Title: The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1
Abstract: Article13 April 2010free access The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1 Yiwei Lin Yiwei Lin Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Yadi Wu Yadi Wu Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Departments of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Junlin Li Junlin Li Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Chenfang Dong Chenfang Dong Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Xiaofeng Ye Xiaofeng Ye Department of Thoracic/Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Young-In Chi Young-In Chi Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author B Mark Evers B Mark Evers Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Department of Surgery, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Binhua P Zhou Corresponding Author Binhua P Zhou Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Yiwei Lin Yiwei Lin Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Yadi Wu Yadi Wu Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Departments of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Junlin Li Junlin Li Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Chenfang Dong Chenfang Dong Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Xiaofeng Ye Xiaofeng Ye Department of Thoracic/Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Young-In Chi Young-In Chi Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author B Mark Evers B Mark Evers Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Department of Surgery, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Binhua P Zhou Corresponding Author Binhua P Zhou Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Author Information Yiwei Lin1,2,‡, Yadi Wu2,3,‡, Junlin Li1,2, Chenfang Dong1,2, Xiaofeng Ye4, Young-In Chi1, B Mark Evers2,5 and Binhua P Zhou 1,2 1Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA 2Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA 3Departments of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, Lexington, KY, USA 4Department of Thoracic/Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA 5Department of Surgery, University of Kentucky College of Medicine, Lexington, KY, USA ‡These authors contributed equally to this work *Corresponding author. Departments of Molecular and Cellular Biochemistry, University of Kentucky School of Medicine, 741 South Limestone, Lexington, KY 40506, USA. Tel.: +859 323 4474; Fax: +859 257 6030; E-mail: [email protected] The EMBO Journal (2010)29:1803-1816https://doi.org/10.1038/emboj.2010.63 There is a Have you seen ...? (June 2010) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Epithelial–mesenchymal transition (EMT) is a transdifferentiation programme. The mechanism underlying the epigenetic regulation of EMT remains unclear. In this study, we identified that Snail1 interacted with histone lysine-specific demethylase 1 (LSD1). We demonstrated that the SNAG domain of Snail1 and the amine oxidase domain of LSD1 were required for their mutual interaction. Interestingly, the sequence of the SNAG domain is similar to that of the histone H3 tail, and the interaction of Snail1 with LSD1 can be blocked by LSD1 enzymatic inhibitors and a histone H3 peptide. We found that the formation of a Snail1–LSD1–CoREST ternary complex was critical for the stability and function of these proteins. The co-expression of these molecules was found in cancer cell lines and breast tumour specimens. Furthermore, we showed that the SNAG domain of Snail1 was critical for recruiting LSD1 to its target gene promoters and resulted in suppression of cell migration and invasion. Our study suggests that the SNAG domain of Snail1 resembles a histone H3-like structure and functions as a molecular hook for recruiting LSD1 to repress gene expression in metastasis. Introduction The increased motility and invasiveness of tumour cells are reminiscent of the processes at the epithelial–mesenchymal transition (EMT), which is essential for morphogenetic events in embryonic development, tissue remodelling, wound healing and metastasis (Wu and Zhou, 2008; Yang and Weinberg, 2008; Thiery et al, 2009). In these EMT processes, epithelial cells acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility. These acquired traits confer cancer cells with the distinct advantages of invasion, metastatic dissemination and acquisition of drug resistance (Wu and Zhou, 2008; Yang and