THE ANATOMICAL RECORD 297:1650–1662 (2014)

SMYD Proteins: Key Regulators in Skeletal and Cardiac Muscle Development and Function 1

SHAO JUN DU,1* XUNGANG TAN,2 AND JIANSHE ZHANG3 Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 2 Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China 3 Department of Bioengineering and Environmental Science, Changsha University, Hunan, China

ABSTRACT Muscle fibers are composed of myofibrils, one of the most highly ordered macromolecular assemblies in cells. Recent studies demonstrate that members of the Smyd family play critical roles in myofibril assembly of skeletal and cardiac muscle during development. The Smyd family consists of five members including Smyd1, Smyd2, Smyd3, Smyd4, and Smyd5. They share two highly conserved structural and functional domains, namely the SET and MYND domains involved in lysine methylation and protein-protein interaction, respectively. Smyd1 is specifically expressed in muscle cells under the regulation of myogenic transcriptional factors of the MyoD and Mef2 families and the serum responsive factor. Loss of function studies reveal that Smyd1 is required for cardiomyogenesis and sarcomere assembly in skeletal and cardiac muscles. Smyd2, on another hand, is dispensable for heart development in mice. However, Smyd2 appears to play a role in myofilament organization in both skeletal and cardiac muscles via Hsp90 methylation. A Drosophila Smyd4 homologue is a muscle-specific transcriptional modulator involved in the development or function of adult muscle. The molecular mechanisms by which Smyd family proteins function in muscle cells are not well understood. It has been suggested that members of the Smyd family may use multiple mechanisms to control muscle development and cell differentiation, including transcriptional regulation, epigenetic regulation via histone methylation, and methylation of proteins other than histones, such as molecular chaperone Hsp90. Anat Rec, 297:1650–1662, C 2014 Wiley Periodicals, Inc. 2014. V

Key words: Smyd1; Smyd2; Hsp90; Unc45b; myofibrillogenesis; sarcomere

INTRODUCTION Muscle fibers are composed of myofibrils, one of the most complex and highly ordered macromolecular assemblies known. Each myofibril is made up of highly organized repetitive structures called sarcomeres, the basic contractile unit in skeletal and cardiac muscles (Laing, 2008). Myofibrillogenesis, the process of sarcomere assembly, is critical for muscle cell differentiation and contraction. Myofibrillogenesis involves hundreds of C 2014 WILEY PERIODICALS, INC. V

Grant sponsors: TEDCO (Maryland Stem Cell Research Fund) and Nature and Science Foundation of China; Grant numbers: NSFC-31230076, NSFC-31128017. *Correspondence to: Shao Jun Du, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 701 E. Pratt St., Baltimore, MD 21202. E-mail: [email protected] Received 2 March 2014; Accepted 28 April 2014. DOI 10.1002/ar.22972 Published online in Wiley Online Library (wileyonlinelibrary. com).

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Fig. 1. Domain organization of the five Smyd proteins. Linear representation of structural domains in Smyd1, 2, 3, 4, 5. The MYND, SET and TPR domains are indicated. The numbers at the end represent the size of each respective Smyd protein in humans.

sarcomeric proteins assembled into a highly organized structure composed of thick and thin filaments as well as Z-disks and M-lines. The assembly of these multiprotein complexes follows ordered pathways that are highly regulated at the transcriptional, translational and posttranslational levels (Sanger et al., 2002, 2010; Sparrow and Sch€ock, 2009). Disruption of these pathways leads to defective myofibril organization and skeletal and cardiac muscle diseases (Ehler and Gautel, 2008). Recent studies have shown that members of the Smyd family, especially Smyd1 and Smyd2, play vital roles in cardiomyogenesis and myofibrillogenesis (Gottlieb et al., 2002; Tan et al., 2006; Just et al., 2011; Donlin et al., 2012; Voelkel et al., 2013; Li et al., 2013). In this review, we will summarize the current understanding of Smyd family proteins in skeletal and cardiac muscles, including protein structure and function, regulation of gene expression and subcellular localization, protein-protein interaction and mechanism of action in muscle cells.

SMYD IS A FAMILY OF SET AND MYND DOMAIN CONTAINING PROTEINS Members of the Smyd family represent a group of proteins that contain the conserved SET and MYND domains (Fig. 1). The SET domain is approximately 130 aa long, originally identified in the Su(var)3–9, Enhancer-of-zeste and Trithorax proteins (Tschiersch et al., 1994; Jenuwein et al., 1998). The SET domain is an evolutionarily conserved catalytic motif responsible for lysine methylation and does so by adding methyl groups to lysine residues of proteins using S-adenosylmethionine (AdoMet) as a donor substrate (Yeates, 2002; Marmorstein 2003; Qian and Zhou, 2006). The MYND domain is a cysteine-rich zinc finger motif originally found in Myeloid translocation protein 8, Nervy, and DEAF-1 (Liu et al., 2007; Matthews et al., 2009). It is defined by seven conserved cysteine residues and a single histidine residue that are arranged in a C4C2HC consensus sequence (Liu et al., 2007). The MYND domain is primarily involved in protein-protein interaction with preference for binding with a proline rich motif (PXLXP). Currently, five members of the Smyd family (Smyd1 to Smyd5) have been identified with diverse biological functions from development regulation to cancer (Leinhart and Brown, 2011). Smyd1, the founding member of the Smyd family, was first identified as a novel gene in

