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Negative regulator of E2F transcription factors links cell cycle checkpoint and DNA damage repair Lili Wanga, Hanchen Chena, Chongyang Wanga, Zhenjie Hua, and Shunping Yana,1 a

College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

DNA damage poses a serious threat to genome integrity and greatly affects growth and development. To maintain genome stability, all organisms have evolved elaborate DNA damage response mechanisms including activation of cell cycle checkpoints and DNA repair. Here, we show that the DNA repair protein SNI1, a subunit of the evolutionally conserved SMC5/6 complex, directly links these two processes in Arabidopsis. SNI1 binds to the activation domains of E2F transcription factors, the key regulators of cell cycle progression, and represses their transcriptional activities. In turn, E2Fs activate the expression of SNI1, suggesting that E2Fs and SNI1 form a negative feedback loop. Genetically, overexpression of SNI1 suppresses the phenotypes of E2F-overexpressing plants, and loss of E2F function fully suppresses the sni1 mutant, indicating that SNI1 is necessary and sufficient to inhibit E2Fs. Altogether, our study revealed that SNI1 is a negative regulator of E2Fs and plays dual roles in DNA damage responses by linking cell cycle checkpoint and DNA repair. E2F

| SNI1 | NSE6 | cell cycle checkpoint | DNA damage repair

T

he ultimate goal of an organism is to pass its genetic information (DNA) to its offspring with high fidelity. However, DNA integrity is constantly assaulted by various exogenous factors (such as UV light, ionizing radiation, ozone, and numerous genotoxic chemicals) and endogenous cues (such as replication errors and metabolic byproducts) (1, 2). It is estimated that every human cell could suffer tens of thousands of DNA lesions per day (3). To cope with this threat, all organisms have evolved elaborate DNA damage response (DDR) mechanisms that include transcriptional reprogramming, checkpoint activation, DNA repair, and apoptosis. Defects in DDR strongly affect development and cause many diseases including cancers (2–5). Checkpoint activation results in a transient arrest of cell cycle progression, allowing cells to have enough time to repair damage before proceeding into the next cell cycle phase. Therefore, checkpoint activation is essential to ensure genome stability (2, 6, 7). A typical eukaryotic cell cycle consists of four distinct phases: G1 (gap 1), S (synthesis), G2 (gap 2), and M (mitosis) phases. During the S phase, the genome is duplicated through DNA replication, whereas during the M phase, the replicated sister chromatids are equally divided between the daughter cells. To ensure faithful DNA replication, cells evolved checkpoints working at distinct stages of the cell cycle including the G1/S checkpoint, the intra-S checkpoint and the G2/M checkpoint. The G1/S transition is a rate-limiting step of cell cycle progression. Therefore, the G1/S checkpoint is also known as the restriction checkpoint (2). The Retinoblastoma (RB)-E2F module is the well-known regulator of the G1/S checkpoint (8–12). E2F represents a family of transcription factors that function together with their dimerization partners DP proteins. The E2F–DP complexes regulate the expression of genes involved in DNA replication to control the onset of S phase. The activities of E2Fs are negatively regulated by the tumor suppressor protein RB, which binds to the transcriptional activation domain of E2Fs. In late G1 phase, RB is phosphorylated by cyclin-dependent kinases (CDKs) and www.pnas.org/cgi/doi/10.1073/pnas.1720094115

subsequently dissociates from E2F transcription factors, which activate the S phase gene expression. The functions of RB-E2F modules are conserved in plants (13–15). The model plant Arabidopsis encodes one RB homolog called Retinoblastoma-related 1 (RBR1) and six E2F homologs (E2Fa-f). E2Fa, E2Fb, and E2Fc are canonical E2Fs, which are capable of forming complexes with RBR1 (16). It is generally accepted that E2Fa and E2Fb are transcriptional activators, while E2Fc is a repressor (17–21). The SMC5/6 complex belongs to the structural maintenance of chromosome (SMC) protein family and is evolutionally conserved in all eukaryotes. Accumulating evidence suggests that the SMC5/6 complex plays multiple essential roles in DNA damage repair (22–25). It is recruited to DNA damage sites and promotes DNA repair through homologous recombination (HR) (26). It also contributes to the rescue of stalled replication forks by stabilizing these structures in recombination-competent configurations, and facilitating the resolution or preventing the formation of certain recombination intermediates. Moreover, the SMC5/6 complex is essential for ribosomal DNA (rDNA) stability (27). In yeast, the SMC5/6 complex is composed of SMC5, SMC6, and six non-SMC elements (NSE1–NSE6) (28). Recently, SNI1 (Suppressor of NPR1-1, Inducible) was identified as the functional counterpart of NSE6 in Arabidopsis despite low sequence similarities to NSE6 (29). In this study, we found that SNI1 is a negative regulator of E2F transcription factors. SNI1 directly binds to the activation domain of E2Fs to repress their transcriptional activities. Genetic evidence strongly indicated that SNI1 is necessary and sufficient to inhibit the functions of E2Fs, reminiscent of RB. We Significance DNA is frequently damaged by both endogenous and exogenous factors. In response to DNA damage, cells activate checkpoints to arrest cell cycle progression, allowing sufficient time for DNA repair. Defects in DNA repair cause many diseases including cancers. The E2F transcription factors are key players in cell cycle progression and are negatively regulated by the tumor suppressor protein Retinoblastoma. In this study, we demonstrated that the DNA repair protein SNI1, a subunit of SMC5/6 complex, is a negative regulator of E2Fs. In addition, this study also suggests that checkpoint and DNA repair are directly linked by SNI1, providing insights into DNA damage responses. Author contributions: L.W. and S.Y. designed research; L.W., H.C., C.W., and Z.H. performed research; L.W., H.C., C.W., and S.Y. analyzed data; and L.W. and S.Y. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. W.G. is a guest editor invited by the Editorial Board. Published under the PNAS license. 1

