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Oncogene (2013) 32, 4883–4891 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc

ORIGINAL ARTICLE CLOCK is a substrate of SUMO and sumoylation of CLOCK upregulates the transcriptional activity of estrogen -a

S Li, M Wang, X Ao, AK Chang, C Yang, F Zhao, H Bi, Y Liu, L Xiao and H Wu

Disruption of the is now believed to associate with a number of hormone-related cancers, such as , in which aberrant -a () signaling is a major contributor. However, the molecular mechanisms underlying the function of core in cancer are still largely undefined. In this study, we showed that circadian locomotor output cycles kaput (CLOCK), a key circadian , can interact with ERa. Furthermore, this interaction was enhanced by estrogen. We also showed that CLOCK can be sumoylated and sumoylation of CLOCK, which is also stimulated by estrogen, had two consequences: (1) it increased the transcriptional activity of CLOCK; and (2) it increased the CLOCK-modulated transcriptional activity of ERa, as shown by increased transcription of cyclin D1. Sumoylation of CLOCK occurred at two lysine residues, K67 and K851. The enhancement of ERa transcriptional activity exerted by wild-type but not mutant (2K/2R) CLOCK in response to estrogen indicated that sumoylation of CLOCK may have an important role in estrogen-dependent signaling. 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay conducted with breast cancer cell lines (MCF-7 and T47D) demonstrated that sumoylation of CLOCK stimulated cell growth and increased the proportion of S phase cells in the cell . The results of this study uncovered new insight into the connection between a major circadian protein and a major estrogen-dependent , providing the basis for further research into the involvement of circadian proteins in breast cancer.

Oncogene (2013) 32, 4883–4891; doi:10.1038/onc.2012.518; published online 19 November 2012 Keywords: CLOCK; ERa; estrogen; sumoylation

INTRODUCTION increased incidence of hormone-related breast cancer as well as 15,16 The steroid hormone estrogen (E2) is essential for normal other types of malignancies. In addition to epidemiological mammary gland physiology, and a potent mammary mitogen.1,2 studies showing a clear connection between disruption of The main mechanism by which E2 regulates transcription is circadian rhythms and breast cancers, numerous studies have through binding with estrogen receptors (ERs), which then form a also reported that the expression levels of the (Per1 and dimer and bind to estrogen-responsive elements (EREs) in the Per2) in both sporadic and familial primary breast tumor regulatory regions of downstream target genes and associate with tissues are significantly lower compared with the levels in normal 17–19 basal transcription factors, coactivators or corepressors to regulate breast tissues. the expressions of these genes.2–4 There are two isoforms of ERs, Most physiological processes in mammals are influenced by ERa and ERb, and both are known to modulate the expressions of circadian rhythms. The mechanism of circadian is genes that are implicated in different cellular processes, including generated by a cell autoregulatory transcription–translation feed- tumorigenesis.5,6 Much investigation has focused on under- back loop. At the molecular levels, circadian rhythm is composed standing the role of ERa in the development and progression of of a set of interlocked clock proteins in which circadian locomotor breast cancer, and it has been shown that aberrant ERa signaling output cycles kaput (CLOCK) and brain and muscle ARNT-like is closely related to the occurrence of ERa-positive breast cancer.7 protein 1 (BMAL1) are the core circadian proteins. CLOCK and ERa has been shown to interact with several coactivators having BMAL1 can associate with each other to form heterodimer, which acetyltransferase activity, including cAMP-response element then bind to E-box elements in the promoters of target genes to binding protein (CREB)-binding protein, p300, and members of the induce the rhythmical transcription of period (Per1, 2 and 3) and p160 family, such as steroid receptor coactivator-1 (SRC-1), SRC-2 (Cry1 and 2) in the positive limb.20–22 CLOCK has an and SRC-3.8,9 The transcriptional activities of all members of the intrinsic histone acetyltransferase activity, so it can initiate p160 family are regulated by their post-translational modifications, chromatin remodeling and create a favorable state for transcrip- such as phosphorylation and sumoylation.10–13 tion.23 The transcripts of Per and Cry genes are then translated into The secretions of endocrine and metabolic hormones occur in their corresponding proteins, PERs and CRYs, in the cytoplasm. a rhythmical manner. Reciprocally circulating levels of these PERs and CRYs form heterodimers and are subsequently trans- hormones influence the circadian rhythms. Disruption of this ported into the nucleus where they can function as potent has a profound effect on health and has inhibitors of CLOCK–BMAL1-driven transcription.24–26 To start a been linked to diverse pathological conditions, including hormone- new cycle, the inhibition of CLOCK–BMAL1 heterodimer has to be related breast and endometrial cancers.14,15 A remarkable released by the proteolytic degradation of PER and CRY.25,26 example is that overnight-shift workers tend to have an Further regulation is achieved by post-translational modification

