Oncogene (2013) 32, 1351–1362 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Interaction with Suv39H1 is critical for Snail-mediated E-cadherin repression in breast cancer C Dong1,2,YWu2,3, Y Wang1,2, C Wang2,4, T Kang5, PG Rychahou2,6, Y-I Chi1, BM Evers1,2,6 and BP Zhou1,2 Expression of E-cadherin, a hallmark of epithelial–mesenchymal transition (EMT), is often lost due to promoter DNA methylation in basal-like breast cancer (BLBC), which contributes to the metastatic advantage of this disease; however, the underlying mechanism remains unclear. Here, we identified that Snail interacted with Suv39H1 (suppressor of variegation 3-9 homolog 1), a major methyltransferase responsible for H3K9me3 that intimately links to DNA methylation. We demonstrated that the SNAG domain of Snail and the SET domain of Suv39H1 were required for their mutual interactions. We found that H3K9me3 and DNA methylation on the E-cadherin promoter were higher in BLBC cell lines. We showed that Snail interacted with Suv39H1 and recruited it to the E-cadherin promoter for transcriptional repression. Knockdown of Suv39H1 restored E-cadherin expression by blocking H3K9me3 and DNA methylation and resulted in the inhibition of cell migration, invasion and metastasis of BLBC. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT, but also paves a way for the development of new treatment strategies against this disease. Oncogene (2013) 32, 1351–1362; doi:10.1038/onc.2012.169; published online 7 May 2012 Keywords: metastasis; EMT; chromatin modifications; transcription; Snail INTRODUCTION plasticity of EMT is determined by a unique gene-expression pattern Breast cancer is the second leading cause of cancer-related death through transcription factor-mediated chromatin modifications, in women with a yearly toll of 440 000 deaths in the United which mainly consist of histone modifications and DNA methyla- States alone.1 Despite exciting progress towards understanding tion.16,17 Post-translational modifications at the N-termini of breast cancer development and progression, and in the histones, including phosphorylation, acetylation, methylation and development of novel therapeutic strategies, breast cancer- ubiquitination, alter chromatin structure to control DNA accessibility. related deaths are primarily due to the ‘incurable’ nature of These modifications can function individually or in combination to metastasis. Therefore, novel therapeutic strategies are required elicit specific effects based on the changes in chromatin structure or to prevent and treat metastatic breast cancer. A better the recruitment of effector proteins that bind to specific patterns of understanding of the molecular events that contribute to tumor histone marks. Histone lysine methylation, in particular, has emerged invasion and metastasis is crucial to the development of such as an important modification due to its central role in transcriptional strategies. regulation. For example, methylated lysines 4, 36 and 79 on H3 The increased motility and invasiveness of metastatic tumor (denoted as H3K4, H3K36 and H3K79) are found in active chromatin, cells are reminiscent of the events that occur at epithelial– whereas methylated H3K9 and H3K27 are generally associated with mesenchymal transition (EMT), which is a characteristic of gene repression. embryonic development, tissue remodeling, wound healing and Loss of H3K4 methylation and gain of H3K9 methylation appear metastasis.2–4 In these EMT processes, epithelial cells acquire most closely associated with DNA methylation,18–21 which fibroblast-like properties and exhibit reduced intercellular provides reinforcement as well as establishment of gene adhesion and increased motility.5–7 EMT also bestows tumor cells silencing. DNA methylation is executed by a family of highly with cancer stem cell-like characters, provides them with resistance related DNA methyltransferase enzymes (DNMT1, DNMT3a and to immunosuppression and confers tumor recurrence.8–11 Two DNMT3b) that transfer a methyl group to the cytosine in a CpG distinct phenomena are commonly associated with EMT. First, EMT dinucleotide which commonly occurs in the promoter region of is provoked by extrinsic signals that cells receive from their genes.22,23 Interestingly, promoter DNA methylation is the microenvironment,4,12 such as signals from tumor growth factor b common cause of loss of E-cadherin expression in basal-like (TGF-b), tumor necrosis factor-a and growth factors.13–15 Asecond breast cancer (BLBC, commonly referred to as triple-negative phenomenon associated with EMT is that it is a reversible process.4 breast cancer), a subtype of breast cancer possesses many EMT When breast cancer cells disseminate to distant sites of