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1720094115/-/DCSupplemental. Published online April 2, 2018.

PNAS | vol. 115 | no. 16 | E3837–E3845

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Edited by Wilhelm Gruissem, ETH Zurich, Zurich, Switzerland, and accepted by Editorial Board Member Julian I. Schroeder March 5, 2018 (received for review November 17, 2017)

propose that SNI1 plays dual roles in DDR by linking checkpoint activation and DNA repair. Results SNI1 Is Required for Root Development. SNI1 is a highly conserved plant protein initially identified for its role in maintaining DNA integrity during plant immune responses (29–31). In addition to the immune phenotypes, the sni1 mutant displays pleiotropic developmental defects, indicating that SNI1 plays very important roles in plants. As shown in Fig. 1 A and B, the primary root length was dramatically reduced in the sni1 mutant compared with wild type (WT). To further dissect the developmental defects of the root meristem, we examined its cellular structure using confocal microscopy. All sni1 roots displayed very strong defects. As shown in Fig. 1C, in contrast to the stereotypical structure in WT, the root meristem structure in the sni1 mutant was highly disorganized. The quiescent center (QC) cells underwent division, and the asymmetric division of the cortexendodermis initials was defective in some roots. These results indicate that the stem cells in sni1 are not properly maintained. To confirm this, we examined the expression of stem cell markers in the sni1 mutant. SHORTROOT (SHR) and SCARECROW (SCR) are key regulators of root radial patterning and stem cell maintenance (32). AGAMOUS-LIKE 42 (AGL42) is a MADS box transcription factor enriched in the QC (33). Compared with the WT, the sni1 mutant showed much lower expression levels of these markers (Fig. 1D). To test the expression of other genes involved in root development, qPCR analysis was performed (SI Appendix, Fig. S1). We found that the expression levels of most of the tested genes (SCR, SHR, PLT1, PLT2, PLT3, PLT4, PIN1, PIN2 PIN3, and PIN4) were greatly reduced in sni1. These results suggest that SNI1 is required for root development.

Fig. 1. SNI1 is required for root development. (A) The sni1 mutant shows a short-root phenotype. WT and the sni1 mutant were grown vertically on 1/2 MS medium. (B) The relative root length in WT and sni1. Data represent mean ± SD (n = 30). The statistical significances were determined using Student’s t test. ***P < 0.001. (C) The root meristem structure is disorganized in sni1. The roots of WT and sni1 were stained by PI and observed by confocal microscopy. (D) The expression (green fluorescence) of stem cell marker SHR:GFP, SCR:GFP, and AGL42:GFP in sni1 are reduced compared with WT. (Scale bars: A, 1 cm; C, 50 μm; D, 20 μm.)