School of Life and Biotechnology, Dalian University of , Dalian, China. Correspondence: Professor H Wu, School of Life Science and Biotechnology, Dalian University of Technology, 2 Ling Gong Road, Dalian 116024, China. E-mail: [email protected] Received 9 May 2012; revised 6 September 2012; accepted 28 September 2012; published online 19 November 2012 Sumoylation in CLOCK regulates ERa’s activity SLiet al 4884 and chromatin remodeling, which are pivotal to maintaining the compared with control. Overexpression of ERa alone without and circadian rhythms. with E2 treatment increased ERE-luc activity (Figure 1e). However, At , few studies have focused on the post-translational overexpression of both ERa and CLOCK resulted in a further modification of CLOCK other than its phosphorylation by protein twofold increase of ERE-luc activity in the presence of E2 kinase C and glycogen synthase kinase-3.27,28 Small ubiquitin treatment. This response was consistent with CLOCK-stimulated like-modifier (SUMO) modification is an important mechanism ERa transcriptional activity in response to E2 treatment. Similarly, for dynamical post-translation regulation of protein functions.29 overexpression of ERa and SRC-1, which is a classical coactivator To our knowledge, there has been no report concerning the for ERa, also increased the level of ERE-luc activity, and repeated sumoylation of circadian proteins, except for BMAL1 and here as a positive control (Figure 1e). Knockdown of CLOCK differentiated embryo-chondrocyte expressed gene1 (DEC1).30,31 expression in MCF-7 cells using the specific small interfering RNA, Both BMAL1 and members of the p160 family transcription siCLOCK-2467, suppressed ERa-mediated transcriptional activity coactivator, which have similar structure with CLOCK, have all while another specific small interfering RNA, siCLOCK-1357, been shown to be sumoylated, so we speculated that CLOCK may showed almost no effect on the transcriptional activity of ERa. also be subjected to sumoylation. This was further confirmed by western blot analysis, which Although a growing number of studies have suggested that showed that siCLOCK-2467 effectively repressed the expression of disruption of the circadian rhythm is associated with breast CLOCK protein, whereas siCLOCK-1375 had no effect on its tumorigenesis, very few studies have investigated the direct expression (Figure 1f). The ability of CLOCK to regulate the involvement of circadian proteins in the estrogen signaling expression of the well-established ERa-targeted gene, cyclin D1, pathway. PER2 expression is now known to be induced by was also examined. Knockdown of CLOCK by siCLOCK-2467 estrogen, and PER2 can bind to ERa and enhance its degradation, decreased the activity of cyclin D1 reporter and expression level of linking the circadian rhythm to ERa signaling pathway.14 CLOCK cyclin D1 (Figures 1g and h). The data from these experiments shares a number of structural features with p160 family proteins, provided support for the upregulation of ERa transcriptional which are classical coactivators of ERa, including a highly activity by CLOCK in the presence of estrogen. conserved basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) domain, interaction domain and a glutamine- CLOCK is modified by SUMO-1 rich C-terminal domain that possesses histone acetyltransferase Previous research has shown that ERa interacts with several activity.23 Based on the structural similarity between CLOCK and cofactors, such as members of the p160 family, and all known p160 family proteins, we proposed that CLOCK could well be a members of p160 family are subjected to sumoylation.10–12 As potential cofactor of ERa. CLOCK has a similar structure with members of the p160 family, In this study, we showed that CLOCK can associate with ERa to we wished to know whether CLOCK is also a target of sumoylation. enhance the transcriptional activity of ERa, and this was promoted As shown in Figure 2a, immunoprecipitated CLOCK from MCF-7 by E2. We also showed that CLOCK could be sumoylated