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A DNA Damage Response Screen Identifies RHINO, a 9-1-1 and TopBP1 Interacting Protein Required for ATR Signaling

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Science  10 Jun 2011:
Vol. 332, Issue 6035, pp. 1313-1317
DOI: 10.1126/science.1203430

Abstract

The DNA damage response (DDR) is brought about by a protein kinase cascade that orchestrates DNA repair through transcriptional and posttranslational mechanisms. Cell cycle arrest is a hallmark of the DDR. We screened for cells that lacked damage-induced cell cycle arrest and uncovered a critical role for Fanconi anemia and homologous recombination proteins in ATR (ataxia telangiectasia and Rad3-related) signaling. Three DDR candidates, the RNA processing protein INTS7, the circadian transcription factor CLOCK, and a previously uncharacterized protein RHINO, were recruited to sites of DNA damage. RHINO independently bound the Rad9-Rad1-Hus1 complex (9-1-1) and the ATR activator TopBP1. RHINO was recruited to sites of DNA damage by the 9-1-1 complex to promote Chk1 activation. We suggest that RHINO functions together with the 9-1-1 complex and TopBP1 to fully activate ATR.

The importance of the DNA damage response (DDR) is underscored by the prevalence of mutations in this pathway found in cancers and developmental syndromes (1). Historically, most DDR genes were identified genetically in yeast as mutants defective in the transcriptional or cell cycle arrest responses to DNA damage. However, yeast lacks many mammalian DDR components. To identify mammalian DDR genes, we developed a high throughput microscopy-based assay using human osteosarcoma (U2OS) cells. Specific proteins were depleted from cells with small interfering RNA (siRNAs), and cells were monitored to measure inappropriate entry into mitosis 18 hours after exposure to 10 Gy of ionizing radiation (IR) (Fig. 1A). Most cells entering mitosis during this prolonged assay incurred damage during S phase [supporting online material (SOM) text and fig. S1]. Cell responses were stratified by the fold change in mitotic index (MI) compared with that of negative control cells: strong (>eight-fold), medium (four- to eight-fold), and weak (two- to four-fold) (Fig. 1B and table S1). Several known genes involved in checkpoint arrest, like Chk1, were not detected due to toxicity. (fig. S2). We rescreened the toxic subset of genes at a lower siRNA concentration and detected an additional 98 genes scoring that included Chk1, PALB2, Wee1, and FANCM (fig. S2D and tables S1 and S2).

Fig. 1

A screen for regulators of DDR signaling. (A) Schematic of the screen. (B) Primary screen statistics. The number of known DDR proteins and potential ataxia telangiectasia mutated (ATM)/ATR substrates are indicated under the header pSQTQ (phospho-SQTQ) for the conserved phosphorylation motif. (C) Secondary screen statistics for 720 candidate genes with and without DNA damage. For each gene, the fraction of siRNAs scoring and the total number of genes scoring is listed. (D) DDR networks identified in primary screen using Ingenuity pathway analysis. (E) ATR pathway signaling integrity after ATR and BRCA2 depletion. Cells collected at the indicated times after IR (10 Gy) were examined for phosphorylation of Chk1 on S317. Smc1 was used as loading control. (F) ATR pathway signaling integrity after ATR, BRCA2 (B2), and BRCA1 (B1) depletion. Cells were collected 1 and 16 hours after IR (10 Gy). Cyclin B1 and PCNA were used as loading controls for the left and right panels, respectively. (G) Depletion of 53BP1 with shRNAs restores cell cycle arrest in BRCA1, FANCM-, FANCL-, and FANCJ-depleted cells but not in ATR- or BRCA2-depleted cells. MI calculated 18 hours after 10 Gy. (H) Chemical inhibition of DNA-PKcs restores cell cycle arrest in BRCA1-, FANCM-, FANCL-, and FANCJ-depleted cells but not in ATR- or BRCA2-depleted cells. The DNA-PK inhibitor was added 2 hours after IR (10 Gy) at a final concentration of 1 μM. MI was calculated as above.

All strong and medium candidates and a subset of prioritized weak candidates, 720 in total, selected for their degree of bypass or phosphorylation in response to DNA damage (2, 3) were chosen for secondary screening. Pools of siRNAs were separated into four individual siRNAs and retested. Effects of >75% of the pools recapitulated with at least one siRNA (Fig. 1C and table S3); 12% of these were eliminated because they increased MI in the absence of damage (Fig. 1C, fig. S3B, and table S3).

