BRG1's association with the promoters of CDKN2A and CDKN1A genes was significantly enhanced in BRCA1 knockdown cells compared with controls (Fig. 4L to toO).O). Together, these results further support the premise that BRG1 contributes to SAHF formation and senescence induced by BRCA1 chromatin dissociation or BRCA1 knockdown through promoting p16INK4a and p21CIP1 expression.

Knockdown of pRB, p16INK4a, and p21CIP1, but not p53, inhibits SAHF formation induced by ectopic BRG1 expression.

We showed that p16INK4a and p21CIP1 expression is induced by BRG1 (Fig. 2F). In addition, a mutant BRG1 (BRG1 K798R) that is deficient in chromatin-remodeling activity is also impaired in SAHF formation (Fig. 2B and andC).C). Further, we showed that the interaction between BRG1 and pRB is required for SAHF formation induced by BRG1 (Fig. 2M and andN).N). However, we failed to observe an increase in p53 expression in BRG1-expressing cells (Fig. 2F). We next sought to directly determine the effects of knockdown of pRB, p16INK4a, p21CIP1, or p53 on SAHF formation induced by ectopic BRG1. To do so, we developed two individual shRNAs corresponding to the human CDKN2A, CDKN1A, pRB, or p53 gene. The knockdown efficacy of these shRNAs was confirmed by immunoblotting using antibodies specific to pRB, p16INK4a, p21CIP1, and p53 (Fig. 5A to toD).D). Interestingly, knockdown of pRB, p16INK4a, or p21CIP1 inhibited SAHF formation induced by ectopic BRG1 expression (Fig. 5E and andF).F). In contrast, knockdown of p53 had no significant effects on SAHF formation induced by ectopic BRG1 expression (Fig. 5E and andF).F). Based on these results, we conclude that knockdown of pRB, p16INK4a, or p21CIP1, but not p53, inhibits SAHF formation induced by ectopic BRG1 expression.

Fig 5
Knockdown of pRB, p21CIP1, or p16INK4a, but not p53, inhibits SAHF formation induced by ectopic BRG1 expression. (A) IMR90 cells were infected with retrovirus encoding BRG1 together with lentivirus encoding the indicated shpRB or the control. Drug-selected ...

BRCA1 knockdown induces both SAHF formation and the DNA damage response (14), and suppression of the DNA damage response impairs SAHF formation (37). We next sought to determine the effects of knockdown of pRB, p16INK4a, p21CIP1, or p53 on SAHF formation induced by BRCA1 knockdown. Notably, knockdown of pRB, p16INK4a, p21CIP1, or p53 inhibited SAHF formation (Fig. 5G to toI).I). Since knockdown of p53 suppresses SAHF formation induced by knockdown of BRCA1 but not ectopic BRG1 (Fig. 5), these data further support the premise that BRG1 functions downstream of BRCA1 knockdown to drive SAHF formation.

DISCUSSION

The role of BRG1 in SAHF formation induced by BRCA1 knockdown or BRCA1 chromatin dissociation.

Here we demonstrated that the interaction between BRG1 and BRCA1 was regulated during RAS-induced senescence (Fig. 1C and andD).D). In addition, chromatin-associated BRG1 levels were increased in cells undergoing senescence induced by oncogenic RAS or BRCA1 knockdown (Fig. 1D and and4K).4K). Consistently, ectopic BRG1 expression drove formation of SAHF and senescence (Fig. 2). Conversely, knockdown of BRG1 inhibited the formation of SAHF induced by oncogenic RAS or BRCA1 knockdown (Fig. 3 and and4).4). Further, we demonstrated that ectopic BRG1 enhanced SAHF formation induced by BRCA1 knockdown, which resulted in a more pronounced senescence phenotype (Fig. 4). However, BRG1 did not dissociate BRCA1 from chromatin (Fig. 2). Interestingly, BRG1's role in promoting SAHF formation is independent of the DNA damage response (Fig. 2F to toH,H, ,3F,3F, and and4D4D to toF;F; see also Fig. S2 and S4 in the supplemental material), while BRCA1 chromatin dissociation or knockdown induces both SAHF formation and DNA damage response (14). Consistently, knockdown of p53 inhibited SAHF formation induced by BRCA1 knockdown but not by ectopic BRG1 (Fig. 5). Together, these results support the premise that BRG1 acts downstream of BRCA1 to promote SAHF formation during senescence (Fig. 6). In addition, they suggest that oncogene-induced BRCA1 chromatin dissociation contributes to the accumulation of DNA damage observed in senescent cells through BRG1-independent mechanisms (Fig. 6). In agreement with this conclusion, we have previously shown that BRCA1-mediated DNA repair is impaired during RAS-induced senescence (14).

Fig 6
A model for the role of BRG1 in regulating SAHF formation and senescence downstream of BRCA1 loss or oncogenic RAS, which triggers BRCA1 chromatin dissociation.

We show that the interaction between BRCA1 and BRG1 is disrupted during senescence, which correlates with the dissociation of BRCA1 from chromatin and an enhanced association of BRG1 with chromatin (Fig. 1). Consistently, the colocalization between BRG1 and BRCA1 was decreased in the nuclei of senescent cells (Fig. 1E). BRCA1 knockdown, which mimics BRCA1 chromatin dissociation and the disruption of the interaction between BRCA1 and BRG1, increased the levels of chromatin-associated BRG1 (Fig. 4K). In contrast, BRG1 overexpression did not affect BRCA1 chromatin association (Fig. 2K). Together, these results suggest that BRCA1 chromatin dissociation and disruption of the BRCA1 and BRG1 interaction, which act upstream of BRG1 during senescence, are associated (Fig. 6). Our data also show that the interaction between BRCA1 and BRG1 was present in the soluble fractions (Fig. 1C). These data are consistent with a model whereby soluble BRCA1 sequesters BRG1 away from chromatin and disruption of the interaction between BRCA1 and BRG1 causes an enhanced association of BRG1 with chromatin during senescence.