and cells was detected by anti-SUMO-1 antibody. The size of this band identified two sumoylation sites. Sumoylation of CLOCK enhanced corresponded to that of SUMO-conjugated CLOCK, and its its transcriptional activity and interaction with ERa, resulting in intensity increased with increasing E2 treatment , although upregulation of ERa activity. Our data have given new insight into the total amount of CLOCK protein remained relatively the involvement of CLOCK in estrogen signaling pathway, with unchanged. Sumoylation was further confirmed in COS-7 cells, ERa as its key interacting transcription factor. when these cells were first transfected with Flag-tagged CLOCK and -SUMO-1, followed by immunoprecipitation carried out with anti-Flag antibody and subsequent western blot analysis with RESULT anti-Myc antibody (Figure 2b). E2 promotes the association of CLOCK with ERa Bioinformatic analysis of the CLOCK protein sequence revealed two putative sumoylation sites, K67 and K851, which are located at As CLOCK shares a similar structure with members of p160 family the bHLH/PAS and C-terminal domain, respectively (Figure 2c). (Figure 2c), all of which are all classical coactivators of ERa, the The sumoylation sites of two members of the p160 family, SRC-1 possibility of CLOCK interacting with ERa was investigated in the and SRC-2, are also shown for comparison purpose. Replacement breast cancer cell line MCF-7, without and with E2 stimulation. of either K67 or K851 with arginine (K67R or K851R mutant) Co-immunoprecipitation experiments showed that CLOCK asso- resulted in decreased sumoylation for the mutant CLOCK ciated with ERa in MCF-7 cells, and this interaction was enhanced compared with wild type (Figure 2d), indicating that K67 and by treatment of the cells with E2 before immunoprecipitation K851 were the sites of sumoylation for CLOCK. However, changing (Figure 1a). Similar co-precipitation of CLOCK and ERa was both lysine residues to arginine (2K/2R mutant) appeared to obtained when the same immunoprecipitation experiment was abolish sumoylation (Figure 2d). To determine how much carried out using COS-7 cells transfected with Flag-tagged CLOCK influence sumoylation exerts on the transcriptional activity of and HA-tagged ERa (Figure 1b). Moreover, the direct interaction of CLOCK, COS-7 cells were transfected with Gal4 promoter-driven ERa and CLOCK was further confirmed using mammalian two- luciferase (Gal4-Luc) construct along with GAL4-DBD or GAL4- hybrid system. Transactivation by pBIND–CLOCK was strongly DBD-CLOCK (wild type or mutant), and the luciferase activity was activated by co-expressing a pACT–ERa fusion protein (Figure 1c). then measured. GAL4-DBD-CLOCK construct bearing wild-type Therefore, we speculated that CLOCK may work as a cofactor CLOCK showed more luciferase activity than the construct bearing participating in the estrogen signaling pathway, and this would any of the three mutants (Figure 2e). These data showed that require the coexpression of ERa and CLOCK in the cell, particularly sumoylation of CLOCK was essential for maintaining a higher level their colocalization in the cell nucleus. Double-label fluorescence of transcriptional activity. immunohistochemistry aiming at detecting the localization of CLOCK and ERa in MCF-7 cells treated with or without E2 showed that after treatment with E2, much more CLOCK and ERa were Sumoylation of CLOCK is reversed by SENP1 colocalized in the nucleus (Figure 1d). This suggested that CLOCK Sumoylation is a dynamic process that can be reversed by sentrin- may directly participate in the estrogen signaling pathway. specific protease (SENP). There are three SENPs present in the The interaction between CLOCK and ERa was further investi- cell nucleus, and these are referred to as SENP1, SENP2 and gated by luciferase reporter assay in MCF-7 cells. Overexpression SENP3.32–35 To determine which of the three SENPs could reverse of CLOCK alone had little effect on the level of ERE-luc activity the sumoylation of CLOCK, COS-7 cells were transfected with