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Since SNI1 is a subunit of the SMC5/6 complex, the phenotypes of sni1 may be attributed to the insufficiency of DNA repair (29). Unrepaired DNA damage will trigger programmed cell death (PCD). Indeed, some sni1 root cells were dead as indicated by the propidium iodide (PI) staining (Fig. 1C). Previous studies have shown that the loss of function of several HRrelated genes including RAD51D, BRCA2, ATR, RAD17, and SWIM, suppressed the sni1 phenotypes (29, 34–36). In these double mutants, the HR pathway is blocked, allowing DNA damage to be repaired through alternative repair pathways. However, based on root length, root meristem structure, and PCD, these mutants only partially suppressed the sni1 mutant (SI Appendix, Fig. S2), suggesting that SNI1 has other functions in addition to DNA repair. SNI1 Is a Target of E2F Transcription Factors. To further study the function of SNI1, we sought to examine SNI1 expression using GENEVESTIGATOR (37). We first analyzed its expression patterns in different tissues and found that SNI1 is highly expressed in tissues where cell division is highly active (such as callus, cell suspension, shoot apex, and root tip). (SI Appendix, Fig. S3C). To confirm the expression patterns, we constructed a vector containing the SNI1 native promoter and genomic sequence in frame with the coding sequence of β-glucuronidase (GUS) and transformed it into the sni1 mutant to obtain SNI1 expression reporter lines (SNI1:SNI1-GUS). The root length of the transgenic plants was restored to WT level (SI Appendix, Fig. S3A), indicating that the construct is biologically active. Consistent with microarray data, we found that SNI1 was highly expressed in shoot apical meristem and root apical meristem (SI Appendix, Fig. S3B), supporting its role in meristem maintenance. We also analyzed SNI1 expression in different mutants and transgenic plants, and we found its expression level was relatively stable. However, it was remarkably elevated in E2Fa-DPa– overexpressing lines (SI Appendix, Fig. S4A). Interestingly, we found that the promoters of SNI1 in both Arabidopsis and rice contain E2F-binding sites (SI Appendix, Fig. S4B), suggesting that SNI1 may be a target gene of the E2Fa transcription factor. To test our hypothesis, we first confirmed the elevated SNI1 expression in E2Fa-DPa–overexpressing lines (35S:E2Fa-DPa) using qRT-PCR (Fig. 2A). To determine whether E2Fs can bind the putative E2F-binding site in the promoter of SNI1, we performed yeast one-hybrid assays. Arabidopsis encodes three canonical E2Fs, among which E2Fa and E2Fb are activators and E2Fc is a repressor (17–21). As shown in Fig. 2B, both E2Fa and E2Fb could bind the oligos containing the potential E2F-binding site of the Arabidopsis SNI1 promoter. To determine whether E2Fs can bind the SNI1 promoter in vivo, we carried out chromatin immunoprecipitation (ChIP) assays followed by qPCR (ChIP-qPCR) using transgenic plants expressing the E2Fa3×FLAG fusion driven by the CaMV 35S promoter. As shown in Fig. 2 C and D, the probes covering the E2F cis element of the SNI1 promoter (P1 and P2), but not the probe in the coding region (P3), were significantly enriched in E2Fa-3×FLAG compared with WT. To test whether E2Fa and E2Fb can activate SNI1 expression, we performed dual-luciferase assays in Nicotiana benthamiana. The reporter vector contained a firefly luciferase (LUC) gene driven by the SNI1 promoter and a renilla luciferase (REN) gene driven by the CaMV 35S promoter. The effector vectors included E2Fs and DPa driven by the CaMV 35S promoter (Fig. 2E). As shown in Fig. 2F, both E2Fa and E2Fb activated SNI1 expression. Collectively, these data demonstrate that E2Fs bind to the SNI1 promoter and activate its expression, suggesting SNI1 is a direct target of E2Fs. Wang et al.

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Fig. 2. SNI1 is a target gene of E2Fa. (A) The expression of SNI1 is dramatically increased in E2Fa and DPa cooverexpressing lines (35S:E2Fa-DPa) compared with WT. The relative expression level of SNI1 was determined through qRT-PCR analysis using ubiquitin 5 (UBQ5) as an internal standard. (B) E2Fa and E2Fb bind to the SNI1 promoter in yeast one-hybrid assays. Three tandem copies of DNA fragment containing E2F-binding site of SNI1 promoter (named as “oligo”) were cloned into pHis2.1 vector. E2Fa and E2Fb were cloned into pGADT7-Rec vector, respectively. pGADT7-Rec-53 and pHis2.1-P53 were used as a positive control. pGADT7-Rec-53 and pHis2.1 were used as a negative control. DDO, double dropout (SD/-Trp/-Leu) medium; TDO, triple dropout (SD/-Trp/-Leu/-His) medium; 3-AT, 3-amino-1,2,4-triazole. (C) Diagram of genomic region of SNI1. P1, P2, and P3 represent the fragments used in ChIP-qPCR in D. (D) E2Fa associates with SNI1 promoter in ChIP-qPCR assays. The percentages of DNA fragments coimmunoprecipitated by antibody relative to the input DNA are shown. Data represent mean ± SE of three biological replicates (n = 3). (E) Schematic representation of the constructs used for dual-luciferase assays. The reporter construct contains the firefly luciferase (LUC) driven by SNI1 promoter, and the Renilla luciferase (REN) driven by the CaMV 35S promoter. The effector constructs contain E2Fa, E2Fb, or DPa driven by the CaMV 35S promoter, respectively. (F) E2Fa-DPa and E2Fb-DPa activate the expression of SNI1 in dualluciferase assays. The reporters and effectors were coexpressed in N. benthamiana, and both REN and LUC activity were measured. The relative LUC activities normalized to the REN activities are shown (LUC/REN). Data represent mean ± SE of three biological replicates (n = 3). The statistical significances were determined using Student’s t test. **P < 0.01, ***P < 0.001; ns, not significant.