Weinberg, 2008; Thiery et al, 2009). Interestingly, EMT is a reversible process (Thiery, 2002; Weinberg, 2008). When cancer cells disseminate to distant sites in the body, they no longer encounter the signals that they experienced in the primary tumour and they can revert to an epithelial state by a mesenchymal–epithelial transition (MET). The phenotypic plasticity of cancer cells at EMT/MET suggests that an epigenetically regulated differentiation programme is involved (Feinberg, 2007). In the eukaryotic nucleus, DNA is packaged with core histones and other chromosomal proteins to form chromatin (Bernstein et al, 2007; Downs et al, 2007; Fraser and Bickmore, 2007). The basic repeating unit of chromatin, nucleosome, is composed of two copies of each of the four core histones, H2A, H2B, H3 and H4, wrapped by 146 base pairs of DNA (Bernstein et al, 2007; Downs et al, 2007; Fraser and Bickmore, 2007). Chromatin harbours not only genetic information encoded in the DNA but also epigenetic information carried by the reversible covalent modifications at the N-terminal tails of histones (Esteller, 2007). Histone modifications, such as acetylation, phosphorylation and methylation, control gene expression by altering chromatin structure (Berger, 2007; Bhaumik et al, 2007; Ruthenburg et al, 2007). Methylation at specific residues of histone H3, in particular, has a major role in the maintenance of the active or silent state of gene expression (Martin and Zhang, 2005; Bhaumik et al, 2007). For example, methylated lysine 9 or lysine 27 on histone H3 (denoted as H3K9 and H3K27) is generally associated with genes the transcription of which is repressed, whereas methylated H3K4, H3K36 and H3K79 are found in active chromatin. Although histone methylation was long considered irreversible, the recent identification of several site-specific histone demethylases provides compelling evidence that this modification is dynamically regulated (Klose and Zhang, 2007; Shi and Whetstine, 2007; Cloos et al, 2008). The reversible methylation of histones allows gene expression to be rapidly switched on at differentiation, while retaining cellular plasticity in response to developmental and microenvironmental signals (Reik, 2007). The mechanism underlying the epigenetic regulation of EMT at metastasis remains unclear, and thus, an understanding of this process will provide important insight into the early steps of cancer metastasis. A hallmark of EMT is the loss of E-cadherin expression (Nieto, 2002; Thiery, 2002), which is often inversely correlated with tumour grade and stage (Cowin et al, 2005). Several transcription factors, such as Snail1, Twist and ZEB1, have been implicated in the transcriptional repression of E-cadherin and in the induction of EMT (Nieto, 2002; Thiery and Sleeman, 2006; Peinado et al, 2007; Yang and Weinberg, 2008). Snail1, a DNA-binding factor, which was identified in Drosophila as a suppressor of the transcription of shotgun (an E-cadherin homologue), controls large-scale cell movement during formation of the mesoderm and neural crest (Nieto, 2002). Expression of Snail1 suppresses E-cadherin expression and induces EMT in MDCK and breast cancer cells, indicating that Snail1 has a fundamental role in EMT and breast cancer metastasis (Batlle et al, 2000; Cano et al, 2000; Zhou et al, 2004). Our findings, and those of others, clearly show that expression of Snail1 correlates with high tumour grade and nodal metastasis and is predictive of a poor outcome in patients with breast cancer (Cheng et al, 2001; Blanco et al, 2002; Zhou et al, 2004; Martin et al, 2005). Besides being a critical regulator of EMT, Snail1, when overexpressed, induces resistance to apoptosis and tumour recurrence in breast cancer (Kajita et al, 2004; Vega et al, 2004; Moody et al, 2005). To better understand the mechanism underlying the epigenetic regulation of EMT at metastasis, we undertook the unbiased approach of tandem array purification (TAP) coupled with mass spectrometry analysis to identify the chromatin-modifying enzymes that interact with Snail1. We found that Snail1 interacted with lysine-specific demethylase 1 (LSD1), a key component of several co-repressor complexes including CoREST, CtBP and HDAC1/2, which remove the methylation of H3K4 (Shi, 2007). In this paper, we characterize the functional interaction of Snail1 with LSD1 and investigate their roles in mediating chromatin modification during metastasis. Results Snail1 interacts with LSD1 through the