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the opposite strand of CD8b in a head-to-head arrangement in the mouse genome, and hereby was initially named Bop for CD8b opposite (Hwang and Gottlieb, 1995). Bop/Smyd1 encodes at least three different mRNA isoforms from alternative splicing. The Smyd1_tv1 and Smyd1_tv2 isoforms, originally named Bop1/Smyd1a and Bop2/Smyd1b, are specifically expressed in muscle cells of mouse and zebrafish embryos (Gottlieb et al., 2002; Tan et al., 2006). Smyd1_tv1 differs from Smyd1_tv2 by containing an extra 39 bp exon 5 encoding a 13 aa sequence within the SET domain. Since the discovery of Bop/Smyd1, several additional Smyd genes have been identified in mouse, human, fish, and other vertebrate genomes, and were subsequently named Smyd2, Smyd3, Smyd4, and Smyd5, respectively (Fig. 1). Overexpression of Smyd2 and Smyd3 has been implicated in tumor cell proliferation (Hamamoto et al., 2004, 2006; Komatsu et al., 2009). Smyd2 and Smyd3 may regulate the activities of tumor suppressors such as p53 and retinoblastoma proteins (RB) by protein methylation (Hamamoto et al., 2004; Huang et al., 2006; Cho et al., 2010; Saddic et al., 2010). These lysine methyltransferases are becoming new drug targets in cancer (Wagner and Jung, 2012). The Smyd family proteins are conserved in vertebrates with all five members identified from fish to human. The Smyd1 and Smyd2 genes are duplicated in zebrafish genome, and are named Smyd1a, Smyd1b, Smyd2a and Smyd2b, respectively (Sun et al., 2008). It has been reported that Drosophila has eleven genes that encode proteins with both SET and MYND domains (Thompson et al., 2008). However, these genes have not been characterized with the exception of Smyd4 (Thompson et al., 2008).

SMYD FUNCTION IN HEART DEVELOPMENT AND CARDIOMYOGENESIS Smyd1 Function in Cardiac Muscle Genetic analyses demonstrate that members of the Smyd family, especially Smyd1, play important roles in heart development and cardiomyogenesis. Smyd1 is expressed in cardiac primordium and heart in developing embryos. Targeted deletion of Smyd1 in mice interferes with cardiomyocyte maturation and proper formation of the right heart ventricle (Gottlieb et al., 2002). The Smyd1 null mouse embryos failed to form right ventricles and died around 10.5 dpc (Gottlieb et al., 2002). Consistent with its critical role in cardiac muscles, knockdown of Smyd1b, one of the two Smyd1 homologous genes in zebrafish, results in no heart contraction and severe myofibril disorganization in cardiac muscles of fish embryos (Tan et al., 2006; Li et al., 2013). The cardiac muscle defect was confirmed in the flatline (fla) zebrafish mutant which carries a nonsense mutation in the Smyd1b gene (Just et al., 2011).

Smyd2 and Smyd3 Function in Cardiac Muscle Cells The highly related paralogue Smyd2 is strongly expressed in muscle cells with highest expression in the neonatal heart. Unexpectedly, Smyd2 is dispensable for heart development in mice. Cardiac specific deletion of Smyd2 has no effect on heart formation in mice (Diehl

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Fig. 2. Smyd1 is required for myosin thick filament organization in skeletal muscles. Anti-myosin antibody (F59) staining shows myosin thick filament organization in slow skeletal muscles of control (A) and in Smyd1 knockdown (B) zebrafish embryos at 24 hpf.

et al., 2010). However, it has been reported that knockdown of Smyd2 in zebrafish embryos impaired cardiac performance (Voelkel et al., 2013). The Smyd2 knockdown fish embryos showed a defective myofibril organization in cardiac muscles. A cardiac defect has also been reported in Smyd3 knockdown zebrafish embryos (Fujii et al., 2011). Knockdown of Smyd3 resulted in pericardial edema with abnormal expression of three heartchamber markers including cmlc2, amhc and vmhc. However, these phenotypes remain to be verified in respective Smyd2 or Smyd3 zebrafish mutants.

SMYD FUNCTION IN SKELETAL MUSCLES Smyd1 is Required for Myofibril Organization in Skeletal Muscles The early embryonic lethality of Smyd1 null mice made it difficult to study Smyd1 function in skeletal muscles. Zebrafish embryos that are permeable to oxygen can survive and develop without cardiac contraction for 4 to 5 days, and thus provide an alternative model for studying Smyd function in skeletal muscles. It has been demonstrated that knockdown of Smyd1 completely blocked myofibril organization in skeletal muscles and resulted in paralyzed zebrafish embryos (Fig. 2, Tan et al., 2006). All key sarcomere structures are disrupted in Smyd1 knockdown embryos including the thick, thin and titin filaments, as well as the M- and Z-lines (Tan et al., 2006; Li et al., 2013). Loss of Smyd1 has no effect on expression of myogenic genes and myoblast formation during development (Tan et al., 2006; Li et al., 2013), suggesting that Smyd1 likely functions at the later

phase of myotube differentiation and myofiber maturation. The muscle defects can be rescued by either the Smyd1_tv1 or the Smyd1_tv2 transgene (Tan et al., 2006), suggesting that these two isoforms share similar biological function in myofibril organization. Cell culture studies demonstrate that over-expression of Smyd1 upregulates muscle-specific marker genes and promotes myoblasts differentiation and myotube formation in C2C12 cells (Li et al., 2009), consistent with its function in myoblast differentiation and myotube maturation. A similar myofibril defect has been confirmed in fla (Smyd12/2) mutant zebrafish embryos (Just et al., 2011). Interestingly, the myofibril defect in fla (Smyd12/2) mutant embryos is restricted to fast muscles when analyzed at 48hpf (Just et al., 2011). This finding differs from a previous report showing a slow muscle defect in Smyd1 knockdown zebrafish embryos around 24 to 28 hpf (Tan et al., 2006). The discrepancy between these two studies is likely due to the different timing of the analyses. The slow muscle defects in early stage embryos might be recovered at the late stages due to the expression of Smyd1a, the second Smyd1 gene with a redundant function in zebrafish (Gao et al., 2014). Consistent with this idea, ectopic expression of Smyd1a is able to rescue the myofibril defect in slow muscles of Smyd1b knockdown embryos at the early stage of embryonic development around 24 to 28 hpf (Gao et al., 2014). Moreover, knockdown of Smyd1a and Smyd1b together results in a stronger slow muscle defect in later stage embryos around 48 to 72 hpf (Gao et al., 2014). The Smyd1 function in myofibril organization appears to be conserved during evolution because ectopic expression of mouse Smyd1 is able to rescue the Smyd1 knockdown