SNI1 Interacts with E2Fs. Given that E2Fs regulate SNI1 expres-

sion and SNI1 may regulate cell cycle progression, we hypothesized that SNI1 can, in turn, regulate E2Fs. Therefore, we sought to explore the possibility that SNI1 interacts with E2Fs. We first carried out yeast two-hybrid (Y2H) assays. However, the assays were unsuccessful because both E2Fa and E2Fb have selfactivation activities and SNI1 has transcriptional repression activity in yeast cells (31), making them not suitable as baits. As an alternative approach, we performed split luciferase assays to test their interactions in N. benthamiana (38). SNI1 was fused with the C-terminal half of luciferase (CLuc) and E2Fs were fused with the N-terminal half of luciferase (NLuc). An interaction between two proteins brings the two halves of the luciferase together, leading to enzymatic activity. As shown in Fig. 3A, SNI1 could interact with both E2Fa and E2Fb. To further confirm their interactions, we carried out coimmunoprecipitation (Co-IP) assays. The E2F-Strep II fusion proteins were coexpressed with SNI1-TAP (containing a 9×Myc tag) or GFP-TAP Wang et al.

in N. benthamiana. After immunoprecipitation using the antiMyc antibody, Western blot was performed using the antiStrep II antibody to detect E2F-Strep II proteins. Both E2Fa and E2Fb could be coimmunoprecipitated by SNI1-TAP, but not the GFP-TAP control, indicating that SNI1 specifically interacts with both E2Fa and E2Fb in vivo (Fig. 3B). To study how SNI1 regulates E2Fs, it is necessary to determine which domain of E2Fs interacts with SNI1. Both E2Fa and E2Fb contain a highly conserved DNA-binding domain, a moderately conserved dimerization domain and a C-terminal transactivation domain (Fig. 3C). In our initial Y2H assays, we found that the N-terminal half of E2Fs does not show selfactivation activity (SI Appendix, Fig. S5A). Therefore, we used Y2H to test whether the N-terminal half of E2Fs could interact with SNI1. As shown in SI Appendix, Fig. S5B, no interaction was detected, implying that the C-terminal domains of E2Fs are required for their interaction with SNI1. Previous studies revealed that the Rb-binding domain (RBBD) in the C-terminal transactivation PNAS | vol. 115 | no. 16 | E3839

Fig. 3. SNI1 interacts with E2Fa and E2Fb. (A) Split luciferase assays. The Agrobacterium carrying the indicated constructs were coinfiltrated into N. benthamiana leaves. The positive luminescence monitored by a CCD camera indicate interaction. (B) Coimmunoprecipitation assays. E2Fa and E2Fb were tagged by Strep II and SNI1 was tagged by TAP (containing 9×Myc tag). GFP-TAP was used as a negative control. The constructs were coexpressed in N. benthamiana leaves. Immunoprecipitation was performed using anti-Myc antibody, and Western blot was performed using either anti-Strep II antibody or anti-Myc antibody. (C) Outline of E2Fa and E2Fb structures highlighting the conserved domains. (D and E) In vitro pull-down assays. GST-RBBD fusion proteins were expressed in E. coli BL21 and purified using glutathione beads. Myc-SNI1 was in vitro translated. Myc-SNI1 could be pulled down by GST-E2Fa RBBD (D) and GST-E2Fb RBBD (E), respectively. Similar amount of GST and GST-RBBD were used as indicated by Ponceau S staining.

