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 receptor-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 circadian rhythm is now believed to associate with a number of hormone-related cancers, such as breast cancer, in which aberrant estrogen receptor-a (ERa) signaling is a major contributor. However, the molecular mechanisms underlying the function of core clock proteins in cancer are still largely undefined. In this study, we showed that circadian locomotor output cycles kaput (CLOCK), a key circadian protein, 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 cycle. The results of this study uncovered new insight into the connection between a major circadian protein and a major estrogen-dependent transcription factor, 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 gene 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 period (Per1 and dimer and bind to estrogen-responsive elements (EREs) in the Per2) genes 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 oscillation 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 histone 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 cryptochrome (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 communication has a profound effect on human 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 Science and Biotechnology, Dalian University of Technology, 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 present, 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, nuclear receptor 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 time, 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.