Oncogene (2013) 4883 – 4891 & 2013 Macmillan Publishers Limited Sumoylation in CLOCK regulates ERa’s activity SLiet al 4885

Figure 1. Evidences for the interaction between CLOCK and ERa.(a) MCF-7 cells were treated with 10 nM E2 after serum starvation for 48 h, and the cells were then collected at the indicated time points and subjected to immunoprecipitation with anti-CLOCK (or anti-ERa antibody) or control immunoglobulin G (IgG) followed by western blot analysis with anti-ERa (or anti-CLOCK) antibody. (b) COS-7 cells were transfected with Flag-tagged CLOCK and HA-tagged ERa. After 24 h of transfection, the cells were subjected to immunoprecipitation with anti-Flag antibody, followed by western blot analysis with anti-HA antibody. (c) Direct interaction between CLOCK and ERa as detected using a mammalian two- hybrid system. CLOCK and ERa were expressed from pBIND-CLOCK and pACT-ERa, respectively, whereas the empty vectors pACT and pBIND were expressed as controls, as indicated with the pG5-luc reporter in COS-7 cells. Cells were transfected with pBIND-ID and pACT-MyoD as a positive control. Luciferase activity was measured after 36 h of transfection. **Po0.01 compared with cells transfected with pACT and pBIND. (d) Colocalization of ERa and CLOCK in MCF-7 nuclei. MCF-7 cells were treated with or without 10 nM E2 for 1 h before treatment with anti-CLOCK and anti-ERa antibodies. CLOCK-antibody complex was visualized with fluorescein isothiocyanate (FITC), whereas ERa-antibody complex was visualized with tetraethyl rhodamine isothiocyanate (TRITC). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). (e) MCF-7 cells were co-transfected with ERE-luciferase construct (300 ng) and either with ERa or CLOCK, ERa and CLOCK or with ERa and SRC-1. Luciferase activity was measured either with or without pre-treatment of the cells with 10 nM E2 for 16 h. (f) MCF-7 cells were transfected with ERE-luciferase only or with ERa, or with ERa and siCLOCK constructs. Luciferase activity was measured either with or without pre-treatment of the cells with 10 nM E2 for 16 h. (g) MCF-7 and T47D cells were transfected with cyclin D1-luc only or together with ERa,orwithERa and siCLOCK constructs. The cells were treated without or with 10 nM E2 for 16 h before subjecting to reporter assay. (h) MCF-7 and T47D cells were transfected with ERa or ERa and siCLOCK constructs and then subjected to serum starvation for 48 h, followed by E2 or no E2 treatment for 4 h before analysis of cyclin D1 expression by real-time PCR. Cyclin D1 mRNA level was normalized with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level. **Po0.01 and *Po0.05 compared with cells transfected with ERa only. Each bar represents the mean±s.d. from three independent experiments.

& 2013 Macmillan Publishers Limited Oncogene (2013) 4883 – 4891 Sumoylation in CLOCK regulates ERa’s activity SLiet al 4886

Figure 2. Sumoylation of CLOCK. (a) MCF-7 cells were treated as described for Figure 1a, and then subjected to immunoprecipitation with anti-CLOCK antibody followed by western blot analysis with anti-SUMO-1 antibody. (b) Extract from COS-7 cells expressing Flag-tagged CLOCK and Myc-tagged SUMO-1 were immunoprecipitated with anti-Flag antibody or control IgG, and then probed with anti-Myc antibody (top). The same membrane was washed and then probed with anti-Flag antibody (bottom). (c) Schematic representation of the primary structures of CLOCK and the predicted sumoylation sites along with the structures of SRC-1 and SRC-2 showing all the known sumoylation sites. bHLH/PAS, a basic helix-loop-helix/Per-Arnt-Sim domain; NRID, a nuclear receptor interaction domain; Q-rich, a glutamine-rich region containing a poly- glutamine stretch. (d) COS-7 cells were transfected with Flag-tagged wild-type or mutant CLOCK (K67R, K851R or 2K/2R) and Myc-SUMO-1, and individual cell extracts were subjected to western blot with anti-Flag or anti-Myc antibody. (e) COS-7 cells were transfected with GAL4-luc reporter and wild-type or mutant CLOCK (K67R, K851R or 2K/2R) that had been fused to GAL4-DBD (200 ng). For control, cells were transfected with GAL4-luc reporter and GAL4-DBD vector only. Cells were collected and luciferase activity was measured 24 h after transfection. Each bar represents the mean±s.d. from three independent experiments. *Po0.05 compared with cells transfected with GAL4-DBD-CLOCK.

