N-Lysine Methyltransferase SMYD
The difference in the positions of the N- and C-terminal lobes in SMYDs results in open and closed structures (Fig. 2). SMYD1 has the most open conformation with the substrate-binding site widely exposed. SMYD3 is found to have the most closed conformation. SMYD2 has an intermediate conformation between SMYD1 and SMYD3. Due to the unique protruding C-terminal helix, SMYD1 structure shows an open-ended “wrench” shape (Fig. 2). The absence of this helix in SMYD2 and SMYD3 results in a clamshell-like structure.
Protein Lysine Methylation
The SMYD protein family methylates both histone and nonhistone proteins (Hamamoto et al. 2004; Huang et al. 2006; Tan et al. 2006; Zhang et al. 2013; Mazur et al. 2014). Histone methylation links the SMYD family to the epigenetic process. Histone methylation is the process in which methyl groups are transferred to amino acids of histone proteins in nucleosomes. Increasing or decreasing transcriptional events can occur due to the methylation of histones; this is dependent on which amino acids are methylated. The histone methylation by SMYD proteins contributes to the regulation of chromatin remodeling and gene accessibility. SMYD1–3 methylate H3K4 which is a methylation site that promotes active transcription (Hamamoto et al. 2004; Sirinupong et al. 2010). The SMYD family does not have an effect on global H3K4 methylation levels but impacts selective promoter regions (Hamamoto et al. 2004; Tan et al. 2006). SMYD2 is found to dimethylate H3 K36 in vitro and repress transcription (Spellmon et al. 2015). The SMYD family also regulates gene expression by recruiting other transcriptional regulators. SMYD1 binds directly to class I and class II histone deacetylases (HDAC) which repress transcription (Gottlieb et al. 2002). SMYD3, being a member of an RNA polymerase complex, interacts with RNA polymerase II and RNA HELZ and plays an important role in transcriptional regulation (Hamamoto et al. 2004).
SMYD proteins are known to methylate nonhistone proteins, such as p53, VEGFR1, retinoblastoma (Rb), and heat shock protein-90 (Hsp90) (Abu-Farha et al. 2011; Jiang et al. 2014). The methylation of these nonhistone proteins expands the role of the SMYD protein family beyond the epigenetic process. The protein methylation may change protein function. SMYD3 methylation of VEGFR1 increases its kinase activity. Methylation of different target lysine residues on Rb and p53 by SMYD2 represses their apoptotic activity (Huang et al. 2006). The protein p53 functions as a tumor suppressor and plays an important role in the suppression of cancer. VEGFR1 is a protein that promotes vasculogenesis and angiogenesis. The protein Rb is a tumor suppressor and serves as a recruiter for chromatin remodeling enzymes. The methylation of these proteins has linked the SMYD protein family to tumorigenesis, cell signaling, and DNA damage response. SMYD2 can also methylate Hsp90 (Abu-Farha et al. 2011). The target lysine residues K209 and K615 are located on the nucleotide-binding domain and dimerization domain, respectively. Hsp90 is a homodimeric, ubiquitous, and essential chaperone composed of three functional domains: the nucleotide-binding domain, middle domain, and dimerization domain. Hsp90 is involved in heat shock response, signal transduction, steroid signaling, and tumorigenesis. The methylation of Hsp90 by SMYD2 may have the potential to regulate Hsp90 dimerization and ATPase activity, thereby impact its chaperone activity and stress response.
The SMYD protein family plays critical roles in heart development. Deletion of SMYD1 in mice interferes with cardiomyocyte maturation and proper formation of the right ventricle (Gottlieb et al. 2002). With the SMYD1 deletion, the mice embryo fails to form the right ventricle and dies around 10.5 days post coitum (dpc). In zebrafish, deletion of SMYD1 results in no heart contraction and severe myofibril disorganization in cardiac muscles (Tan et al. 2006). SMYD2 is most expressed in the newborn heart. Deletion of SMYD2 has no apparent effect on mice (Spellmon et al. 2015). However, the knockdown of SMYD2 in zebrafish leads to impaired cardiac performance and defective myofibril organization in cardiac muscles (Spellmon et al. 2015). Deletion of SMYD3 in zebrafish produces a cardiac defect (Fujii et al. 2011). SMYD3 knockdown produces pericardial edema with abnormal expression of three heart chamber markers including cmlc2, amhc, and vmhc. The current results have provided supporting evidence of the importance of the SMYD protein family in heart development.
