Emerin Suppresses Notch Signaling by Restricting the Notch Intracellular Domain to the Nuclear Membrane

Emerin Suppresses Notch Signaling by Restricting the Notch Intracellular Domain to the Nuclear Membrane Byongsun Lee a, Tae-Hee Lee b and Jaekyung Shim a,b* a Department of Molecular Biology, Sejong University, Seoul 05006, Republic of Korea b Laboratory for Cancer & Stem Cell Biology, Plant Engineering Institute, Sejong University, Seoul 05006, Korea Contact Information: *All correspondence should be addressed to Jaekyung Shim , Ph.D. Department of Molecular Biology, Sejong University, 98, Kunja-Dong, Kwangjin-Gu, Seoul 05006, Korea Phone: 82-2-3408-3944, Fax: 82-2-3408-4336, E-mail: [email protected] Running Title: Inhibition of Notch signaling by emerin Keywords: Emerin, Notch signaling, nuclear membrane Abbreviations: CSL, CBF1/suppressor hairless/Lag-1; NEXT, Notch1 extracellular truncation; NICD, Notch intracellular domain; ADAM, A disintegrin and metalloproteinase; CoA, Coactivators; CoR, Corepressors; PM, Plasma membrane; NE, Nuclear envelope; NGPS, Néstor- Guillermo progeria syndrome; LEM, LAP2-emerin-MAN1; TM, Transmembrane ABSTRACT Emerin is an inner nuclear membrane protein that is involved in maintaining the mechanical integrity of the nuclear membrane. Increasing evidence supports the involvement of emerin in the regulation of gene expression; however, its precise function remains to be elucidated. Here, we show that emerin downregulated genes downstream of Notch signaling, which are activated exclusively by the Notch intracellular domain (NICD). Deletion mutant experiments revealed that the transmembrane domain of emerin is important for the inhibition of Notch signaling. Emerin interacted directly and colocalized with the NICD at the nuclear membrane. Emerin knockdown induced the phosphorylation of ERK and AKT, increased endogenous Notch signaling, and inhibited hydrogen peroxide-induced apoptosis in HeLa cells. Notably, the downregulation of barrier-to-autointegration factor (BAF) or lamin A/C increased Notch signaling by inducing the release of emerin into the cytosol, implying that nuclear membrane-bound emerin acts as an endogenous inhibitor of Notch signaling. Taken together, our results indicate that emerin negatively regulates Notch signaling by promoting the retention of the NICD at the nuclear membrane. This mechanism could constitute a new therapeutic target for the treatment of emerin-related diseases. 1. Introduction The nuclear lamina establishes the mechanical support for the nucleus and provides a platform for protein interactions that contribute to gene regulation, DNA replication, and genome stability [ 1- 4]. Nuclear proteins that bind to the lamina include emerin, MAN1, lamin-associated polypeptide 2 (LAP2), and LEMD, which scaffold potentially hundreds of proteins [5, 6]. Multiple human diseases are caused by loss of individual nuclear lamina proteins, highlighting the importance of this network [2, 7]. Emerin was originally identified as a 35 kDa protein encoded by the EMD gene, which is located on the human X-chromosome. Emery-Dreifuss muscular dystrophy (EDMD) consists of X- linked EDMD (X-EDMD) and autosomal dominant EDMD (AD-EDMD) [4]. X-EDMD is caused by mutations in EMD (encoding Emerin) located on chromosome Xq28 and AD-EDMD is caused by mutations in LMNA (encoding Lamin A) located on chromosome 1q11-q23 [8-10]. EDMD is characterized by skeletal muscle wasting and cardiac defects, and mutations in the emerin gene that cause X-EDMD result in the loss of the emerin protein [11, 12]; however, the role of emerin loss in this disease has not been precisely elucidated. In addition to its known function in supporting the mechanical integrity of the nuclear membrane, emerin plays a role in the regulation of gene expression. Emerin interacts with many proteins, including nuclear lamins, germ cell-less (GCL), nesprin-1α, and BAF [13-16]. In particular, GCL proteins localize to the nuclear envelope and bind directly to emerin with high affinity [14, 17, 18]. The emerin bound GCL can also interact with the DP3 subunit of E2F-DP heterodimers and represses E2F-DP-dependent gene expression [17, 18]. However, emerin downregulation leads to the mislocalization of GCL from the nuclear envelope and the concomitant increase in E2F-DP-dependent gene expression, suggesting that emerin plays an important role in transcriptional repression by GCL. Similarly, the transcriptional repressor Btf (Bcl- 2-associated transcription factor), which is highly expressed in skeletal muscle [19, 20], interacts with emerin to exert its inhibitory role on transcription [21], suggesting that Btf may be relevant to EDMD. These findings indicate that emerin can negatively modulate gene expression through the recruitment of transcriptional repressors. Emerin