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Fig. 3. GFP expression in head muscles of a zebrafish embryo directed by the Smyd1 promoter. GFP expression was analyzed in Tg(smyd1:gfp) transgenic zebrafish embryos at 5 days post fertilization (Du et al., 2006). The smyd1 prompter directs GFP specific expression in extraocular muscles controlling eye movement and cranial cepgalic muscles including the intermandibularis posterior (imp), intermandibularis anterior (ima), interhyoideus (ih), hyohyoideus (hh) and sternohyoideus (sh). Ventral view, head to the left.

defects in zebrafish embryos (Gao et al., 2014). On a similar line, knockdown of Smyd1 in Xenopus results in a muscle defect with paralyzed frog embryos (Kawamuta et al., 2008).

et al., 2013). These phenotypes are yet to be verified in loss of function Smyd2 zebrafish mutants.

Smyd3 Function in Skeletal Muscles Smyd2 Function in I-Band Organization Knockdown studies in zebrafish indicate that Smyd2 also plays an important role in development of skeletal muscles (Donlin et al., 2012). It has been shown that knockdown of Smyd2a leads to defective myofibril organization in the Z-disk and I-band regions of skeletal muscles (Donlin et al., 2012). The myofibril defects in Smyd2 knockdown embryos appear weaker compared with the loss of Smyd1 phenotype in zebrafish. Unlike loss of Smyd1, which leads to complete disruption of sarcomere organization, thick and thin filaments, as well as M-band regions, appear normal in the absence of Smyd2 (Donlin et al., 2012). Interestingly, a recent study showed that knockdown of Smyd2a causes developmental delay and aberrant tail formation in zebrafish (SeSe

It has been reported that knockdown of Smyd3 in zebrafish embryos resulted in abnormal expression of myogenic markers including MyoD (Fujii et al., 2011), suggesting that Smyd3 may play a role in muscle development. This is in contrast to loss of Smyd1, which has no effect on expression of myogenic genes and myoblast formation during development (Tan et al., 2006; Li et al., 2013). A recent study has implicated Smyd3 in skeletal muscle atrophy. This study showed that induced skeletal muscle atrophy via dexamethasone administration specifically upregulates Smyd3 mRNA levels in tibialis anterior muscles, while Smyd1, Smyd2, Smyd4, and Smyd5 transcripts were unaltered or negatively affected by Dex administration (Proserpio et al., 2013). Smyd3 regulates skeletal muscle atrophy through mediating the recruitment of transcriptional cofactors at the myostatin

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gene, a negative regulator of skeletal muscle growth (Proserpio et al., 2013). Smyd3 depletion by shRNA prevents muscle loss and fiber size decrease in a mouse model of dexamethasone-induced skeletal muscle atrophy.

Smyd4 Function in Drosophila Muscles A Drosophila Smyd4 (dSmyd4) homologue is a musclespecific transcriptional modulator involved in the development or function of adult muscle (Thompson and Travers, 2008). dSmyd4 expression is observed in visceral, cardiac and somatic muscle precursors and in late embryogenesis dSmyd4 is strongly expressed in the somatic musculature. Knockdown of dSmyd4 expression using muscle-specific RNAi results in eclosion failure, leading to embryonic lethality predominantly at the late pupal stage (Thompson and Travers, 2008). The eclosion failure is likely caused by defective muscle formation because eclosion requires peristaltic movement of the abdominal muscles to enable flies to escape from the pupal case, suggesting an important role for dSmyd4 in muscle development in Drosophila. The biological function of Smyd4 in vertebrate remains to be determined.

REGULATION OF SMYD GENE EXPRESSION IN MUSCLE CELLS Members of the Smyd family are expressed in developing somites, cardiac and skeletal muscles (Kawamura et al., 2008; Sun et al., 2008; Fujii et al., 2011). Among the five Smyd genes in vertebrates, Smyd1 expression is highly restricted in the heart and skeletal muscle tissues. Expression analysis using the Tg(smyd1:gfp) transgene revealed that Smyd1 is also expressed in extraocular muscles controlling eye movement and cranial cepgalic muscles in zebrafish (Fig. 3; Du et al., 2006). Collectively, these data suggest that Smyd1 is expressed in striated muscles.

Regulation of Smyd1 Expression in Cardiac Muscle The molecular regulation of Smyd1 muscle-specific expression has been well characterized comparing with other Smyd genes. It has been demonstrated that cardiac expression of Smyd1 is directly regulated by MEF2C and serum response factor (SRF) (Phan et al., 2005; Li et al., 2009a). Smyd1 expression is significantly attenuated in the heart of SRF conditional knockout mice (Niu et al., 2008). Smyd1 may serve as a downstream factor of SRF in myofibrillogenesis regulation. Consistent with this idea, it has been shown that SRF regulates gene expression of contractile proteins and orchestrates nascent sarcomerogenesis in cardiomyocytes (Balza and Misra, 2006; Niu et al., 2008).