domain of E2F is responsible for their interactions with RB (39). Therefore, we hypothesized that SNI1 also interacts with E2Fs through RBBD. To test this, we performed in vitro pull-down assays. Compared with the GST control, both GST-E2Fa-RBBD and GST-E2Fb-RBBD could pull down Myc-tagged SNI1 protein (Fig. 3 D and E), suggesting that SNI1 directly binds to RBBD of E2Fs. These data strongly support that SNI1 interacts with E2Fs. SNI1 Represses the Transcriptional Activities of E2Fs. Since SNI1 can bind to the transactivation domains of E2Fs, we further hypothesized that SNI1 represses the transcriptional activities of E2Fs. Interestingly, in our attempts to test the interactions between SNI1 and E2Fs through Y2H assays, we found that SNI1 could inhibit the self-activation activities of E2Fs and E2Fs-RBBD (SI Appendix, Fig. S5 C and D), which supports with our hypothesis. To confirm this activity of SNI1 in planta, we first E3840 | www.pnas.org/cgi/doi/10.1073/pnas.1720094115

performed ChIP-qPCR assays to test whether SNI1 could bind to the promoters of E2F target genes. Since SNI1 is highly expressed in callus, we conducted ChIP assays using the callus of SNI1-TAP line (SNI1:SNI1-TAP) described previously (29, 34). As shown in Fig. 4A, the promoters of E2F target genes (MCM3, ORC1B, CDC6, ORC3) (40, 41) were significantly enriched in the ChIP DNA of SNI1:SNI1-TAP compared with that of WT. The enrichment could not be detected for the CYCB1;1 promoter, a G2/M specific gene that is not targeted by E2Fs. These results indicate that SNI1 associates with the promoters of E2F target genes. To further test whether SNI1 represses transcriptional activities of E2Fs in planta, we carried out dual luciferase assays. In these experiments, the reporter vectors contained the LUC gene driven by the promoters of ORC1B and CDC6, two well-established E2F target genes (40, 41). The effector vectors contained E2FaDPa, E2Fb-DPa, and/or SNI1 driven by the CaMV 35S promoter Wang et al.

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Fig. 4. SNI1 represses the transcriptional activities of E2F transcription factors. (A) SNI1 associates with the promoters of E2F-target genes. ChIP-qPCR assays were performed using anti-Myc antibody in the callus of SNI1:SNI1-TAP or WT. The promoter fragments containing E2F consensus motif of E2F target genes (MCM, ORC1B, CDC6, and ORC3) were quantified, and the percentages of coimmunoprecipitated DNA by antibody relative to the input DNA were shown. Actin7 and CYCB1:1 were used as negative controls. Data represent mean ± SE of three biological replicates (n = 3). (B) Schematic representation of the constructs used in dual-luciferase assays. (C and D) SNI1 represses the transcriptional activities of E2Fa (C) and E2Fb (D) as indicated by dual-luciferase assays, respectively. The promoters of E2F-target genes (ORC1B and CDC6) were cloned into the reporter vectors. E2Fa, E2Fb, DPa, or SNI1 were cloned into the effector vectors, respectively. Data represent mean ± SE of three biological replicates (n = 3). The statistical significances were determined using Student’s t test. **P < 0.01, ***P < 0.001; ns, not significant.

(Fig. 4B). As shown in Fig. 4 C and D, the E2F–DP complexes could activate the expression of the reporters, indicating that the Arabidopsis E2F can function properly in the heterologous tobacco system. As expected, the activation was repressed when SNI1 was coexpressed with E2Fa and E2Fb. Both SNI1 and E2Fs are highly conserved in plants. Therefore, we were interested to know whether SNI1 represses E2Fs in other plant species, such as rice (Oryza sativa). As shown in SI Appendix, Fig. S6, OsSNI1 interacted with OsE2F1 in vivo and repressed the transcriptional activity of OsE2F1. Taken together, these results suggested that SNI1 represses the transcriptional activities of E2Fs and this mechanism is well conserved in plants. SNI1 Recruits Histone Deacetylases to Repress E2Fs. Previous studies

reported that loss of SNI1 function results in increased histone 3 acetylation (AcH3) (31), suggesting that SNI1 may recruit histone deacetylases (HDACs) to repress gene expression. There are 16 HDACs in Arabidopsis (42), and we tested their interactions with SNI1 using Y2H assays. We found that HADC6 and HDAC7, which belong to class I HDAC, could interact with SNI1 (Fig. 5A). To confirm their interactions in vivo, we carried Wang et al.

out split luciferase assays. As expected, both HDAC6 and HDAC7 could indeed interact with SNI1 (Fig. 5B). To test whether HDAC6 and HDAC7 contribute to the transcriptional repression activity of SNI1, we performed dual-luciferase assays. Compared with SNI1 alone, coexpression of SNI1 and HDAC6 or HDAC7 further repressed the expression of E2F target genes ORC1B and CDC6 (Fig. 5 C and D). These results supported the idea that SNI1 can recruit HDACs to repress the transcriptional activities of E2Fs. Overexpression of SNI1 Suppresses the Phenotype of E2Fa and DPa Overexpressing Plants. Our biochemical data strongly suggest that