GAL4-DBD-CLOCK and either one of the three Flag-tagged SENPs, sumoylation of CLOCK (Figures 1a and 2a). This suggested that and the level of luciferase activity was measured. Only COS-7 cells the interaction between CLOCK and ERa may also be governed by co-transfected with CLOCK and SENP1 showed reduced level of the sumoylation of CLOCK. To demonstrate this, MCF-7 cells were reporter activity, about 50% compared with cells transfected with transfected with Flag-tagged wild-type or the double mutant CLOCK alone (Figure 3a). In contrast, the level of reporter activity CLOCK 2K/2R, followed by immunoprecipitation and western blot in COS-7 cells co-transfected with CLOCK and either SEPN2 or analysis performed with anti-Flag antibody and anti-ERa antibody, SENP3 was similar to that of cells transfected with CLOCK alone. respectively. ERa was detected only in the extract of cells Thus, only SENP1 was able to reverse the sumoylation of CLOCK overexpressing wild-type CLOCK, whereas cells overexpressing through its desumoylation activity. Mutant SENP1 (R630L/K631M), 2K/2R showed no detectable level of ERa (Figure 4a), demonstrat- which lost its catalytic activity had no effect on the sumoylation of ing the impact of sumoylation on the interaction between CLOCK CLOCK. In all cases, the levels of CLOCK were similar as shown by and ERa, which was consistent with the fact that sumoylation of western blot, consistent with the reduction of reporter activity CLOCK promoted its interaction with ERa. In addition, the effect of being caused by the action of SENP1, which reflected the CLOCK sumoylation on the interaction between CLOCK and ERa sumolyation status of CLOCK. In addition, the reduction of was also examined using nuclear and cytosolic extracts prepared luciferase activity caused by SENP1 occurred in a dosage- from MCF-7 cells that had been treated without and with E2 for dependent manner (Figure 3b). The amount of sumoylated CLOCK 30 min. The two extracts were first subjected to immunoprecipita- in cells transfected with wild-type SENP1 was reduced compared tion using anti-ERa antibody, and then to re-immunoprecipitation with that transfected with a mutant SENP1 (Figure 3c), and this using anti-CLOCK antibody, followed by western blot with anti- provided further support for the SENP1-mediated inhibition of SUMO-1 antibody. As shown in Figure 4b, a single band was CLOCK transcriptional activity through desumoylation. detected by anti-SUMO-1 antibody, and only for the nuclear extract. This band was attributed to SUMO–CLOCK, which appeared to be much more intense after the cells had been Sumoylation of CLOCK influences its interaction with ERa treated with E2. This result suggested that interaction between In the aforementioned experiment, treatment of MCF-7 cells with CLOCK and ERa was restricted to the nucleus, and further E2 promoted the association of CLOCK with ERa as well as the confirmed the promotion of CLOCK sumoylation by estrogen.

Oncogene (2013) 4883 – 4891 & 2013 Macmillan Publishers Limited Sumoylation in CLOCK regulates ERa’s activity SLiet al 4887

Figure 4. Sumoylation of CLOCK influences its interaction with ERa. (a) MCF-7 cells were transfected with Flag-tagged wild-type or mutant CLOCK (2K/2R) for 24 h. The cells were starved of serum for 48 h, and then treated with or without 10 nM E2 for 30 min. The proteins of the cell extracts were immunoprecipitated by anti-Flag antibody and then probed with anti-ERa antibody. (b) MCF-7 cells were treated as above (Figure 1a). Cytosolic and nuclear fractions of the cell extract were prepared and subjected to immunoprecipita- tion with anti-ERa antibody and then to re-immunoprecipitation (Re-IP) with anti-CLOCK antibody, followed by western blot analysis with anti-SUMO-1 antibody.

endogenous CLOCK activity (that was associated with ERa)by mutant CLOCK, which appeared to interfere the transcriptional activity of ERa. Nevertheless, the result did show that activation of ER-mediated signaling by CLOCK closely associated with the sumoylation of CLOCK. Cyclin D1 is known to promote the proliferation of MCF-7 Figure 3. Repression of CLOCK transcriptional activity by desumoy- cells.36 MCF-7 cells co-transfected with ERa, cyclin D1-luciferase lation. (a) COS-7 cells were transfected with GAL4-DBD-CLOCK and wild-type CLOCK showed the highest level of luciferase (200 ng) or GAL4-DBD and GAL4-luc reporter construct together with Flag-SENP1, Flag-SENP1 mut, Flag-SENP2 or Flag-SENP3. Cells activity in the presence of E2 treatment (Figure 5b). Furthermore, were collected and luciferase activity was measured 24 h after the double mutant CLOCK appeared to have no effect on cyclin- transfection. (b) Similar experiments as described in a were D1-luciferase activity, either with or without E2 treatment. Similar performed but with increasing dosages of Flag-SENP1 or Flag- result was also obtained for real-time PCR analysis, where a higher SENP1 mut (20, 100 and 300 ng). *Po0.05 and **Po0.01 compared level of cyclin D1 mRNA was observed for cells transfected with with cells transfected with GAL4-DBD-CLOCK only. Each bar wild-type CLOCK than the double mutant CLOCK in the presence represents the mean±s.d. from three independent experiments. of E2 treatment (Figure 5c). In all three experiments, no difference (c) COS-7 cells were transfected with Flag-CLOCK, SUMO-1, SENP1 or was detected among the cells overexpressing ERa only or together SENP1 mut, followed by western blot with anti-Flag antibody. with wild-type or mutant CLOCK in the absence of E2 treatment.