SNAG domain To identify the potential proteins that interact with Snail1, we generated a stable HEK293 cell line expressing dual-tagged Snail1 (Figure 1A). After enriching the nuclear extracts, we carried out a two-step sequential protein purification process with Flag and hemagglutinin (HA) affinity columns (Shi et al, 2003). The final immunocomplexes were separated on SDS–PAGE and subjected to silver staining (Figure 1A). Bound proteins were excised from the gel and subjected to mass spectrometry analysis. Three known proteins that interact with Snail1, GSK-3β, β-Trcp and PRMT5 (Zhou et al, 2004; Hou et al, 2008), were found in the complexes, which validated the specificity of this system. Lysine-specific demethylase 1 (Shi, 2007), an H3K4 demethylase, was also identified as a protein that associated with Snail1. Six trypsin-digested peptides from a protein with a molecular weight of about 110 kDa were perfectly matched to the protein sequence of LSD1 (Figure 1A). Figure 1.Snail1 interacts with LSD1 through the SNAG domain. (A) The schematic diagram shows the stable expression of dual-tagged Snail1 in HEK293 cells (top and middle panels). The Snail1 complex was isolated by two-step immunopurification, separated on SDS–PAGE and visualized by silver staining. A protein with molecular weight close to 110 kDa was excised and identified as LSD1 by mass spectrometry (bottom panel). (B) Flag-tagged LSD1 and HA-tagged wild-type or SNAG-deleted Snail1 were co-expressed in HEK293 cells. After immunoprecipitation, bound Snail1 or LSD1 was examined by western blotting. (C) Endogenous Snail1 and LSD1 were immunoprecipitated from PC3, HCT116, SKBR3 and MDA-MB231 cells and bound endogenous LSD1 and Snail1 were examined by western blotting. (D) GFP-tagged Snail1 was expressed in HEK293 cells. After fixation, the cellular localization of Snail1 (green) and LSD1 (red) was examined by immunofluorescent staining. Scale bar=50 μm. Download figure Download PowerPoint To validate the physical interaction of Snail1 with LSD1, we co-expressed Snail1–HA and Flag–LSD1 in HEK293 cells and conducted a co-immunoprecipitation experiment. After immunoprecipitating LSD1, we detected the associated Snail1, and vice versa (Figure 1B), indicating that these two molecules are associated. Interestingly, deletion of the SNAG (Snail1/GFI) domain (Batlle et al, 2000; Nieto, 2002; Peinado et al, 2004; Barrallo-Gimeno and Nieto, 2009), a highly conserved repressive domain present at the N-terminus of several transcription factors (such as Snail1 and GFI1), significantly reduced the interaction of Snail1 with LSD1, indicating that the SNAG domain is required for this interaction (Figure 1B). We also immunoprecipitated endogenous Snail1 and LSD1 from PC3, HCT116, MDA-MB 231 and SKBR3 cells and detected the presence of endogenous LSD1 and Snail1, respectively (Figure 1C). Consistent with these data, when GFP–Snail1 was expressed in HEK293 cells, we found that Snail1 was co-localized with endogenous LSD1 in the nucleus (Figure 1D). Taken together, our results indicate that Snail1 interacts with LSD1 and that the SNAG domain of Snail1 is required for mediating their interaction. The SNAG domain is essential for the stability of Snail1 In addition to Snail1 and GFI1, several other transcription factors, such as Slug, Scratch, insulinoma-associated protein IA-1 (Insm1) and Ovo-like 1 (OVOL1), also contain a similar SNAG domain at their N-terminus (Figure 2A). This motif is highly conserved among species and is important for the repressive activity of these transcription factors in mammalian cells (Batlle et al, 2000; Nieto, 2002; Peinado et al, 2004; Barrallo-Gimeno and Nieto, 2009). We, along with others, previously showed that Snail1 is a highly unstable protein and is regulated by protein stability and subcellular localization (Dominguez et al, 2003; Zhou et al, 2004; Yook et al, 2005). To test whether the SNAG domain regulates the protein stability, subcellular localization and repressive activity of Snail1, we generated a Snail1 mutant with the SNAG domain deleted (ΔSNAG–Snail1) and a SNAG-fused destabilized d2-GFP (SNAG–d2-GFP; Figure 2B). Consistent with previous findings, deletion of the SNAG domain significantly reduced the repressive activity of Snail1 on the E-cadherin promoter, whereas adding the SNAG domain to d2-GFP had no effect (Figure 2B), suggesting that the SNAG domain is required but is not sufficient for the transcriptional repressive activity of