SNI1 inhibits E2Fs. To further support our hypothesis, we used genetic approaches. Previous studies have shown that cooverexpression of E2Fa and DPa resulted in increased endoreplication level and retarded growth (17). Therefore, we sought to test whether overexpression of SNI1 can suppress these phenotypes. We generated transgenic plants overexpressing E2Fa, DPa, and SNI1 (35S:E2Fa-DPa-SNI1) by crossing homozygous 35S:E2Fa plants with plants coexpressing DPa and SNI1 (35S:DPaSNI1). The qPCR analysis confirmed that E2Fa, DPa, or SNI1 PNAS | vol. 115 | no. 16 | E3841

Fig. 5. SNI1 recruits HDAC6 and HDAC7 to repress the transcriptional activity of E2Fs. (A) SNI1 interacts with HDAC6 and HDAC7 in Y2H assays. pGBKT753 was cotransformed with pGADT7-RecT as a positive control, and pGBKT7-Lam was cotransformed with pGADT7-RecT as a negative control. DDO, double dropout (SD/-Trp/-Leu) media. QDO, quadruple dropout (SD/-Trp/-Leu/-His/-Ade) media. (B) SNI1 interacts with HADC6 and HDAC7 in split-luciferase assays. (C and D) The repression activity of SNI1 on E2Fa (C) and E2Fb (D) is enhanced by HDAC6 and HDAC7 as indicated by dual-luciferase assays. The expression of E2Fa- and E2Fb-target genes (ORC1B and CDC6) was used as reporter. Data represent mean ± SE of three biological replicates (n = 3). The statistical significances were determined using Student’s t test. *P < 0.05, **P < 0.01.

were all overexpressed in these plants (SI Appendix, Fig. S7B). Consistent with previous studies, in the 35S:E2Fa-DPa seedlings, the roots were shorter than WT and the cotyledons were curled and smaller (17). In contrast, the 35S:E2Fa-DPa-SNI1 seedlings were similar to WT (Fig. 6 A and B and SI Appendix, Fig. S7A). In accordance with the root length, the sizes of root meristem were reduced in 35S:E2Fa-DPa and restored in 35S:E2Fa-DPa-SNI1 plants (SI Appendix, Fig. S7C). We also compared the endoreplication level in these plants through flow cytometry analysis. As expected, the increased endoreplication level in 35S:E2Fa-DPa was restored to WT level when SNI1 was cooverexpressed (Fig. 6C and SI Appendix, Fig. S8). Furthermore, the elevated expression of E2F target genes in 35S:E2Fa-DPa was also suppressed by overexpressing SNI1 (Fig. 6D). These data suggest that overexpression of SNI1 is sufficient to inhibit the functions of E2Fa. E3842 | www.pnas.org/cgi/doi/10.1073/pnas.1720094115

Loss of E2F Function Suppresses the sni1 Mutant. Similar to 35S: E2Fa-DPa plants, the sni1 mutant also showed a short-root phenotype (Figs. 1A and 7A). In addition, we found that the endoreplication level in sni1 was also increased through flow cytometry analysis (Fig. 7D and SI Appendix, Fig. S9), suggesting that the phenotypes in sni1 may be due to the lack of repression on E2Fs by SNI1. Therefore, we tested whether loss of E2F function could suppress the sni1 mutant. To this end, we crossed sni1 with the e2fa e2fb double mutant. However, the root length of the sni1 e2fa e2fb triple mutant was similar to that of the sni1 mutant (SI Appendix, Fig. S10). This reminded us that Arabidopsis contains another canonical E2F transcription factor, E2Fc. Although E2Fc was proposed to be a repressor while E2Fa and E2Fb to be activators (17–21), it was recently reported that E2Fa, E2Fb, and E2Fc are functionally redundant (43). Indeed, we found that SNI1 can also interact with E2Fc in the split luciferase assays Wang et al.