Sumoylation of CLOCK has a role in ER signaling Effect of CLOCK sumoylation on MCF-7 cell proliferation Sumoylation of CLOCK regulated its transcriptional activity and its The effect exerted by sumoylation of CLOCK on the growth of association with ERa. To determine whether sumoylation of MCF-7 and T47D cells was examined. Consistent with higher CLOCK also affects the transcriptional activity of ERa, MCF-7 cells transcription levels of cyclin D1, cells overexpressed wild-type were co-transfected with ERE-luciferase and ERa only, or ERE- CLOCK showed more growth than cells that overexpressed the luciferase, ERa and wild-type or mutant CLOCK, and the levels of double mutant CLOCK 2K/2R in the presence of E2 treatment reporter activity for these cells were determined after subjecting (Figure 6a), suggesting that the proliferation of these cells was to E2 or no E2 treatment. As expected, luciferase activity was subjected to regulation by CLOCK-modulated transcription factors, highest for E2-treated cells that overexpressed both ERa and wild- including ERa, whose activity is known to influence the prolifera- type CLOCK (Figure 5a). Overexpression of the double mutant tion of breast cancer cells. Furthermore, the regulation of these CLOCK and ERa resulted in as much as 35% drop in reporter transcription factors by CLOCK was also dependent on activity compared with overexpression of ERa alone in the the sumoylation status of CLOCK. To further examine whether presence of E2 treatment, and this may due to interference of the effect of the sumoylation status of CLOCK on the proliferation

& 2013 Macmillan Publishers Limited Oncogene (2013) 4883 – 4891 Sumoylation in CLOCK regulates ERa’s activity SLiet al 4888 through regulating other transcription factors, but also had a direct effect on the cell cycle by maintaining a greater percentage of the cell in the S phase.

DISCUSSION Although more and more studies have indicated a connection between the disruption of the circadian rhythm and tumorigen- esis, especially hormone-related tumorigenesis such as the development of breast cancer, it is not known whether CLOCK is directly involved in the modulation of such process. CLOCK works as a core transcription factor that has a crucial role in the regulation of circadian rhythm. However, CLOCK also regulates the expression of several non-circadian proteins, such as AhR and Cyp1a1.23,37 In this study, we demonstrated that in the human breast cancer cell line, CLOCK participated in the estrogen signaling pathway through acting as a regulator of ERa, and that this interaction between CLOCK and ERa was stimulated by E2. A possible mechanism whereby CLOCK may regulate ERa is through enhancing ERa-mediated transactivation activity, and E2 appeared to reinforce this effect. In estrogen-mediated signaling, E2 exerts its effects through the ERs, ERa and ERb. Cai et al.38 identified a novel way in which estrogen signaling is regulated by demonstrating that ERb is subjected to control by circadian clock proteins, and that this regulation is important for determining the cellular response to estrogens. Our data showed that the transcriptional activity of CLOCK also responded to the presence of estrogen, and it is reasonable to suggest that CLOCK is also involved in the estrogen signaling pathway through interacting with ERa. In MCF-7 cells, stimulation with E2 can promote this interaction within 30 min (Figure 1a). According to the classical estrogen pathway, E2 induces the formation of a classical genomic ER–ERE complex that then activates the expressions of estrogen-dependent genes. In this process, ER recruits