Snail1. Interestingly, ΔSNAG-Snail1 became less stable than wild-type Snail1, whereas SNAG-d2-GFP became stabilized in comparison with d2-GFP (Figure 2C). The failure of ΔSNAG-Snail1 to suppress E-cadherin promoter activity (Figure 2B) was not due to the instability of this molecule, as treatment with MG132 did not enhance the suppressive function of this mutant (Supplementary Figure S1). To further extend this finding, we investigated the degradation of these proteins with treatment of the protein synthesis inhibitor, cycloheximide, for various time intervals. We found that the protein level of ΔSNAG–Snail1 significantly decreased after 1 h in comparison with that of wild-type Snail1 (Figure 2D). In contrast, SNAG–d2-GFP became more stable than d2-GFP (Figure 2E), indicating that the SNAG domain is critical for maintaining the protein stability of Snail1, in addition to its transcriptional repressive function. Figure 2.The SNAG domain is essential for the stability of Snail1. (A) Sequence alignment of the SNAG domain from several transcriptional repressors. The consensus sequence is shown in red and the lysine and arginine residues are highlighted in blue. (B) Scheme showing the SNAG constructs used in this study (top panel). WT or SNAG-deleted Snail1, d2-GFP or SNAG-d2-GFP was co-expressed with the E-cadherin promoter luciferase construct in MCF7 cells. After 48 h, luciferase activity was measured by using a Dual-Luciferase Reporter Assay (Promega) (mean±s.d. of three separate experiments; bottom panel). (C) WT and SNAG-deleted Snail1, d2-GFP and SNAG–d2-GFP were expressed in HEK293 cells and analysed by western blotting. (D) WT or SNAG-deleted Snail1 was expressed in HEK293 cells then treated with cycloheximide (10 μg/ml) for different time intervals. The level of Snail1 was analysed by western blotting. Densitometry results from three independent experiments were statistically analysed and plotted (bottom panel). A representative western blotting experiment is shown in the top panel. (E) d2-GFP or SNAG-fused d2-GFP was expressed in HEK293 cells and treated with cycloheximide as described above. The level of d2-GFP was analysed by western blotting. Densitometry results from three independent experiments were statistically analysed and plotted (bottom panel) and a representative blot is shown on the top panel. Download figure Download PowerPoint We next examined whether the SNAG domain affects the subcellular location of these proteins. Although the intensity of SNAG–d2-GFP was enhanced because of its increased stability, d2-GFP and SNAG–d2-GFP were distributed equally in the nucleus and cytoplasm (Supplementary Figure S2A). We also expressed wild-type Snail1– and ΔSNAG–Snail1–GFP in HEK293 cells and found that both were localized predominantly in the nucleus (Supplementary Figure S2B). Although ΔSNAG–Snail1 has significantly decreased protein stability, the loss of the SNAG domain did not affect its nuclear localization. We also performed subcellular fractionation analysis and found that the addition or deletion of the SNAG domain did not change the subcellular localization of d2-GFP or Snail1, respectively (Supplementary Figure S3). Taken together, our results indicate that the SNAG domain is critical in controlling the protein stability but not the subcellular location of Snail1. The SNAG domain of Snail1 interacts with LSD1 by mimicking the structure of the histone H3 tail We also expressed d2-GFP and SNAG–d2-GFP in HEK293 cells. Immunoprecipitation of SNAG–d2-GFP, but not of d2-GFP, revealed the association of endogenous LSD1, indicating that the SNAG domain is sufficient for Snail1 to interact with LSD1 (Figure 3A). Lysine-specific demethylase 1 is a demethylase for H3K4me2 (Shi, 2007). Although it can bind to the histone H3 tail for demethylation, it does not contain a DNA-binding motif. The manner in which LSD1 binds to a specific promoter chromatin remains unclear. The X-ray structure of the LSD1–CoREST–Histone H3 peptide complex has been determined (Forneris et al, 2007, 2008). Forneris et al (2008) demonstrated that residues of Arg2, Thr6, Arg8, Lys9 and Thr11 of histone H3 (highlighted with blue dots at the top of Figure 3B) are critical for establishing the contact interactions of histone H3 within the catalytic cavity of LSD1. We noticed that the sequence of the SNAG domain is highly similar to that of the N-terminus of histone H3 and contains arginine- and lysine-rich residues (Figure 3B). Interestingly, the SNAG