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(SI Appendix, Fig. S11). Therefore, SNI1 interacts with all three canonical E2Fs (E2Fa/b/c). In contrast, the noncanonical E2Fs (E2Fd/e/f) did not interact with SNI1 (SI Appendix, Fig. S11). These results further indicated that SNI1 interacts with E2Fs through the RBBD domain. After crossing sni1 with the e2fa e2fb e2fc triple mutant, we performed genotyping to obtain high order mutants. Like sni1 e2fa e2fb, the root length in sni1 e2fa e2fc and sni1 e2fb e2fc were similar to sni1 (SI Appendix, Fig. S10). For unknown reasons, we were unable to obtain the sni1 e2fa e2fb e2fc quadruple mutant. Nevertheless, we found that the short-root phenotype was completely suppressed in the sni1 e2fa e2fb/+ e2fc mutant (Fig. 7 A and B). Although E2Fb was heterozygous in the sni1 e2fa e2fb/+ e2fc mutant, we could not detect the expression of E2Fb in the mutant (SI Appendix, Fig. S12). The disorganized root meristem structure, the increased endoreplication level, and PCD phenotypes in sni1 were also fully suppressed in the sni1 e2fa e2fb/+ e2fc mutant (Fig. 7 C and D). Taken together, these results suggest that loss of E2F function can suppress the defects of the sni1 mutant. Discussion DDR is a fundamental mechanism used by all organisms to maintain genome stability. In response to DNA damage, cells activate checkpoints to arrest cell cycle progression and allow cells enough time to repair DNA. In this study, we provided strong biochemical and genetic data to demonstrate that the DNA repair protein SNI1 is a molecular link between checkpoint activation and DNA repair (SI Appendix, Fig. S14). SNI1 interacts with and inhibits E2F transcription factors to stop cell cycle progression. However, as a subunit of the SMC5/6 complex, SNI1 promotes the DNA damage repair process. Therefore, we Wang et al.

propose that SNI1 plays dual roles in DDR to ensure efficient DNA damage repair to maintain genome stability. In the sni1 mutant, both checkpoint activation and DNA damage repair mechanisms are defective. As a result, DNA damage cannot be properly repaired, leading to cell death and severe growth defects in the sni1 mutant. In the sni1 ssn mutants, the DNA damage repair efficiency is improved by blocking the HR pathway and promoting other repair pathways such as nonhomologous end joining (NHEJ) (29). However, the checkpoint is still defective due to the lack of E2F inhibition by SNI1 in sni1 ssn. Therefore, cells may not have enough time to repair DNA. In addition, it is likely that there are more potential SSN genes in Arabidopsis and disruption of multiple SSN genes may suppress sni1 better than a single ssn mutant. Since some SSN genes such as RAD17 and RAD51 are E2F target genes (41), the expression of multiple SSN genes may be simultaneously disrupted in the sni1 e2fa e2fb/+ e2fc mutant. Therefore, both checkpoint activation and DNA damage repair mechanisms are recovered in the sni1 e2fa e2fb/+ e2fc mutant, which coordinately contribute to suppression of sni1. As E2Fs are required for cell death during plant immune responses (43), loss of E2F function may also directly account for the suppression of cell death in sni1. Although the sni1 e2fa e2fb/+ e2fc mutant is similar to WT under normal growth condition, it is hypersensitive to DNAdamaging agents hydroxyurea (inducing replication stress) and camptothecin (inducing double-strand breaks), which further support that SNI1 plays important role in DNA damage repair (SI Appendix, Fig. S13). In the SMC5/6 complex, while SMC5, SMC6, and NSE1– NSE4 subunits are highly conserved in all eukaryotes, NSE5 and NSE6 are divergent even between fission yeast and budding yeast (44–46). Through biochemical and genetic studies, SNI1 and its PNAS | vol. 115 | no. 16 | E3843

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Fig. 6. Overexpression of SNI1 suppresses E2Fa and Dpa overexpressing plants. (A) The phenotypes of WT, E2Fa, and DPa cooverexpressing (35S:E2Fa-DPa) and E2Fa, DPa, and SNI1 cooverexpressing (35S:E2Fa-DPa-SNI1) plants. (B) The length of the primary roots of the indicated plants. Data represent mean ± SD (n = 30). The statistical significances were determined using Student’s t test. (C) Quantitative analysis of the flow cytometry data. Data represent mean ± SD (n = 3). (D) The expression levels of E2F-target genes (ORC1B, CDC6, ORC3, and RNR) in the indicated plants through qRT-PCR analysis using UBQ5 as an internal standard. Data represent mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Fig. 7. Loss of E2F function inhibits the phenotypes of the sni1 mutant. (A) The phenotypes of WT, sni1, sni1 e2fa e2fb/+ e2fc, and the e2fa e2fb e2fc mutant. (B) The length of the primary roots of the indicated plants. Data represent mean ± SD (n = 30). The statistical significances were determined using Student’s t test. ***P < 0.001. (C) The root meristem structure of the indicated plants. (Scale bars: A, 1 cm; C, 50 μm.) (D) Quantitative analysis of the flow cytometry data of the indicated plants. Data represent mean ± SD (n = 3).