Regulation of Smyd1 Expression in Skeletal Muscle The skeletal muscle expression of Smyd1 is controlled by the SRF and myogenic transcriptional factors of MyoD and Myogenin family (Phan et al., 2005; Du et al., 2006; Li et al., 2009). These transcriptional factors bind directly to the Smyd1 promoter region and synergisti-

cally activate Smyd1 expression in C2C12 cells (Blais et al., 2005; Li et al., 2009a). Overexpression of SRF and myogenin significantly increases Smyd1 expression in C2C12 cells (Li et al., 2009a). It has been shown that IGF-1 stimulates Smyd1 expression through SRF response element in C2C12 cells (Wang et al., 2010). Collectively, these studies indicate that Smyd1, a key regulator of myogenic differentiation, acts as a downstream target of muscle regulatory factors such as SRF, MyoD and myogenin.

SUBCELLULAR LOCALIZATION OF SMYD FAMILY PROTEINS Members of the Smyd protein family show a broad pattern of subcellular localization. Smyd1, Smyd2, and Smyd3 are particularly abundant in the cytoplasm, consistent with the lack of nuclear localization signal (NLS) in these proteins. However, their nuclear localization has been reported (Sims et al., 2002; Hamamoto et al., 2004), suggesting that Smyd1 may be transported into the nucleus by association with other factors, and thus, they may have dual functions in the nucleus and cytoplasm.

Smyd1 Nuclear and Cytosolic Localization The nuclear localization of Smyd1 has also been observed in C2C12 myoblasts in vitro (Sims et al., 2002). In addition, the Smyd1 nuclear localization has been detected in murine heart sections by immunohistochemistry (Just et al., 2011). Interestingly, Smyd1 shows a nucleus to the cytosol translocation during myoblast differentiation into myotubes in vitro (Sims et al., 2002). The predominant cytoplasmic localization is consistent with in vivo studies using transgenic zebrafish expressing a myc-tagged Smyd1 or Smyd1-GFP fusion protein. Immunostaining or direct GFP observation showed that Smyd1 is mainly located in the cytosol of myoblasts and myotubes, little or no nuclear localization of Smyd1 could be detected during skeletal muscle development in zebrafish embryos (Li et al., 2011). The dynamic subcellular localization of Smyd1 suggests that it play multiple functions depending on its localization.

Smyd1 Sarcomeric Localization It has been shown that Smyd1 protein localizes to the M-lines of sarcomeres in differentiated skeletal and cardiac muscle fibers (Just et al. 2011; Li et al., 2011). The sarcomeric localization has been observed with myctagged Smyd1 or Smyd1-GFP fusion proteins in zebrafish embryos (Li et al., 2011), and the endogenous Smyd1 protein in murine cardiac muscle cells (Just et al., 2011). Smyd1b encodes two mRNA isoforms, Smyd1_tv1 and Smyd1_tv2, from alternative splicing. Strikingly, the sarcomeric M-line localization is specific for the longer isoform (Smyd1_tv1) containing the extra 13 aa encoded by exon 5 (Li et al., 2011). The sarcomeric localization of Smyd1_tv1 requires the highly conserved Phe223 and Ser225 residues within the 13 aa. Substitution of Phe223 or Ser225 with alanine significantly reduced of Smyd1 sarcomeric localization. However, replacing Ser225 with threonine (S225T) retained the sarcomeric localization (Li et al., 2011). Serine and

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Fig. 4. Three-dimensional (3D) structural comparison of the TPR motifs in Smyd1 and Hsp70-Hsp90 organizing protein (HOP). The 3D structure of Smyd1 (green) was compared with structures in the Protein Data Bank using the Dali server (http://ekhidna.biocenter.helsinki. € m, 2010). The results showed that fi/dali_server; Holm and Rosenstro

the Smyd1 C-terminal domain contains three motifs that share similar antiparallel arrangement of a-helices found in the TPR motifs of HOP (red). The PDB codes are 3N71 and 3FWV for Smyd1 and HOP, respectively.

threonine residues are potential targets for phosphorylation. However, it remains to be determined whether Ser225 is modified by post-translational modification, and its role in regulating Smyd1 localization. In contrast to Smyd1_tv1, the Smyd1_tv2 isoform which lacks the 13 aa shows no or weak sarcomeric localization (Li et al., 2011), suggesting that the sarcomeric localization of Smyd1 is regulated by alternative splicing. The sarcomeric localization appears to be dynamically regulated during mofibrillogenesis; slowing down myofibrillogenesis by a partial knockdown of Unc45b reveals that Smyd1-GFP is associated with nascent myosin on the thick filaments (Just et al., 2011). The biological significance of this dynamic sarcomeric localization is not known. It has been shown that myosin chaperones such as Hsp90 and Unc45b shuttle from the Z-line to the A-band under fiber stress conditions. It has been suggested that Hsp90 and Unc45b may play a regulatory role in maintaining the sarcomere integrity when myosin homeostasis is disturbed (Etard et al., 2008). It has been shown that Smyd1 plays a regulatory role in myosin folding and sarcomere assembly (Tan et al., 2006; Just et al., 2011; Li et al., 2013). Knockdown or mutation of Smyd1 in zebrafish results in increased myosin pro-

tein degradation and disruption of sarcomere organization. The dynamic localization of Smyd1 on the M-line and A-band may suggest a direct role for Smyd1 in sarcomere organization and fiber stress response.