domain of Snail1 contains almost identical residues at four of these five positions (Arg3, Arg8, Lys9 and Ser11). To identify the critical residues on the SNAG domain required for interaction with LSD1, we performed alanine scan mutagenesis on the SNAG domain of Snail1 (Figure 3C). Among the 15 Snail1 mutants screened, we found that mutations at Pro2, Arg3, Lys9 and Pro10 decreased the protein stability of Snail1 (Figure 3C; Supplementary Figure S4). However, treatment with proteasome inhibitor, MG132, restored the protein stability of these mutants (Supplementary Figure S4), indicating that these four residues are critical for controlling the protein stability of Snail1. This is consistent with the finding that the SNAG domain is important for the protein stability of Snail1 (Figure 2). Similar to the SNAG deletion mutant of Snail1, mutation of these four residues did not alter the nuclear localization of Snail1 (Figure 3D). Figure 3.The SNAG domain of Snail1 interacts with LSD1 by mimicking the structure of the tail of histone H3. (A) d2-GFP or SNAG–d2-GFP was expressed in HEK293 cells. After immunoprecipitation of d2-GFP, bound endogenous LSD1 was examined by western blotting. (B) The schematic diagram shows the sequence alignment of the SNAG domain with the histone H3 tail. The conserved sequence is highlighted in yellow and arginine and lysine resides are shown in red. Residues of Arg2, Thr6, Arg8, Lys9 and Thr11 of histone H3 (highlighted in blue dots on the top) are important for establishing the critical interaction of histone H3 within the catalytic cavity of LSD1 (Forneris et al, 2008). Triangles at the bottom represent the residues on the SNAG domain that are important for interacting with LSD1. (C) Schematic diagram shows the position of the alanine mutations in the SNAG domain (top panel). The Snail1 mutants were expressed in HEK293 cells and the level of Snail1 was examined by western blotting (bottom panel). (D) EGFP-tagged WT or mutant Snail1 was expressed in HEK293 cells and the subcellular localization of Snail1 (green) was visualized by immunofluorescence microscopy (DAPI for nuclei, red). Scale bar=20 μm. (E) WT or mutant Snail1 was expressed in HEK293 cells treated with MG132 (10 μM) for 6 h. Endogenous LSD1, WT or Snail1 mutants were immunoprecipitated, and the bound Snail1 or endogenous LSD1 was examined by western blotting, respectively. Input lysates were shown in the bottom panel. (F) A ball-and-stick model structure of the LSD1–SNAG–Snail1 complex (green) superposed to the LSD1–histone H3 peptide complex (yellow; PDB access code 2V1d) showing the position of key interacting residues and the substrate lysine residue (Lys4). The LSD1 molecule is shown as a partially transparent surface representation. Download figure Download PowerPoint We next examined the interaction of these 15 mutants with LSD1 by immunoprecipitating endogenous LSD1. We found that Pro2, Arg3, Ser4, Phe5, Arg8 and Lys9 mutants completely lost their ability to interact with LSD1 (Figure 3E; data not shown). The loss of interaction of these mutants with LSD1 was not due to the instability of these mutants, as cells were treated with the proteasome inhibitor, MG132, to prevent Snail1 from degrading (input lysates on Figure 3E). Consistent with these data, when Snail1 was immunoprecipitated, the association of these mutants with LSD1 was also abolished (Figure 3E). Interestingly, mutants that cannot interact with LSD1 also lost their ability to inhibit E-cadherin promoter luciferase activity, suggesting that the interaction with LSD1 is critical for the suppressive function of Snail1 (Supplementary Figure S5). We also performed protein modelling analysis on the basis of the structure of the LSD1–CoREST–Histone H3 complex. We found that the SNAG domain of Snail1 adopted a conformation that was superimposed by the histone H3 tail at the catalytic cavity of LSD1 (Figure 3F). Noticeably, Arg3, Arg8 and Lys9 of the SNAG domain of Snail1 participate in similar critical contacts within the catalytic cavity of LSD1, compared with those of the histone H3 tail. This is consistent with the finding that these mutants lose their interaction with LSD1 and their suppressive function on the E-cadherin promoter. As methylation of arginine and lysine residues has been reported on other non-histone proteins (Huang and Berger, 2008), and because Arg3, Arg8 and Lys9 in the SNAG domain of Snail1 are critical for the interaction with LSD1, we speculate that