associated protein ASAP1 were identified to be the first NSE6 and NSE5 in multicellular organisms (29). Both SNI1 and ASAP1 are highly conserved in plants. In our study, rice SNI1 could also interact with and repress the rice E2F (SI Appendix, Fig. S6), indicating that this mechanism is conserved in plants. Although animals do not contain SNI1 homologs, it has been recently reported that SLF2 (SMC5/6 localization factors 2) is an NSE6 protein in human cells (46). It will be interesting to see whether SLF2 can interact with and inhibit E2Fs. In eukaryotes, there are six SMC proteins (SMC1–6), which form three distinct complexes and regulate nearly all aspects of chromosome biology. Cohesin (containing SMC1 and SMC3) maintains sister-chromatid cohesion and is required for proper chromosome segregation. Condensin (containing SMC2 and SMC4) mediates chromosome condensation. The SMC5/6 complex plays multiple essential roles in DNA repair (22, 28, 47). While both cohesin and condensin are also involved in checkpoint activation (24, 48–51), it is unknown whether the SMC5/ 6 complex has such functions. Recently, it was reported that AtMMS21, the NSE2 subunit of SMC5/6 complex, also regulates cell cycle (52). This study tested the interactions between AtMMS21 and eight cell cycle regulators (CDKA, CYCB1; 1, RBR, E2Fa, E2Fb, E2Fc, DPa, and DPb) and found that AtMMS21 interacted with DPa. AtMMS21 competes with E2Fa for DPa and affects the subcellular localization of E2Fa/DPa. Taken together with our study, these data suggest that the SMC5/6 complex can also regulate cell cycle through multiple mechanisms. RB is a well-known regulator of E2Fs both in plants and in animals (8–12). In this study, we found that SNI1 is also an E2F regulator. While SNI1 expression is activated by E2Fs, the SNI1 protein inhibits the E2F transcriptional activities, indicating that SNI1 and E2Fs form a negative feedback loop. Interestingly, E3844 | www.pnas.org/cgi/doi/10.1073/pnas.1720094115

SNI1 shares several similar characteristics with RB. First, both SNI1 and RB can interact with the RBBD domain of E2Fs and inhibit their transcriptional activities (53) (Figs. 3 and 4). Second, both SNI1 and RB are target genes of E2Fs (54) (Fig. 2). Third, neither SNI1 nor RB has clear biochemical activities (31, 53). Notably, when SNI1 was first reported, it was shown to have a short stretch of sequence homology with the mouse RB protein although the full-length SNI1 sequence is not homologous to any known proteins (30). It is also worthwhile to note that there are three RB proteins in humans. In contrast, Arabidopsis only has one RB homolog although they have similar number of E2Fs (13, 15). Based on these lines of evidence, it is tempting to propose that SNI1 is an RB-like protein. Recently, two independent research groups demonstrated that RBR1 directly regulates DNA repair (55, 56). The rbr1 mutant is hypersensitive to DNA-damaging agents. RBR1 could interact with DNA repair proteins BRCA1 and RAD51 and could be recruited to DNA damage sites. While these two studies found that the cell cycle regulator is directly involved in DNA repair, our study revealed that the DNA repair protein is directly involved in cell cycle regulation. Therefore, these studies indicate that directly linking cell cycle checkpoints and DNA repair is a general mechanism that ensures efficient DNA repair to maintain genome stability. Further studies are required to identify additional regulators. Materials and Methods Materials. The sni1 mutant was described in ref. 30. The 35S:E2Fa and 35S:DPa lines were described in ref. 17. The e2fa e2fb e2fc triple mutant were described in ref. 43. Methods. Y2H assays were performed by using the Matchmaker Gal4 TwoHybrid System (Clontech). Yeast one-hybrid assays were carried out using

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Rad CFX 96 detection system. Detailed methods are described in SI Appendix, SI Materials and Methods.

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the Matchmaker Yeast One-Hybrid System (Clontech). For in vitro pull-down assays, the GST fusion proteins were purified from Escherichia coli BL21(DE3), and the Myc-SNI1 was in vitro translated using TNT Coupled Wheat Germ Extract System (Promega). Coimmunoprecipitation assays were performed as described previously with some modifications (57). Immunoprecipitations were performed using anti-Myc antibody. The split luciferase assays were performed as described (38). The dual luciferase assays were performed as described (58). ChIP assays were performed as described previously with some modifications (59). Flow cytometry analysis was performed using BD FACS Calibur flow cytometer. The qPCR analyses were performed using Bio-

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ACKNOWLEDGMENTS. We thank Xinnian Dong, Lieven De Veylder, and John Withers for critical reading of the manuscript and Philip Benfey, Shui Wang, Yi Tao, Mingyi Bai, and Hongtao Liu for sharing materials. The work was supported by National Science Foundation of China Grants 31571253 and 31771355, Huazhong Agricultural University Scientific & Technological Self-innovation Foundation Grant 2014RC004, the Fundamental Research Funds for the Central Universities Grant 2662015PY064, and a Thousand Talents Plan of China-Young Professionals grant (to S.Y.).