Smyd2 and Smyd3 Subcellular Localization The subcellular localization of Smyd2 and Smyd3 is not well characterized in muscle cells. Similar to Smyd1, a sarcomeric localization of Smyd2 has been observed with a GFP-tagged Smyd2 protein expressed in primary chick skeletal myocytes (Donlin et al., 2012). Smyd2 appears as tightly spaced double bands flanking a-actinin striations at the sarcomeric I-band (Donlin et al., 2012). The Smyd2 I-band localization is probably mediated by the direct binding with titin N2A-domain (Donlin et al., 2012; Voelkel et al., 2012). A similar sarcomeric localization is found with the endogenous Smyd2 in human diaphragm muscle (Donlin et al., 2012). The sarcomeric localization appears to be dynamically regulated as well because endogenous Smyd2 is found in the cytoplasm of C2C12 myoblast cells (Donlin et al., 2012). Immunostaining revealed that Smyd3 localization in both nucleus and cytosol in proliferating Huh7

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TABLE 1. Smyd1 interaction proteins Protein names (Gene ID)

Method of detection

Reference

HDAC2 histone deacetylase 2 (Gene ID: 3066) HDAC3 histone deacetylase 3 (Gene ID: 8841) H3 histone, family 3C (Gene ID: 440093) Heat shock protein 90a1 (Gene ID: 30591) Unc45b (Gene ID: 266640) FK506 binding protein 8 Tetratricopeptide repeat domain protein 33 (Gene ID: 23548) skNAC (Gene ID: 4666)

Co-IP Co-IP Two-hybrid Co-IP Co-IP Two-hybrid, Co-IP Two-hybrid

Gottlieb et al., 2002 Gottlieb et al., 2002 Weimann et al., 2013 Li et al., 2013 Li et al., 2013 Park et al., 2010 Weimann et al., 2013

Two-Hybrid, Co-IP

Myosin, heavy chain 7B (cardiac muscle, beta) (Gene ID: 57644) Myosin(rabbit muscle, Fluka, 70045) Adenylosuccinate synthase (Gene ID: 159)

Affinity Capture-Luminescence Two-hybrid Pulldown assay Affinity Capture-Luminescence Two-hybrid Two-hybrid Two-hybrid, Co-IP Two-hybrid

Sim et al., 2002 Park et al., 2010 Weimann et al., 2013

Oxoglutarate dehydrogenase-like (Gene ID: 55753) Helicase U3 small nucleolar ribonucleoprotein, homolog A (Gene ID: 10813) RNA binding motif protein 4B (cardiac muscle, beta) (Gene ID: 150572) Coiled-coil domain containing protein 113 (Gene ID: 29070) WD repeat domain protein 77 (Gene ID: 79084) Mical Leukocyte receptor cluster member 8 (Gene ID: 114823)

cancer cells (Hamamoto et al., 2004). Its subcellular localization in muscle cells is however not known. The dynamic localization of Smyd2 and Smyd3 likely correlates with their biological functions. The nuclear localization, for example, may involve epigenetic regulation of muscle gene expression via histone lysine methylation. The cytosolic and sarcomeric localization suggests that Smyd2 and Smyd3 may have additional nonnuclear function in the cytosol of muscle cells.

STRUCTURES OF SMYD FAMILY PROTEINS The 3D Structures of Smyd1, Smyd2, and Smyd3 The 3D crystal structures of Smyd1, Smd2, and Smyd3 have been determined by using the crystal complex with an analog of the S-adenosyl methionine (SAM) methyl donor cofactor. The shape of the Smyd1 structure resembles an openended wrench, with two thick "grips" separated by a deep concave opening (Sirinupong et al., 2010). The putative histone binding site is located at the bottom of the concave opening, suggesting that both the N- and C-terminal grips are involved in substrate recognition (Sirinupong et al., 2010). The crystal structure of Smyd2 reveals a comparable pocket and a long groove for target protein binding, and conformational flexibility of the autoinhibitory c-terminal domain (Xu et al., 2011a; Jiang et al., 2011; Wang et al., 2011; Ferguson et al., 2011). SmyD2 appears to be a conformational “intermediate” between an open form of SmyD1 and a close form of SmyD3 which has a deep and narrow substrate binding pocket (Xu et al., 2011b; Foreman et al., 2011; Sirinu-

Just et al., 2011 Weimann et al., 2013 Weimann et al., 2013 Park et al., 2010 Weimann et al., 2013

Affinity Capture-Luminescence Two-hybrid Two-hybrid

Weimann et al., 2013

Two-hybrid Two-hybrid, Co-IP Two-hybrid

Weimann et al., 2013 Park et al., 2010 Weimann et al., 2013

Weimann et al., 2013

pong et al., 2011). Although the Smyd1, 2 and 3 are similar in the overall structure, they differ substantially in the potential substrate-binding site. The binding site of Smyd3 consists mainly of a deep and narrow pocket, while those of Smyd1 and Smyd2 consist of a comparable pocket and a long groove. The differences in the substrate-binding site might account for the observed divergence in the specificity and methylation state of the substrates (Xu et al., 2011a,b).