Molecular mechanisms by which BRG1 promote SAHF formation.

BRG1 is known to regulate heterochromatin structure. Indeed, BRG1 interacts with pRB, which is known to play a key role in regulating SAHF formation (7, 22). Here we show that the interaction between BRG1 and pRB was enhanced during senescence and that formation of SAHF induced by ectopic BRG1 required its interaction with pRB (Fig. 1F and and2L2L to toN).N). Consistently, knockdown of pRB suppressed SAHF formation induced by ectopic BRG1 expression (Fig. 5). However, the BRG1 ΔRB mutant did not affect upregulation of p16INK4a and p21CIP1 (Fig. 2O). In contrast, a chromatin-remodeling-activity-deficient BRG1 mutant (BRG1 K798R) that failed to induce p16INK4a and p21CIP1 expression was unable to drive SAHF formation (Fig. 2B and andC).C). These data suggest that the BRG1 and pRB complex acts downstream of p16INK4a and p21CIP1, whose expression requires BRG1's chromatin-remodeling activity (Fig. 6). Indeed, both p16INK4a and p21CIP1 are known regulators of pRB (38). Together, these data support the notion that the complex of BRG1 and pRB functions downstream of p16INK4a and p21CIP1 to promote SAHF formation (Fig. 6).

It has been demonstrated that p16INK4a and pRB are necessary for SAHF formation during senescence induced by oncogenic RAS (7). However, p53 and p21CIP1 are not necessary for SAHF formation in this context (7). In contrast, both p53 and p21CIP1 are required for SAHF formation induced by knockdown of adenovirus E1A-associated p400 protein (9). Here we show that ectopic BRG1 induced the expression of p21CIP1 and p16INK4a but had no effects on p53 expression (Fig. 2F). This is consistent with a previous report showing that BRG1-induced upregulation of p21CIP1 is independent of p53 (24). Notably, the association of BRG1 with the promoters of the CDKN2A and CDKN1A genes, which encode pRB regulators p16INK4a and p21CIP1, was enhanced during senescence induced by oncogenic RAS or BRCA1 knockdown (Fig. 3G to toJJ and and4L4L to toO).O). In addition, we demonstrated that knockdown of BRG1 suppressed SAHF formation, which correlates with suppression of expression of both p16INK4a and p21CIP1 induced by BRCA1 knockdown or oncogenic RAS (Fig. 3 and and4).4). Further, knockdown of p16INK4a, p21CIP1, or pRB, but not p53, inhibited SAHF formation induced by ectopic BRG1 expression (Fig. 5). Together, these data support a model whereby BRG1 promotes SAHF formation via its interaction with pRB, which is regulated by the upregulation of p16INK4a and p21CIP1 through its chromatin-remodeling activity (Fig. 6).

BRG1, DNA damage, and SAHF.

Here we showed that ectopic BRG1 drives SAHF formation and that this is independent of the DNA damage response by multiple markers (Fig. 2F to toH;H; see also Fig. S2 in the supplemental material). These DNA damage markers include expression of γH2AX, p53, and serine 15-phosphorylated p53 (Fig. 2F). Consistently, BRG1 did not affect the extent of DNA damage in these cells, as measured by the comet assay (Fig. 2G and andH).H). Conversely, knockdown of BRG1 inhibited SAHF formation induced by oncogenic RAS or BRCA1 knockdown with no overt effects on the DNA damage response (Fig. 3F and and4H4H to toJ;J; see also Fig. S4 in the supplemental material). Interestingly, previous evidence suggests that formation of SAHF limits the extent of DNA damage induced by oncogenic RAS (37). Likewise, it has been demonstrated that ectopic p16INK4a drives SAHF formation. However, p16INK4a-induced senescence displays little DNA damage response (39). Together, our data suggest that DNA damage is not necessary for SAHF formation.

In summary, we showed that the interaction between BRG1 and BRCA1 is regulated during senescence. BRG1 is required for SAHF formation and senescence induced by BRCA1 knockdown or oncogenic RAS, which triggers BRCA1 chromatin dissociation. This correlates with an increase in chromatin-associated BRG1 levels. The association of BRG1 with the promoters of CDKN2A and CDKN1A genes is enhanced during senescence and correlates with the upregulation of p16INK4a and p21CIP1 in a chromatin-remodeling-activity-dependent manner. In addition, the interaction between BRG1 and pRB is enhanced during senescence and SAHF formation induced by ectopic BRG1 requires its interaction with pRB. Further, p16INK4a and p21CIP1 act upstream of pRB to mediate SAHF formation induced by ectopic BRG1 (Fig. 6). Thus, our current report sheds new light on how BRG1 regulates SAHF formation and senescence downstream of BRCA1 loss or oncogenic RAS that triggers BRCA1 chromatin dissociation.

Supplementary Material

Supplemental material:

ACKNOWLEDGMENTS

We thank Katherine Aird, Benjamin Bitler, and Michael Amatangelo for critical reading of the manuscript and other members of the laboratory for discussions and suggestions.

This work was supported by a NIH/NCI grant (R01CA160331 to R.Z.) and, in part, by a DOD award (OC093420 to R.Z.). Support of core facilities used in this study was provided by Cancer Center Support Grant CA010815 to The Wistar Institute.

Footnotes

Published ahead of print 25 February 2013

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01744-12.

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