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HMG20A Is Required for SNAI1-Mediated Epithelial to Mesenchymal Transition

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HMG20A Is Required for SNAI1-Mediated Epithelial to Mesenchymal Transition Oncogene (2015) 34, 5264–5276 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE HMG20A is required for SNAI1-mediated epithelial to mesenchymal transition S Rivero1, M Ceballos-Chávez1, SS Bhattacharya2 and JC Reyes1 HMG20A is a high mobility group (HMG) domain containing protein homologous to HMG20B, a core subunit of the Lys-specific demethylase 1/REST co-repressor 1 (LSD1-CoREST) histone demethylase complex. Here, we show that HMG20A can replace HMG20B and, therefore, they are mutually exclusive subunits of the complex. Both proteins interact through a coiled-coil domain with BHC80, another subunit of the LSD1-CoREST complex. To investigate the functional differences between the two proteins, we performed transcriptomic analysis of HMG20A- and HMG20B-depleted cells. Analysis of the misregulated genes in HMG20A- knockdown cells evidenced a high proportion of genes related to the epithelial-to-mesenchymal transition (EMT) process. EMT occurs during embryonic development or during the course of malignant cancer progression and consists in the dynamic and reversible transitions between epithelial and mesenchymal phenotypes. We show that HMG20A together with LSD1 are required for SNAI1-dependent repression of epithelial genes and for (transforming growth factor β) TGF-β-triggered EMT. Importantly, HMG20A-depleted cells displayed reduced binding of LSD1 to epithelial gene promoters and increased methylation of lysine 4 of histone H3, suggesting a role of HMG20A in recruiting or in stabilizing the complex at the chromatin. SNAI1 and the TGF-β-related transcription factor SMAD4 were found to be associated with the LSD1-CoREST complex containing HMG20A. Furthermore, we show that HMG20A-depleted cells displayed reduced motility and invasion activity. Finally, we show that expression of HMG20A correlates positively with mesenchymal markers and negatively with epithelial markers in human tumor samples. Taken together, our data demonstrate that HMG20A is essential for the mesenchymal phenotype. Oncogene (2015) 34, 5264–5276; doi:10.1038/onc.2014.446; published online 2 February 2015 INTRODUCTION gene expression and protein stability and by promoting its 1 Cellular plasticity is the ability of some differentiated cell types to migration to the nucleus. Both EMT and MET require extensive reorganization of the reversibly change their phenotype. An example of cellular 7,8 plasticity is the process by which epithelial cells can downregulate epigenetic information of the cells. In fact, master transcription epithelial characters and acquire mesenchymal characteristics. regulators of these processes recruit multiprotein complexes that fi This process, known as epithelial-to-mesenchymal transition introduce or remove modi cations of chromatin-associated (EMT), is a key process during embryonic development as well histones. For example, SNAI1 represses transcription of epithelial as in some pathological situations, including cancer and organ genes, such as CDH1 (encoding E-cadherin), by recruiting – chromatin-modifying machineries including the Polycomb repres- fibrosis.1 4 In human cancer, EMT is an important process sive complex 2 and the Lys-specific demethylase 1/REST co- conducive to tumor invasion and dissemination. The reverse – repressor 1 (LSD1-CoREST) complex.9 11 LSD1 is the first reported transformation, the mesenchymal-to-epithelial transition (MET), is 12 fi histone demethylase and interacts directly with the SNAG required for the formation of organs in the nal destinations of domain of SNAI1. LSD1 is involved in gene repression as a part of embryonic migratory cells and for establishment of overt 2,5 the LSD1-CoREST complex mediating the demethylation of metastases at distant sites in tumors. H3K4me1/213 and in gene activation associated with nuclear During EMT, cells perform an extensive reorganization of cell receptors through demethylation of H3K9me1/2.14 Consistent junction complexes, cytoskeletal architecture and interactions with its function as SNAI1 co-repressor, LSD1 is highly expressed in with the extracellular matrix. Cells increase their motility and estrogen receptor-negative tumors, which display mesenchymal invasion properties and become more resistant to drugs. These gene signatures.15 Furthermore, blocking SNAI1-LSD1 interaction transformations require large changes in gene expression suppresses the motility and invasiveness of cancer cells of controlled by master regulators, including SNAIL, TWIST and different origins.16 The core of the LSD1-CoREST complex 1 zinc-finger E-box-binding (ZEB) transcription factors. SNAI1 (also comprises CoREST, HDAC1-2, BHC80 (also called PHF21A) and called SNAIL1 or SNAIL) is one of the most important factors HMG20B (also called BRAF35).13,17–22 HMG20B contains a high involved in EMT.6 SNAI1 is a repressor of epithelial genes and an mobility group (HMG) domain in the amino terminal half of the activator of mesenchymal genes. Multiple signaling pathways protein and was first identified as an interactor of BRCA2.23 The including transforming growth factor β (TGF-β), WNT, Notch and role of HMG20B in the LSD1-CoREST complex is unclear.21 MAPKs cooperate in the initiation of EMT, by increasing SNAI1 HMG20A is a paralogous of HMG20B. Both proteins share the 1Molecular Biology Department, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain and 2Cell Therapy and Regenerative Medicine Department, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Seville, Spain. Correspondence: Dr JC Reyes, Molecular Biology Department, CABIMER-CSIC, Av. Americo Vespucio, Seville 41092, Spain. E-mail: [email protected] Received 30 July 2014; revised 28 November 2014; accepted 5 December 2014; published online 2 February 2015 HMG20A is required for EMT S Rivero et al 5265 HMG domain and a sequence with predicted coiled-coil structure targets HMG20B. If HMG20A binds the complex through HMG20B, in their carboxy terminus.24 It has previously been reported that then depletion of this protein would impair association of HMG20A HMG20A is a functional antagonist of HMG20B in the process of with the complex. However, knockdown of HMG20B did not affect neural differentiation.24,25 We have also shown that HMG20A the association of HMG20A with the complex (Figure 1d). These forms homodimers and heterodimers with HMG20B, but HMG20B data suggested that HMG20A and HMG20B bind independently to is not able to form homodimers. Furthermore, we showed that the complex. Then, we wondered whether the LSD1-CoREST HMG20A-HMG20B heterodimerization impairs sumoylation of complex can contain both factors at the same time or whether, HMG20B which is essential for its function.25 However, some alternatively, their interaction with the complex is mutually proteomic analyses have reported substoichiometric amounts of exclusive. To investigate these possibilities, the LSD1-CoREST HMG20A in the complex,17,18,20 while other works do not detect complex was immunoprecipitated using anti-LSD1 antibodies the protein as a subunit of LSD1-CoREST complex.13,19,21,22 To under normal conditions or upon overexpression of HA-HMG20A clarify this situation, we have investigated the presence of or Flag-HMG20B. Interestingly, overexpression of HMG20A dis- HMG20A in the LSD1-CoREST complex and its functional placed endogenous HMG20B from the LSD1-CoREST complex and consequences. Here, we show that HMG20A is able to substitute increased the level of HMG20A associated with the complex HMG20B in the LSD1-CoREST complex and therefore that HMG20A (Figure 1e). As a control, we verified that the mutant HMG20AΔcc and HMG20B are mutually exclusive subunits of the core complex. was not able to displace endogenous HMG20B from the complex Gene expression profiling of cells depleted of HMG20A or (Supplementary Figure S1). Conversely, overexpression of HMG20B HMG20B demonstrated that both proteins have common as well provoked the substitution of endogenous HMG20A by HMG20B in as specific targets. Interestingly, many differentially regulated the complex (Figure 1f). These data suggest that the interaction of genes in the absence of HMG20A were related to EMT and MET HMG20A or HMG20B with the complex is mutually exclusive. processes. Importantly, we show that HMG20A is required for Consistently, in vitro pull-down experiments showed that HMG20A SNAI1-dependent repression of epithelial genes and for TGF-β- interacts with BHC80 (Figure 1g), the same subunit of the LSD1- mediated EMT. The LSD1-CoREST complex containing HMG20A CoREST complex responsible for the interaction with HMG20B interacts with SNAI1 and with SMAD4. Consistent with these data, (Figure 1g and ref. 26). Furthermore, HMG20B was more efficiently HMG20A was required for mesenchymal phenotypes including retained by the GST-BHC80 coated beads than HMG20A (Figure 1g), cell motility and invasion activity. Taken together, these data which may account for the lower amount of HMG20A normally demonstrate that HMG20A is an important factor required for associated with the LSD1-CoREST complex. Therefore, our data promoting and maintaining mesenchymal characters. indicate that HMG20A can replace HMG20B in the LSD1-CoREST complex (Figure 1h). In addition, the fact that HMG20A can also 25 RESULTS form homodimers and heterodimers with HMG20B suggests the existence of a complex equilibrium to determine the proportion of HMG20A and HMG20B are alternative subunits
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