The C-Terminal TPR Motifs A unique feature of the Smyd1, Smyd2 and Smyd3 structures is the presence of a TPR motif-containing C-terminal domain (CTD). It has been shown that the CTD domain plays a regulatory role in modulating the methyltransferase activity of Smyd proteins. Biochemical analysis showed that deletion of the CTD in Smyd1 and Smyd3 increased their histone methyltransferase activity in vitro (Sirinupong et al., 2010, 2011). However, functional analyses in vivo showed that deleting the C-terminal domain abolished Smyd1 function in myofibril organization (Just et al., 2011). The molecular mechanism underlying the importance of C-terminal domain is not clear. Structural and sequence analyses revealed that Smyd1, Smyd2 and Smyd3 C-terminal domains contain three TPR motifs. Each TPR motif contains 34 amino acids that forms an anti-parallel a-helix. The TPR motifs in Smyd1 share similar structure with the TPR motifs found in many Hsp90 co-chaperones such as HOP, CyP40, FKBP51/52 and p23 (Fig. 4). The TPR motifs are involved in interaction with the Hsp90 C-terminus MEEVD sequence. We suggest that Smyd family proteins may serve as Hsp90

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co-chaperones and play a regulatory role in Hsp90 activity. Consistent with this idea, it has been shown that Smyd2 methylates Hsp90, and Hsp90 methylation by Smyd2 is critical for Hsp90 function in titin filament organization (Donlin et al., 2012; Voelkel et al., 2012). Alternatively, interaction with Hsp90 may be important for the methyltransferase activity of Smyd proteins. It has been shown that addition of Hsp90 enhances histone methylation by Smyd1, Smyd2 and Smyd3 in vitro (Hamamoto et al., 2004; Tan et al., 2006; Abu-Farha et al., 2008).

SMYD FAMILY INTERACTING PROTEINS Smyd1 Interacting Proteins Mapping protein–protein interactions is an invaluable tool for understanding protein function and potential mechanisms of actions. The protein interaction network for Smyd1 has been characterized by multiple approaches, including yeast two-hybrid screening, affinity capture-luminescence, Co-IP assay and followed by high-throughput mass spectrometry (Park et al., 2012; Weimann et al., 2013). Over twenty proteins have been identified that interact with Smyd1 (Table 1). The Smyd1 interactome covers a diverse group of proteins. These include histone protein such as histone H3 and histone modification enzymes such as HDACs, molecular chaperone Hsp90 and co-chaperone Unc45b, and muscle specific proteins such as myosin and skNAC. The findings that Smyd1 interacts with H3 histone and HDACs are consistent with the idea that Smyd1 may function in the nucleus to regulate gene expression. On the other hand, association with myosin and Hsp90 supports the notion that Smyd1 may function in the cytosol to control myosin folding and thick filament assembly.

skNAC is a Major Smyd1 Binding Protein skNAC represents a major Smyd1 interacting protein in cultured skeletal myoblasts (Sim et al., 2002; Park et al., 2010). The functional significance of its interaction with skNAC is not clear. skNAC is a muscle-specific isoform of NAC (nascent polypeptides associated complex) which plays a critical role in the proper maturation of newly synthesized proteins. skNAC is markedly upregulated after skeletal muscle injury (Munz et al., 1999). Knockdown of skNAC expression resulted in defective sarcomere organization in skeletal muscles of zebrafish embryos and myoblasts in culture (Li et al., 2009b; Berger et al., 2012). Genetic studies in mice demonstrate that skNAC plays an important role in cardiac development and skeletal muscle growth and regeneration. Targeted deletion of skNAC in mice results in ventricular hypoplasia with decreased cardiomyocyte proliferation, and reduced postnatal skeletal muscle growth and impaired regenerative capacity (Park et al., 2010). The molecular mechanism by which skNAC functions in muscle cells is not known. skNAC has been reported as a muscle-specific transcriptional factor of the myoglobin promoter and involved in normal differentiation along the myogenic lineage and in the regulation of myoblast fusion (Yotov and St-Arnau, 1996). Similar to Smyd1, skNAC is also translocated from the nucleus to the cytoplasm during C2C12 myoblast differentiation

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into myotubes in vitro (Sim et al., 2012). The dynamic subcellular localization suggests that skNAC may have dual roles in the nucleus and cytosol. Interestingly, a recent study showed that skNAC depletion perturbs sarcomerogenesis by enhancing calpain 1 and 3 activity (Berkholz et al., 2013). It has been shown that loss of skNAC or Smyd1 results in significant increase of myosin degradation (Li et al., 2009b; 2013; Just et al., 2011). It will be interesting to test whether calpain 1 and 3 are involved in myosin degradation in Smyd1 deficient embryos and whether blocking Smyd1 function would increase calpain 1 and 3 activity in muscles.

Smyd1 Interacts with Hsp90 and Unc45b Co-IP analyses showed that Smyd1 binds to Hsp90 (Li et al., 2013). This is consistent with the presence of TPR motifs required for Hsp90 binding at the Smyd1 Cterminal domain. The functional significance of Smyd1 binding to Hsp90 is not clear. Hsp90 is strongly expressed in developing somites and skeletal muscles of zebrafish embryos. Cellular and biochemical analyses showed that Hsp90 is a key molecular chaperone required for myosin folding and assembly (Srikakulam and Winkelmann, 2004). Genetic studies in zebrafish have demonstrated that Hsp90a1 is essential for myofibril assembly in skeletal muscles (Etard et al., 2007; Du et al., 2008; Hawkins et al., 2008). Loss of Hsp90a1 results in a complete disruption of sarcomere organization in skeletal muscles. Interestingly, Co-IP assay showed that Smyd1 also associates with Unc45b (Li et al., 2013), a myosin chaperone and Hsp90 cochaperone that plays a key role in myosin folding and sarcomere assembly (Barral et al., 2002; Landsverk et al., 2007; Wohlgemuth et al., 2007; Etard et al., 2007; Srikakulam et al., 2008; Gazda et al., 2013). Loss of Unc45b or Hsp90a1 function results in a similar increased myosin degradation and sarcomere disorganization as in Smyd1 deficient zebrafish embryos (Bernick et al., 2010; Just et al., 2011; Li et al., 2013). Expression of SmyD1, Unc45b and Hsp90a1 appears to be coregulated. Knockdown of Smyd1 or mutation results in a significant upregulation of Hsp90a1 and Unc45b expression in skeletal muscles, indicating a close relationship among Smyd1, Hsp90a1, and Unc45b (Just et al., 2011; Li et al., 2013). Collectively, these studies suggest that Smyd1 may play a concerted role with Hsp90 and Unc45b to control myosin folding, degradation, and assembly into sarcomeres during myofibrillogenesis.