DDR mutations often cause sensitivity to DNA damage; therefore, sensitivity to mitomycin C (MMC) was assessed after depletion by siRNAs (fig. S3C). Of these, 53% that were detected with at least two siRNAs in the checkpoint assay also were detected with two or more siRNAs in the MMC-sensitivity assay (97 genes). These genes were further characterized with Dharmacon’s On target plus siRNAs and were tested for checkpoint function, MMC-sensitivity, and homologous recombination (HR) efficiency (4) (fig. S4A and table S4; see SOM text for details). This high confidence list was enriched in genes annotated for the biological categories of DNA replication, recombination, and repair, as well as nucleic acid metabolism and cancer relevance (fig. S4B).

Bioinformatic analysis revealed a strong enrichment for the ATR [ataxia telangiectasia and Rad3-related], Fanconi anemia (FA), and HR pathways (Fig. 1D and fig. S4C). This seemed counterintuitive because double-strand breaks (DSBs) remain unrepaired in the absence of HR, and DDR signaling should persist until the repair process is complete. However, examination of Chk1 phosphorylation kinetics indicated that, in the absence of the breast cancer susceptibly proteins BRCA1 and BRCA2, ATR signaling was not sustained (Fig. 1, E and F). Although BRCA1 regulates Chk1 phosphorylation (5), we observed no defect in Chk1 activation 1 hour after exposure of cells to IR, but we observed a defect in maintenance of the activation (Fig. 1F). BRCA1 acts upstream of BRCA2, which has a direct role in promoting Rad51 filament formation (1). Although HR is the major DSB repair pathway in S and G2 phases, other competing repair pathways such as nonhomologous end-joining (NHEJ) also contribute. To examine whether the removal of signaling DNA ends by NHEJ repair blocks HR in BRCA-depleted cells, we inhibited NHEJ by depleting 53BP1 with short hairpin RNA (shRNA) or with an inhibitor of DNA-dependent protein kinase catalytic subunit (NU7441). We chose 53BP1 because of its role in promoting ligation of DNA ends that are not in close proximity (1), a characteristic of ends that remain unrepaired for a long period of time. MI was decreased in BRCA1-depleted cells under both conditions (Fig. 1, G and H), suggesting that NHEJ processes the breaks and that this allows restart of the cell cycle. We did not observe a rescue of the BRCA2 defect, suggesting that either the processed DSB intermediates that accumulate in these cell lines are not repaired by NHEJ or that BRCA2 has an additional role in the DDR to promote arrest. Inhibiting NHEJ also suppressed the signaling defects in cells depleted of FANCM, FANCL, and FANCJ (Fig. 1, G and H). These results are consistent with the finding that loss of 53BP1 partially rescues the HR defect and damage sensitivity of BRCA1 mutant cells, but not BRCA2 mutants, and of sensitivity to damage in FA-deficient cells (69).

Twenty-four genes were selected based on the secondary assays to assess their localization to sites of damage, a hallmark of DDR proteins (10). U2OS cells stably expressing green fluorescent protein (GFP) fusions were created and subjected to laser-induced DNA damage, marked by γ-H2AX costaining. Three proteins (INTS7, CLOCK, and MGC13204, an orphan protein) showed colocalization with γ-H2AX in the form of laser stripes (Fig. 2A) but did so independently of H2AX (fig. S5A).

Fig. 2

Characterization of several DDR candidates. (A) Analysis of DNA damage localization. U2OS cells expressing GFP fusions to INTS7, Clock, and RHINO were striped with a UV laser and costained with γ-H2AX to identify damaged cells. (B) Cell cycle arrest of RHINO with individual siRNAs at 30 nM, 18 hours after 10 Gy, is shown. SiRNAs 9 and 11 result in statistically significant increases in MI with P values of 8 x 10−5 and 0.05, respectively, determined by a two-tailed t test. (C) HR results using individual RHINO siRNAs normalized to the negative control (FF). (D) The multicolor competition assay (MCA) was used to assess DNA damage sensitivity of the indicated depleted genes in response to the indicated damaging agents. (E) Mass spectrometry analysis of Flag-HA RHINO after IR. Genes are ranked according to the ratio of the number of peptides versus the molecular weight of total protein. (F) Interaction of 9-1-1, TopBP1, Rad18, and Ubc13 with RHINO. Human embryonic kidney (293T) cells expressing GFP alone or Flag-HA-RHINO were treated with 10 Gy and harvested 4 hours later, and analyses for association by immunoblotting were carried out. The asterisk in the Rad9 panel represents the immunoglobulin G (IgG) heavy chain migrating above endogenous Rad9.