the body, characteristics and characterized by an aggressive, metastasis- they no longer encounter the signals that they experienced in the prone phenotype that associates with poor clinical outcome.24–28 primary tumor, and they can revert to an epithelial state via a A hallmark of EMT is the loss of E-cadherin expression.6,29 Snail is mesenchymal–epithelial transition. The phenotypic and cellular the key transcriptional repressor for downregulating E-cadherin in 1Department of Molecular and Cellular Biochemistry, The University of Kentucky College of Medicine, Lexington, KY, USA; 2Markey Cancer Center, The University of Kentucky College of Medicine, Lexington, KY, USA; 3Department of Molecular and Biomedical Pharmacology, The University of Kentucky College of Medicine, Lexington, KY, USA; 4Department of Biostatistics, The University of Kentucky College of Medicine, Lexington, KY, USA; 5State Key Laboratory of Oncology in South China, Guangzhou, China and 6Department of Surgery, The University of Kentucky College of Medicine, Lexington, KY, USA. Correspondence: Dr BP Zhou, Department of Molecular and Cellular Biochemistry, Markey Cancer Center, The University of Kentucky College of Medicine, BBSRB Room B336, 741 South Limestone, Lexington, KY 40506-0509, USA. E-mail: [email protected] Received 14 February 2012; revised 26 March 2012; accepted 2 April 2012; published online 7 May 2012 E-cadherin repression mediated by Snail and Suv39H1 C Dong et al 1352 EMT.5,6,29 Although Snail is highly expressed in BLBC for repressing We previously showed that the SNAG domain adopted a E-cadherin expression, the sequential event and the molecular conformation similar to that of the histone H3 tail for recruiting mechanism leading to DNA methylation at the E-cadherin the chromatin modifying enzyme LSD1.30 Because Suv39H1 was promoter in BLBC remain not well characterized. In our previous identified as a protein associated with the SNAG peptide of Snail, study, we found that Snail interacted with lysine-specific we performed a co-immunoprecipitation experiment to assess demethylase 1 (LSD1) and recruited LSD1 to the E-cadherin whether the SNAG domain of Snail is required for the interaction promoter to specifically remove H3K4 methylation.30 However, with Suv39H1. We found that deletion of the SNAG domain H3K4 demethylation is known to be an initial step in gene significantly reduced the interaction of Snail with Suv39H1, repression,31 suggesting that an intermediate step is required to indicating that the SNAG domain is required for the interaction bridge H3K4 demethylation to the DNA methylation on the of Snail with Suv39H1 (Figure 2a). We noticed that the sequence of E-cadherin promoter. Here, we showed that Snail interacted with the SNAG domain is highly similar to that of the N-terminus of Suv39H1 (suppressor of variegation 3-9 homolog 1), a key histone H3 that surrounds the lysine 9 residue, a substrate of component of methyltransferase responsible for H3K9me3. We Suv39H1 (Figure 2b). To identify the critical residues within the reported that this functional interaction is critical for H3K9me3 SNAG domain required for interaction with Suv39H1, we and DNA methylation at the E-cadherin promoter in BLBC. performed alanine-scan mutagenesis on the SNAG domain of Snail (Figure 2c). Among the 12 Snail mutants screened, we found that mutations at Phe5, Lys9 and Ser11 completely lost their ability RESULTS to interact with Suv39H1. The loss of interaction of these mutants The SNAG domain of Snail interacts with the catalytic domain of with Suv39H1 was not due to the variation of protein stability as Suv39H1 about equal amounts of Snail was immunoprecipitated (input Several transcription factors, such as Snail, Slug, GFI, insulinoma- lysates on Figure 2c). Together, these results indicate that the associated protein IA-1 (Insm1) and Ovo-like 1 (OVOL1), contain an SNAG domain, particularly several key amino-acid residues SNAG (Snail/GFI) domain at the N-terminus, which is important for surrounding lysine 9, was required for the interaction of Snail their repressive activity in mammalian cells.32 We previously with Suv39H1. demonstrated that the SNAG domain adopted a conformation Suv39H1 contains several functional domains, including an similar to that of the histone H3 tail for interaction with LSD1,30 N-terminal bromo domain, a catalytic region containing a Pre-SET, suggesting that this domain is critical in recruiting chromatin SET and Post-SET domain that is responsible for its enzymatic repressive complexes for gene silencing. To further identify the methyltransferase activity (top panel, Figure 2d). To identify the protein complex associated with the SNAG domain of Snail, we region responsible