Smyd2 and Smyd3 Interacting Proteins Proteomic analyses have been performed to generate protein interaction network for Smyd2, Smyd3, and Smyd5 (Abu-Farha et al., 2008, 2011). The data reveal that the three Smyd proteins associate with both unique and shared subsets of proteins (Abu-Farha et al., 2011). Overall, Smyd-bound proteins can be divided into three main functional groups. The first group represents proteins involved in chromatin remodeling and histone modification, consistent with their functions in epigenetic regulation of gene expression. The second group represents proteins related to DNA replication and repair. This is in agreement with previous findings of Smyd2-mediated p53

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methylation in regulating DNA repair (Huang et al., 2006). The third group represents molecular chaperones such as Hsp90 and associated co-chaperones. CoIP studies confirmed that Smyd2 and Smyd3, but not Smyd5 form a protein complex with Hsp90 (Abu-Farha et al., 2008, 2011). The interaction data are consistent with the presence of TPR motifs in the C-terminal domain in Smyd2 and Smyd3 but not Smyd5. Recent studies demonstrate that Hsp90 methylation by Smyd2 plays an important role in stabilization of the sarcomeric I-band region (Donlin et al., 2012). Interestingly, a direct interaction between Smyd2 and the titin N2A domain has been confirmed by yeast twohybrid and GST pull-down assays (Donlin et al., 2012), indicating that Smyd2 may be directly involved in the I-band organization.

MECHANISMS OF FUNCTION The molecular mechanisms by which Smyd family proteins function in muscle cells are not well understood. Biochemical studies indicate that Smyd1, Smyd2, and Smyd3 could methylate lysine residues in histone H3 proteins in vitro, suggesting that they may regulate gene expression via histone modification (Hamamoto et al., 2004; Tan et al., 2006; Brown et al., 2006; Sirinupong et al., 2010). However, subcellular analyses revealed that Smyd proteins are mainly localized in the cytoplasm and are able to methylate proteins other than histones, arguing that Smyd proteins may have nonnuclear functions.

Histone Methylation and Transcriptional Regulation by Smyd1 The methyltransferase activity appears to be critical for Smyd1 function in myofibril organization. Mutating five conserved residues (203CWPNC207) in the SET domain involved in lysine methylation completely abolished Smyd1 function in myofibril organization (Tan et al., 2006). Histone methylation plays a vital role in transcriptional regulation. Supporting a potential nuclear role for Smyd1 in transcriptional regulation, Smyd1 represses gene transcription in a histone deacetylase (HDAC) dependent fashion in an in vitro assay (Gottlieb et al., 2002). The target genes of Smyd1 are yet to be identified. It has been shown that Smyd1 is required for Hand2 and Irx4 expression in the heart, and functions in a transcriptional network to control ventricular cardiomyocyte growth and differentiation (Gottlieb et al., 2002). However, it is not clear whether Smyd1 directly regulates Hand2 and Irx4 gene expression. A recent study questioned whether or not histone methyltransferase activity is required for Smyd1 function in myofibril organization (Just et al., 2011). Just et al. showed that substitution of the conserved Tyr247 residue in Smyd1 SET domain with phenylalanine had no effect on Smyd1 function in myofibril assembly (Just et al., 2011). The Tyr247 residue is highly conserved among all five members of the Smyd family, and has been implicated in histone methylation in Smyd2 and Smyd3 proteins (Brown et al., 2006). Tyr247Phe substitution might abolish Smyd1 histone methylation activity, however, this has not been tested directly. Therefore, it

is still an open question whether histone methylation is required for Smyd1 function in muscle cells.

Transcriptional Regulation by Smyd2, Smyd3, and Smyd4 It has been reported that Smyd2 associates with the Sin3 repression complex and is involved in transcriptional repression in an in vitro assay system (Brown et al., 2006). Smyd2 has a potential role in the transcriptional regulation of genes associated with protein translation. Moreover, Smyd2, similar to Smyd3, interacts with RNA Polymerase II as well as to the RNA helicase. It is, however, not clear whether transcriptional repression by Smyd2 plays a role in muscle cells. A recent study has implicated Smyd3 in skeletal muscle atrophy (Proserpio et al., 2013). Smyd3 regulates skeletal muscle atrophy through activation of myostatin gene expression which plays an inhibitory role in skeletal muscle growth (Proserpio et al., 2013). A Drosophila smyd4 homologue has been shown to act as a muscle-specific transcriptional modulator. dSmyd4 interacts selectively with Ebi, a component of the dHDAC3/SMRTER co-repressor complex and repressed transcription and recruited class I histone deacetylases (Thompson and Travers, 2008).

Hsp90 Methylation by Smyd2 In addition to transcription regulation and histone methylation, it has been demonstrated that Smyd family proteins are capable of methylating proteins other than histones and play important regulatory roles in protein function. Smyd2 is capable of methylating Hsp90, and the Hsp90 methylation is modulated by its cochaperones (Abu-Farha et al., 2011; Donlin et al. 2012). Methylated Hsp90, Smyd2, and the sarcomere protein titin form a complex, which contributes to the stability of titin and the sarcomere in both skeletal and cardiac muscles (Donlin et al., 2012; Voelkel et al., 2013). It has been increasingly recognized that post-translational modifications control diverse activities of Hsp90 chaperone in protein folding and protein complex assembly by stipulating substrate specificity, subcellular localization and selective co-chaperone binding. It remains to be determined how methylation of Hsp90 by Smyd2 affects Hsp90 function and whether Smyd1 can also methylate Hsp90 and control Hsp90 function in myosin folding and sarcomere assembly.