methylation of these three residues may regulate their interaction with LSD1. To test this idea, we immunoprecipitated Snail1 and subjected it to western blot analysis using antibodies against H3K4, H3K9 and H3K27 methylation, as well as against pan-lysine and pan-arginine methylation (Supplementary Figure S6; data not shown). Unfortunately, we were unable to detect the methylation of Snail1 because of the lack of a specific methylation antibody on these three residues. Together, our results indicate that the SNAG domain of Snail1 interacts with LSD1 by adopting a conformation similar to that of the N-terminal tail of histone H3. The amine oxidase domain of LSD1 is responsible for its interaction with Snail1 The N-terminal one-third of LSD1 contains a SWIRM (Swi3p, Rsc8p and Moira) domain that is commonly found in chromatin-remodelling complexes with unknown functions (Figure 4A). The C-terminal two-thirds of LSD1 comprise an amine oxidase (AO) domain that shares extensive sequence homology with FAD-dependent AO. To identify the region that is responsible for the LSD1 interaction with Snail1, we generated LSD1 domain-deletion mutants (Figure 4A) and co-expressed them with Snail1 in HEK293 cells. As expected, immunoprecipitation of full-length LSD1 revealed the association with Snail1. A small C-terminal deletion mutant of LSD1 and the AO domain retained the ability to interact with Snail1 (lanes 2 and 4; Figure 4B; Supplementary Figure S7). The N-terminal region of the SWIRM domain, however, was completely incapable of interacting with Snail1 (lane 3; Figure 4B). In the reciprocal immunoprecipitation experiment, immunoprecipitation of Snail1 revealed the associations between full-length and small C-terminal-deleted LSD1 and the AO domain, but not with the SWIRM domain (Figure 4C). These results indicate that the AO domain is required for the interaction of LSD1 with Snail1. Consistent with this result, when wild-type and deletion mutants of glutathione-S-transferase (GST)–LSD1 were pulled down from cell lysates, the SWIRM domain of LSD1 failed to interact with Snail1 (lane 3; Figure 4D), confirming that the AO domain but not the SWIRM domain is required for mediating the interaction of LSD1 with Snail1. Figure 4.AO domain of LSD1 is responsible for its interaction with Snail1. (A) The cartoon and schematic diagram shows the structure of LSD1 and the different deletion constructs used in this study. (B, C) HA-tagged Snail1 and Flag-tagged full-length or deletion mutants of LSD1 were co-expressed in HEK293 cells treated with MG132 for 6 h. After immunoprecipitation, bound Snail1 (B) and LSD1 (C) were examined by western blotting. (D) GST-tagged full-length or deletion mutants of LSD1 were incubated with lysate from HEK293 cells expressing Snail1. The GST pull-down complex was eluted with SDS–PAGE buffer and about one-tenth of these elutents were analysed for the association of Snail1 by western blotting (bottom panel). The rest of the elutents were examined for the presence of purified GST–LSD1 by Coomassie staining (top panel). (E) Flag-tagged AO domain, AO domain without Tower (AOΔTower) or Tower domain of LSD1 was co-expressed with Snail1 in HEK293 cells treated with MG132 for 6 h. After immunoprecipitation, the bound deletion mutants of LSD1 and Snail1 were examined by western blotting. Download figure Download PowerPoint Although the AO domain of LSD1 shares high sequence homology with other FAD-containing AOs, it contains a unique 100-amino-acid insertion that forms a tower-like structure (tower domain) protruding away from the AO domain (yellow insertion; Figure 4A; Chen et al, 2006; Stavropoulos et al, 2006; Yang et al, 2006; Forneris et al, 2007). The tower domain divides the AO domain of LSD1 into two functional lobes, the FAD-binding lobe and the substrate-binding and -recognition lobe. CoREST binds to the tower domain and allosterically modulates the interaction of the FAD-binding lobe with the substrate-binding lobe of LSD1 and thus controls the demethylase activity of LSD1 (Chen et al, 2006; Stavropoulos et al, 2006; Yang et al, 2006; Forneris et al, 2007). To further narrow down the region that is required for the association of the AO domain with Snail1, we generated a tower domain-only LSD1 and a tower-deleted AO domain (AOΔTower) by linking FAD- and substrate-binding lobes with a polyglycine linker as described previously (Figure 4A; Chen et al, 2006). When these deletion mutants were co-expressed with Snail1 in