The SMYD protein family plays crucial roles in muscle development. SMYD1 is specifically expressed in muscle cells under the regulation of myogenic transcriptional factors including the MyoD and Mef2 families and the serum responsive factor (Spellmon et al. 2015). Deletion of SMYD1 in zebrafish leads to complete blockage of myofibril organization in skeletal muscles, producing paralyzed embryos (Tan et al. 2006). All key sarcomere structures are disrupted in SMYD1-depleted zebrafish, including actin, myosin, and M-lines. SMYD1 is also expressed in extraocular muscles controlling movement of the eye and cranial cephalic muscles in zebrafish. SMYD2, SMYD3, and SMYD4 also play a role in muscle development (Spellmon et al. 2015). Knockdown of SMYD2 results in defective myofibril organization at Z-lines and I-bands. However, SMYD2 knockdown does not lead to complete disruption of the muscle developmental process. SMYD3 knockdown in zebrafish embryos leads to abnormal expression of myogenic markers including MyoD (Fujii et al. 2011). A recent study shows that SMYD3 is related to skeletal muscle atrophy (Proserpio et al. 2013). By regulating the expression of the myostatin gene, SMYD3 inhibits myogenesis and muscle cell growth and differentiation. SMYD4 is found in the visceral, cardiac, and somatic muscle precursors in Drosophilia Melanogaster during late embryogenesis (Thompson and Travers 2008). Knockdown of SMYD4 results in eclosion failure, causing disruptions at the late pupal stage. The eclosion failure is likely due to defective muscle development due to the necessity of abdominal muscles to escape the pupal case.
The SMYD protein family is a contributing factor to the development of cancer. Overexpression of SMYD2 and SMYD3 has a tumor-promoting effect (Spellmon et al. 2015). SMYD2 is widely overexpressed in many cancers. In leukemia, the expression level of SMYD2 is almost eight times higher than normal bone marrow controls. The high expression level of SMYD2 is associated with low survival rates in leukemia patients. SMYD2 overexpression is also associated with esophageal squamous cell carcinoma (Spellmon et al. 2015). Knockdown of SMYD2 in esophageal squamous cell carcinoma inhibits tumor cell growth, with overexpression of SMYD2, and promotes proliferation. SMYD2 connects to cancer possibly through methylation of nonhistone targets. As stated earlier with the ability to methylate p53 and Rb, SMYD2 could contribute to the dysregulation of these tumor suppressor proteins. Protein p53 regulates the cell cycle; with a mutation it will lead to overgrowth and tumor formation. Protein Rb is also a tumor suppressor gene that prevents cell overgrowth. Any dysregulation of these proteins could lead to cancerous effects.
SMYD3 is overexpressed in over 15 types of cancers (Spellmon et al. 2015). Overexpression of SMYD3 upregulates a number of genes corresponding to oncogenes, homeobox genes, and genes that contribute to the cell cycle. It is shown that SMYD3 upregulates the expression of the matrix metalloproteinase MMP-9 through H3K4 trimethylation. MMP-9 is involved in tumor progression and metastasis by stimulating cell migration, tumor invasion, and angiogenesis. Suppression of SMYD3 results in reduced MMP-9 gene expression. Methylation of nonhistone substrates also connects SMYD3 to cancer. MAP3K2 methylation by SMYD3 upregulates MAP kinase signaling contributing to the tumorigenesis of Ras-driven carcinomas (Mazur et al. 2014). Ras is a family of oncogenes that is activated in a large fraction of human cancers. The methylation of VEGFR1 by SMYD3 may promote angiogenesis and enable the cancerous tumor with sufficient access to blood and oxygen consumption (Spellmon et al. 2015).