CLOCK (checkpoint bypass with 7 of 8 siRNAs; MMC sensitivity with 6 of 8 siRNAs) is a basic helix-loop-helix transcription factor that participates in circadian rhythm regulation. Numerous examples of cross-talk between DNA damage, cell cycle regulation, and circadian rhythms have been described (11). Loss of CLOCK or its partner, BMAL1, in mice results in chronic enhanced sensitivity to DNA cross-linking agents, previously presumed to be due to misregulation of transcriptional targets (12). However, localization of CLOCK to sites of DNA damage suggests that it has a more direct role in the DDR.

INTS7 is a member of the Integrator complex that interacts with RNA polymerase II to assist in small nuclear RNA processing (13). Depletion of INTS7 resulted in bypass of cell cycle arrest (6 of 7 siRNAs) and sensitivity to MMC (3 of 7 siRNAs). One component of the Integrator complex, INTS3, associates with two related DNA single-strand binding proteins, SSB1 and SSB2, to form a complex recruited to damage sites and is required for checkpoint function (14, 15). INTS7 also interacted with SSB1 (fig. S5B).

Depletion of MGC13204, referred to hereafter as RHINO, led to checkpoint bypass and sensitivity to MMC, IR, and camptothecin (Fig. 2D). Moreover, RHINO localized to foci in response to IR (fig. S6A), to MMC, and to ultraviolet (UV) laser stripes (Fig. 2A). The extent of RHINO depletion strongly correlated with both checkpoint and HR defects (Fig. 2, B and C, and figs. S6B and S8), which were suppressed by expression of an siRNA (#9)–resistant RHINO cDNA and partially suppressed by the wild-type (WT) cDNA (fig. S7).

Mass spectrometry analysis of proteins immunoprecipitated with Flag- hemagglutinin (HA)–tagged RHINO identified peptides for each subunit of the 9-1-1 complex (Rad9, Rad1, and Hus1), which acts as a damage-specific clamp that binds the ATR activator TopBP1, the E3 ubiquitin ligase Rad18, and the E2 conjugating enzyme Ubc13 (Fig. 2E). Western blotting independently verified these interactions (Fig. 2F). To determine whether these components controlled RHINO’s damage recruitment, we depleted TopBP1, Rad18, or Rad17 (which loads 9-1-1) and quantified RHINO localization to UV-induced DSBs. Rad17 depletion caused a 40% reduction in RHINO recruitment (fig. S9A). UV laser striping analysis of WT and mutant mouse embryo fibroblasts (MEFs) expressing RHINO directly demonstrated the requirement of the 9-1-1 complex in RHINO recruitment (Fig. 3A and fig. S9B). For this reason, we named the orphan protein MGC13204 as RHINO (Rad9, Rad1, Hus1 interacting nuclear orphan).

Fig. 3

Requirement of RHINO for DDR signaling. (A) Hus1-dependent RHINO recruitment to sites of DSBs. MEFs (WT, RAD18−/−, HUS1−/−, and HUS1−/− reconstituted with HUS1) expressing GFP-RHINO had DSBs introduced by UV laser treatment, and RHINO recruitment was assessed. (B) RHINO is required for Chk1 phosphorylation. Cells after ATR and RHINO depletion were treated with the indicated IR doses, collected at the indicated times, and immunoblotted. (C) Restoration of RHINO expression rescues DDR signaling. Three days after siRNA transfection, cells were infected at a multiplicity of infection of 2 with a retrovirus expressing the Flag-HA negative control or Flag-HA-RHINO. Eighteen hours later, cells were treated with 5 Gy and were harvested 4 hours later. Ran was used as a loading control.

Rad18 and Ubc13 function in postreplication repair, which controls ubiquitination of proliferating cell nuclear antigen (PCNA) (16). Depletion of Rad18, but not RHINO, decreased UV-induced monoubiquitination of PCNA (fig. S10). Because TopBP1 and 9-1-1 have critical roles in ATR activation and phosphorylation of Chk1 (17), we investigated whether RHINO influenced IR-induced phosphorylation of Chk1. Indeed, depletion of RHINO led to a reduced phosphorylation of Chk1 (Fig. 3B) that was rescued by reexpression of RHINO (Fig. 3C). Together, these data suggest a role for RHINO in ATR-mediated activation of Chk1 driven by TopBP1 and 9-1-1.