several transcription cofactors, such as members of the p160 family, CARM1 and p300 to regulate the expression of downstream genes.39–41 BMAL1 may also take part in this process, as coexpression of CLOCK and BMAL1 promoted this activation effect (data not shown). Many proteins involved in transcription are subjected to sumoylation, which either activate or inhibit their activities, or change their subcellular locations, and these will in turn lead to changes in the statuses of the proteins they regulate.42,43 In this Figure 5. Sumoylation of CLOCK upregulates the transcriptional study, we showed that CLOCK can be sumoylated, and identified activities of ERa and its downstream target gene. (a) MCF-7 cells two sumoylation sites, K67 and K851. Sumoylation of CLOCK was were transfected with ERE-luciferase or with ERa, or with ERa and promoted by E2 treatment. Sumoylation of CLOCK was important Flag-tagged wild-type or mutant CLOCK (2K/2R). Luciferase activity was measured either with or without pre-treatment of the cells with for its regulation of ERa transcriptional activity, as MCF-7 cells 10 nM E2 for 16 h. (b) MCF-7 cells were transfected with cyclin D1-luc overexpressing the double mutant CLOCK 2K/2R (which had lost or with ERa, or with ERa and Flag-tagged wild-type or mutant both sumoylation sites) had reduced level of ERa-controlled CLOCK (2K/2R). The cells were treated without or with 10 nM E2 for reporter gene activity compared with MCF-7 cells overexpressing 16 h before subjecting to reporter assay. Each bar represents the wild-type CLOCK (Figure 5a). The expression of either wild-type mean±s.d. from three independent experiments. (c) MCF-7 cells CLOCK or 2K/2R did not change the overall expression of ERa were transfected with ERa only or ERa with Flag-tagged wild-type or (data not shown). Therefore, the observed enhancement in ERa mutant CLOCK, and then subjected to serum starvation for 48 h, activity was not due to increased level of ERa protein, and may followed by E2 or no E2 treatment for 4 h before analysis of cyclin D1 possibly be the result of the interaction that occurred between expression by real-time PCR and normalized with a glyceraldehyde ERa and CLOCK, especially sumoylated CLOCK, the status of which 3-phosphate dehydrogenase (GAPDH) mRNA level. *Po0.05 and **Po0.01 compared with cells transfected with ERa only. was also confirmed by SENP1, which effectively reversed the sumoylation process. The attachment of SUMO to a protein often causes a change in MCF-7 cells would manifest through its effect on the cell cycle, the conformation of the protein, thereby affecting its association cells transfected with either wild-type or mutant CLOCK 2K/2R with other proteins. The sumoylation sites of CLOCK were located were subjected to flow cytometry analysis to evaluate the cell in the bHLH/PAS (K67) and C-terminal (K851) regions. Analysis of cycle profile of asynchronous cells. Overexpression of wild-type the primary structure of CLOCK in different species of organisms CLOCK resulted in an overall increase in the percentage of S phase showed that K67 and K851 are conserved residues. SRC-1, a classic cells, with a corresponding reduction in the percentage of cells in coactivator of ER and , is sumoylated at G0/G1 phase compared with control cells (Figure 6b). Over- K732 and K774, and both sites are located in the protein–protein expression of 2K/2R had little effect on the cell cycle. Thus, interaction domain. SRC-1 that has lost both sumoylation sites has sumoylation of CLOCK not only increased cell proliferation markedly reduced interaction with progesterone receptor,12