Smyd1 and Smyd2 Bind to Sarcomeric Proteins Recent studies showed that Smyd1b binds to myosin (Just et al., 2011), whereas Smyd2 associates with titin protein (Donlin et al., 2012). Myosin binding appears to be critical for Smyd1 function. Deletion of the myosin binding domain in Smyd1 abolished its biological function (Just et al., 2011). It has been long observed that the myosin S1 domain contains methylated lysines (Hardy and Perry, 1969; Huszar and Elzinga, 1969; Kuehl and Adelstein, 1969; Hardy et al., 1970a,b). Myosin methylation markedly increases the ATPase activity of myosin S1 fragment (White and Rayment, 1993; Bivin et al., 1994), suggesting that myosin methylation may enhance proper folding of myosin head domain and thus its ATPase activity. In addition to myosin, several other muscle proteins,

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such as actin and creatine kinase have been shown to be methylated at lysine residues (Tong and Elzinga, 1983; Iwabata et al., 2005). Protein methylation has distinct effects on protein stability, which can lead to either the stabilization or de-stabilization of the proteins (Yang et al., 2009; Egorova et al., 2010). The functional significance of muscle protein methylation and the methyltransferase(s) involved in the process are not clear. It would be interesting to test whether Smyd1 and Smyd2 are directly involved in muscle protein methylation to control sarcomere assembly.

CONCLUDING REMARKS AND FUTURE DIRECTIONS Although members of the Smyd family have been implicated in regulation of skeletal and cardiac muscle formation, the molecular mechanisms underlying their functions in muscle cells are not well understood. This is, in part, due to the dynamic subcellular localization of Smyd family proteins in both nucleus and cytosol, suggesting that they may have multiple functions depending on their subcellular localization. Their nuclear localization and histone methyltransferase activity are consistent with their potential function in regulating gene expression via histone modification. However, the direct gene targets of Smyd proteins involved in muscle development have yet to be identified. Systematic genomic approach such as ChIP-sequencing can be used to identify the potential target genes. It has been increasingly recognized that Smyd proteins can methylate proteins other than histones in the cytosol. Structural and biochemical analyses reveal that Smyd 1, 2 and 3 possess the characteristic TPR motifs found in many Hsp90 co-chaperones. Smyd1, 2, and 3 bind to Hsp90 which plays an important role in myosin folding and sarcomere assembly. Could Smyd proteins serve as Hsp90 co-chaperones to control Hsp90 function in muscle cells? It has been shown that co-chaperones play critical roles in regulating Hsp90 activity and binding specificity. Consistent with this idea, Smyd2 methylates Hsp90 and controls Hsp90 function in titin filament organization. Methylation is a post-translational modification that can affect numerous features of proteins, notably subcellular localization, turnover, activity, and molecular interactions (Zhang et al., 2012). A recent study has uncovered a new group of distantly related lysine methyltransferases that preferentially interact with molecular chaperones to regulate their activity (Cloutier et al., 2013). Interestingly, a growing amount of evidence links the protective action of Hsp90 chaperone to mechanisms related to posttranslational modifications of soluble nuclear factors as well as histones directly involved in transcriptional regulation (Erlejman et al., 2014). It has been demonstrated that Smyd2 directly methylates estrogen receptor a (ERa), a Hsp90 client protein, and represses ERa target gene expression (Zhang et al., 2013). It will be interesting to determine whether the chaperone function regulated by Smyd-Hsp90 interaction is connected with their nuclear function in histone modification and transcriptional regulation. In addition to heat shock proteins, it is known that Smyd proteins interact with many additional nuclear and cytosolic proteins. The functional significance for

most of the interactions is not clear. Some of these proteins may represent the methylation substrates. A systematic proteomic approach can be used to identify Smyd methylation target proteins. It should be noted that in addition to their functions in muscle cells, members of the Smyd family especially Smyd2 and Smyd3 have been identified as putative oncogenes involved in the proliferation of cancer cells (Hamamoto et al., 2004; Huang et al., 2006). It has been shown that methylation of retinoblastoma tumor suppressor (Rb) by Smyd2 enhances cell cycle progression (Saddic et al., 2010; Cho et al., 2012). In addition, Smyd2 may function as a putative oncogene by methylating p53 and repressing its tumor suppressive function (Huang et al., 2006), thus making Smyd2 an attractive drug target (Ferguson et al., 2011). Similar to Smyd2, Smyd3 has been shown to regulate cancer cell phenotypes by epigenetic regulation of metalloproteinase MMP-9 and androgen receptor gene transcription of (Van Aller et al., 2012; Cock-Rada et al., 2012; Liu et al., 2013). Moreover, Smyd3 promotes myocardin-related transcription factor-A-mediated transactivation of myosin regulatory light chain 9 (MYL9) and migration of MCF-7 breast cancer cells (Luo et al., 2014). Strikingly, a recent study presents an intriguing new aspect of Rb function in muscle cells (Araki et al., 2012). Data from this study showed that cytoplasmic translocation of the retinoblastoma protein disrupts sarcomeric organization in muscle cells (Araki et al., 2012; Cossu et al., 2013). Therefore, knowledge gained from studying the oncogenic functions of Smyd proteins could lead to discovery of new molecular pathways of Smyd function that may be involved in muscle cell differentiation, and vice versa, better understanding the mechanisms by which Smyd proteins function in muscle cell differentiation may provide important insights into their oncogenic actions in tumor formation.

ACKNOWLEDGEMENTS The authors thank Nick Du for proof reading the article.

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