RHINO orthologs exist in vertebrates, with high conservation in the N- and C-terminal regions (fig. S11A). The N terminus has similarity to the APSES (ASM-1, Phd1, StuA, EFG1, and Sok2) domain (18) (fig. S11B), a DNA binding domain homologous to the KilA-N domain found in eukaryotic viruses. To identify TopBP1 and 9-1-1 interaction regions, we mutated a conserved 7 amino acid region in the APSES domain [referred to as SWV for the first 3 amino acids of the motif (fig. S11)] and made C- and N-terminal truncations (Fig. 4A). The SWV mutation specifically blocked the interaction with the 9-1-1 complex but not TopBP1 (Fig. 4, B and D). Although the N-terminal, but not the C-terminal fragment bound 9-1-1, neither bound TopBP1 (Fig. 4, B and D). The SVW mutant was completely defective in localization to sites of damage, and the N-terminal fragment was partially impaired (Fig. 4, C and D, and fig. S12A), suggesting that the C-terminal region functions in localization to DNA damage. The interaction between RHINO and 9-1-1 is critical, because the SWV mutant RHINO failed to complement the checkpoint or HR defects (Fig. 4D and fig. S12). The RHINO N-terminal fragment partially rescued both phenotypes, consistent with its partial localization phenotype.

Fig. 4

RHINO interaction with 9-1-1 is critical for the DDR. (A) Schematic representation of the different mutant and truncated versions of RHINO. (B) RHINO binds 9-1-1 and TopBP1 independently. Extracts from 293T cells expressing either a Flag-HA negative control, Flag-HA-RHINO, or the indicated mutants were immunoprecipitated and immunoblotted with the indicated antibodies. The asterisk represents the IgG heavy chain. (C) RHINO localization requires 9-1-1. HeLa cells expressing WT GFP-RHINO or the indicated mutants were treated with the UV laser to introduce DSBs and assessed for RHINO localization. (D) Synopsis of the behavior of different mutants of RHINO.

Depletion of Rad17, RHINO, or TopBP1 results in a partial defect in HR, although not as strong as that caused by ATR depletion (fig. S4A and S13). Depletion of both Rad17 and RHINO leads to a similar HR defect to that caused by individual depletion, suggesting that RHINO may mediate the 9-1-1’s role in HR (fig. S13). Together, these data suggest that the 9-1-1/RHINO/TopBP1 axis promotes DDR activation in response to lesions occurring during S phase (fig. S14).

We have uncovered a large number of genes and pathways required to maintain prolonged cell cycle arrest in response to DNA damage occurring in S phase. We uncovered an unexpected role for the HR and FA pathways in promoting cell cycle arrest. Although this may in part reflect differential funneling of repair intermediates into NHEJ, the inability of NHEJ inhibition to rescue the signaling defect from BRCA2 depletion suggests the possibility that cells may retain the capacity to signal if they process lesions through the HR pathway. There are two identified single-stranded DNA (ssDNA) binding complexes required for DDR signaling replication protein A (RPA) and the SSB1 and SSB2 complexes. The HR pathway, which replaces RPA with another ssDNA binding protein, Rad51, may represent a third (fig. S15 and SOM Text). We were unable to further test this possibility because of extreme toxicity of Rad51 depletion. However, it makes sense that cells would evolve the capacity to sense ongoing repair, because it is the completion of repair that is the relevant biological event with respect to turning off the DDR.

We also defined a new component of the Rad17/9-1-1/TopBP1 pathway, RHINO. TopBP1 recruitment to sites of damage through the Rad17/9-1-1 complex is important for activation of ATR (19). This recruitment has been suggested to occur through association of the BRCT repeats of TopBP1 with constitutively phosphorylated Rad9 (20). However, experiments in Xenopus have indicated that there is a 9-1-1–dependent but Rad9-phosphorylation–independent mechanism that also operates to recruit TopBP1 (21). The association of RHINO with both 9-1-1 and TopBP1 through independent domains on RHINO suggests that it may bridge these to help recruit TopBP1 to the 9-1-1 complex. Whether RHINO is involved in this second mechanism, and why two different mechanisms operate, remains to be determined, but it is possible that they are each used in response to different classes of lesions or to bolster long-term signaling. Alternatively, RHINO could allosterically regulate TopBP1 to promote its association with, or activation of, ATR/ATR interacting protein (ATRIP).

Supporting Online Material

www.sciencemag.org/cgi/content/full/332/6035/1313/DC1

Materials and Methods

SOM Text

Figs. S1 to S15

Tables S1 to S4

References

References and Notes

  1. Acknowledgments: We thank the Institute of Chemistry and Cell Biology-Longwood for screening assistance. We also thank K. Hofmann, A. Ciccia, A. Bredemeyer, B. Adamson, A. Burrows, A. Smogorzewska, and A. Brass for helpful discussions. This work was supported by NIH grants to S.J.E. and J.W.H. C.C.R. is the recipient of a long-term European Molecular Biology Organization fellowship. S.J.E. is a Howard Hughes Medical Institute investigator. W.H. is a paid consultant for Millennium Pharmaceuticals and its program in oncology.
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