Oncogene (2013) 4883 – 4891 & 2013 Macmillan Publishers Limited Sumoylation in CLOCK regulates ERa’s activity SLiet al 4889

Figure 6. Sumoylation of CLOCK promotes the growth of breast cancer cell lines. (a) MCF-7 and T47D cells were transfected with Flag-tagged wild-type or mutant CLOCK, and then treated without or with 10 nM E2 for the indicated . The cells were then subjected to 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay performed according to the manufacturer’s instructions (Key Gen). *Po0.05 compared with cells transfected with control vector. (b) MCF-7 cells were transfected with control vector, Flag-tagged wild-type or mutant CLOCK. Flow cytometry analysis of cell-cycle distribution of MCF-7 cells after 36 h of growth in the presence of 10 nM E2.

suggesting that the extent of sumoylation of SRC-1 has a role in It appears that the expression of CLOCK is positively correlated receptor–coactivator interaction. As for SRC-2, one of its sumoyla- with the malignancy of breast cancer. In this study, the tion sites, K239, is also located in the bHLH/PAS domain, and transcriptional activity of ERa and the growth of the two breast mutant form of SRC-2 in which this K239 has been replaced with cancer cells, MCF-7 and T47D, promoted by CLOCK were E2 arginine has decreased transactivation activity.10 Similarly, K67 of dependent, as cell lines are ERa-positive cell lines. This may CLOCK is located in the region that mediates CLOCK’s association suggest that stimulation of ERa by CLOCK may represent an with other proteins, such as BMAL1, PER1 and CRY1, and this important mechanism for the growth of ER-positive breast cancer. interaction may be affected by the loss of sumoylation at K67 as As for the higher level of CLOCK observed for ERa-negative tumors demonstrated by the loss of transcriptional activity for the K67R previously reported,16 there may exist another unknown pathway mutant. K851 lies almost at the C-terminal end of CLOCK, similar to participated by CLOCK, which has yet to be determined. the location of K1378 of SRC-1 (see Figure 2c). K1378 is another A number of tumors related to the action of hormone are sumoylation site of SRC-1, and sumoylation of SRC-1 at this site closely associated with a disorder of circadian rhythm. Although also enhances its transactivation activity.12 Although sumoylation we have shown here that CLOCK can work as a coactivator in ER at K851 of CLOCK also exerted a moderate effect on the activity of signaling pathway and sumoylation of CLOCK had crucial roles in CLOCK, and hence, on the interaction between CLOCK with ERa this progress, the exact mechanism pertaining to the nature of the and possibly other proteins, it is unlikely that protein–protein circadian rhythm is unknown. Elucidating the relationship interaction is a key factor controlled by sumoylation at this site. between clock proteins and ER network will further uncover the Sumoylation of CLOCK altered the level of ERa transcriptional molecular mechanism between circadian disruption and breast activity probably through enhancing its binding with ERa cancer, and this may provide better insight for conceiving a way to (Figure 4a). In the case of cyclin D1 expression, wild-type CLOCK combat cancers based on the manipulation of CLOCK–ERa- increased its expression by 40% in the presence of E2 treatment, mediated signaling pathway. but no change was observed for the mutant CLOCK 2K/2R (Figure 5b). These data suggested that change in the expression of cyclin D1 was induced through ERa, which can be regulated by sumoylated CLOCK. However, whether sumoylation of CLOCK has MATERIALS AND METHODS a direct effect on the expression of cyclin D1 or other proteins Plasmids and antibodies involved in E2 signaling pathway remain to be determined. Rabbit polyclonal anti-Gal4-DBD, anti-Flag, anti-CLOCK, anti-ERa and anti- ER-positive breast cancers generally have a better prognosis Myc antibodies and mouse monoclonal anti-Gal4-DBD and anti-SUMO-1 and are often responsive to anti-estrogen therapy, which is the antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat polyclonal anti-CLOCK antibody was obtained from Santa Cruz first example of a successful therapy that targets a nuclear Biotechnology. Mouse monoclonal anti-Flag antibody (M2) was purchased receptor, ER.44 Unfortunately ER-negative breast cancers are more 45 16 from Sigma (St Louis, MO, USA). aggressive and unresponsive to anti-estrogens. Hoffman et al. CLOCK mutants (K67R, K851R and K67R/K851R) were generated using a found that patients with ER-negative tumors had even higher site-directed mutagenesis kit according to the manufacturer’s instructions levels of CLOCK expression than patients with ER-positive tumors. (Stratagene, La Jolla, CA, USA). Flag-tagged CLOCK (wild type and mutants)

& 2013 Macmillan Publishers Limited Oncogene (2013) 4883 – 4891 Sumoylation in CLOCK regulates ERa’s activity SLiet al 4890 were amplified by PCR and subcloned into 3 Â Flag-pCMV10 vector at the All statistical analyses of data were preformed with analysis of variance HindIII and BamHI sites. method. Data are given as mean±s.d., and significance was considered at either P-value o0.05 or 0.01 level. Cell culture, reporter assays and small interfering RNA constructs The human breast cancer cell line MCF-7 and African green monkey kidney cell line COS-7 had been used in our previous study.11 They were cultured CONFLICT OF INTEREST in Dulbecco’s modified Eagle’s medium (Invitrogen, Auckland, New The authors declare no conflict of interest. Zeeland) containing 10% fetal bovine serum (Hyclone, Beijing, China). As for T47D cells, it was maintained in RPMI-1640 medium (Invitrogen) supplemented with 0.2 U/ml insulin and 10% fetal bovine serum. Cells ACKNOWLEDGEMENTS were plated in 24-well plates and transfected with the appropriate plasmid We are grateful to Dr Miche`le Teboul (Universite´ de Nice-Sophia Antipolis) and Dr Urs construct using Lipofectamine 2000 (Invitrogen) according to the Albrecht (University of Fribourg) for their present of mouse CLOCK and GAL4-DBD- manufacturer’s specification. Five after transfection, the medium CLOCK constructs. This work was supported by grants (31171353, 31271500 to HW) was replaced with phenol red-free medium containing 10% charcoal– from National Natural Science Foundation of China and grants (973 program: dextran-treated fetal bovine serum for 48 h, followed by treatment with or 2011CB504201 to HW) from the Ministry of Science and Technology of China. without 10 nM 17b-estradiol (E2) for 16 h. Cells were then subjected to luciferase and Renilla activity assays using a dual luciferase kit (Promega, Madison, WI, USA). 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