also functions as a negative regulator of gene expression by trapping transcriptional activators at the nuclear membrane. For example, the direct interaction of emerin with β-catenin causes the downregulation of Wnt signaling [22]. In the absence of emerin, the levels of nuclear β- catenin increase, resulting in the upregulation of target genes [22, 23]. Emerin induces the nuclear envelope localization of LIM Domain Only 7 (LMO 7), a transcriptional activator for myogenic differentiation, and suppresses its transcriptional function [24]. The mislocalization of transcriptional activators could represent a new addition to the list of already well-known regulatory mechanisms of gene expression such as transcription, translation, and post-translational modification; however, few studies have explored this regulatory mechanism. In the present study, we examined the role of emerin as a transcriptional regulator and assessed its effect on gene expression using a transcription factor profiling assay covering 84 genes. We identified Notch signaling as a potential target pathway regulated by emerin. In Notch signaling, a Notch receptor interacts extracellularly with its canonical ligand on a contacting cell and is cleaved proteolytically by metalloproteinases and secretases. The NICD released from the receptor translocates to the nucleus, where it interacts with a CBF1/suppressor hairless/Lag-1 (CSL) family DNA-binding protein, resulting in the initiation of transcription of Notch target genes involved in several biological functions, such as development, growth, differentiation, and survival [25]. Here, we show that emerin can modulate Notch signaling by inducing the retention of the NICD at the nuclear membrane; thus, emerin may participate in some genetic laminopathy disorders, at least in part through the modulation of Notch signaling. 2. Materials and methods 2.1. Cell culture and transfection HeLa cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM (Welgene, South Korea) supplemented with 10% FBS and 1% penicillin-streptomycin (Welgene, South Korea). Transfection was performed using the Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY) according to the manufacturer's instruction. The transfected cells were cultured for 24–72 h, washed with DPBS, and harvested with lysis buffer (#FNN0011; Life technology, Grand Island, NY). 2.2. Antibodies For immunoblotting, primary antibodies specific for emerin (1:5000, #sc-15378,), BAF (1:100, #sc- 166324), lamin A/C (1:5000, #sc-20681), ERK1 (1:3000. #sc-93), pERK (1:2500, #sc-7383), pAKT/Thr 308 (1:3000, #sc-16646), AKT (1:3000, #sc-8312), pSTAT3 (1:2500, #sc-8059), and STAT3 (1:3000, #sc-482) were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA). NICD antibody (1:3000, #4147S) was obtained from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA). Primary antibodies specific for HA (1:5000, #G036), Flag (1:1000, #G191), α-tubulin (1:5000, #G094), and β-actin (1:5000, #G043) were purchased from Applied Biological Materials (Applied Biological Materials, Richmond, BC, Canada). Primary antibodies specific for GAPDH (1:5000, #csb-ma000071m0m) and GST (1:3000, #csb- ma000031m0m) were purchased from Cusabio (Cusabio, Wuhan, China). HES1 (1:1000, #ab 5702) and HES5 (1:1000, #ab 5708) antibodies were purchased from Millipore (Millipore, Darmstadt, Germany). FITC (1:500, # 209-095-082) and TRITC (1:500, # 209-025-082) antibodies were purchased from Jackson Immuno Research Laboratories (Jackson Immuno Research Laboratories, West Grove, PA). 2.3. Plasmid constructs Human emerin and NICD cDNA were provided by 21C Frontier Human Gene Bank (South Korea). The emerin cDNA was amplified by PCR and inserted into the restriction enzyme sites of HA- pcDNA3 for biochemical studies. The amplified full-length NICD cDNA was inserted into the restriction enzyme sites of pEGFP-C1 (Clontech, Mountain View, CA) for immunocytochemistry. Alternatively, both amplified genes were inserted into the restriction enzyme sites of pcDNA3 for biological assays. To generate the GST-NICD or GST-YTHDC1 fusion protein, the coding region of each cDNA was amplified by PCR and inserted into the restriction enzyme sites of pGEX-4T-1 (GE Healthcare, Marlborough, MA). For construction of emerin deletion mutants or NICD deletion mutants, the corresponding regions were amplified by PCR and inserted into the restriction enzyme sites of HA-pcDNA3. 2.4. Transcription factor profiling assay Total RNA was isolated using Trizol reagent (Life Technologies), and 1 μg of total RNA was used for cDNA synthesis. The human transcription factor profiling PCR array was performed according to the manufacturer’s protocol (#PAHS-075ZC-2; Qiagen, Valencia, CA). Data were obtained using the manufacturer’s software. 2.5.

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