SMYD4 has been associated with breast cancer (Spellmon et al. 2015). SMYD4 represses the expression of the platelet-derived growth factor receptor (PDGFR-α) gene in breast cancer cell lines. High expression of PDGFR-α promotes tumorigenesis. This suggests a role of SMYD4 as a tumor suppressor gene in breast cancer. Recent studies show several somatic mutations in SMYD proteins, which include missense, nonsense, insertions, and deletions (Kudithipudi and Jeltsch 2014). Some of these mutations target the catalytic SET domain and could lead to gain or loss of function. A mutation in SMYD4 could induce the same effects as SMYD2 or SMYD3, leaving it without a tumor suppressor effect. Therefore, the somatic mutation of the SMYD protein family may represent additional pathways for its involvement in the development of cancer.
There is supporting evidence of the SMYD protein family playing a role in the immune system and estrogen receptor (ER)-mediated expression. SMYD5 is shown to work in the genetic program of the immune response in Drosophilia and vertebrates (Stender et al. 2012). The methylation of histone H4K20 by SMYD5 represses the inflammatory response by the suppression of toll-like receptor 4 (TLR-4)-mediated expression in macrophages. The role of other SMYD family members in immunity is unknown, though they are all expressed in macrophages (Stender et al. 2012). SMYD3 is involved in estrogen receptor-mediated expression through the H3K4 methyltransferase activity (Kim et al. 2009). SMYD3 directly interacts with the ligand-binding domain of ER, functions as a cofactor, and upregulates ER activity. SMYD3 is required for ER-regulated gene transcription in estrogen signaling pathways. Downregulation of SMYD3 represses the expression of ER target genes and reduces the H3K4 methylation level at their promotor regions. ER is a sequence-specific transcription factor that regulates a cascade of gene targets whose products mediate the initiation, development, and metastasis in breast cancer cells. Overexpression of SMYD3 has been found in breast cancer tissues and breast cancer cell lines (Spellmon et al. 2015). This, together with the regulatory role of SMYD3 in ER-mediated gene transcription, has established a potential mechanistic link between SMYD3 and breast cancer.
Additional roles of the SMYD protein family await to be explored. SMYD5 expression is high in brain, skeletal muscle, digestive system, and salivary glands (Uhlen et al. 2010). Immunohistology staining shows SMYD5 is present in squamous epithelium, gallbladder, kidney tubule, and intestinal lumen. Knockout of SMYD5 in mice shows elevated hyperactivity possibly demonstrating an uncharacterized neuropathic disorder (Ayadi et al. 2012). SMYD2 may have multiple functions. SMYD2 knockout mice show significant phenotypes in the behavior, reproductive system, renal system, immune system, and digestive and alimentary system (Uhlen et al. 2010). No significant phenotype is observed for SMYD3 knockout mice, but its gene expression is found to be associated with aging and autoimmune diseases such as rheumatoid arthritis.
SMYD protein family contributes to one of the seven classes of SET domain-containing lysine methyltransferases (Calpena et al. 2015). It contains five protein members: SMYD1, SMYD2, SMYD3, SMYD4, and SMYD5. These proteins can be grouped into three classes based on domain structures: SMYD3 (containing SMYD1 and SMYD2), SMYD4, and SMYD5. All SMYD proteins contain a SET domain interrupted by a MYND zinc finger. They also contain a C-terminal TPR motif, with the exception of SMYD5, which does not contain a TPR, and SMYD4 which contains two TPR regions. The TPR and MYND domains are key regulators in protein–protein interactions. The SET domain is required for methylation of both histone and nonhistone proteins. The SMYD protein family plays important roles in many cellular processes such as the cell cycle, chromatin remodeling, transcription, and signal transduction. SMYD proteins contribute to epigenetic changes during myogenesis and cardiomyocyte differentiation. However, there is much to be discovered regarding this functionally important protein family. SMYD proteins are of therapeutic interest due to the growing list of diseases linked to SMYD overexpression or dysfunction. Continuing knowledge of the SMYD protein family could provide valuable drug targets against cancer and cardiovascular disease.
- Ayadi A, Birling MC, Bottomley J, Bussell J, Fuchs H, Fray M, et al. Mouse large-scale phenotyping initiatives: overview of the European Mouse Disease Clinic (EUMODIC) and of the Wellcome Trust Sanger Institute Mouse Genetics Project. Mamm Genome. 2012;23:600–10. doi: 10.1007/s00335-012-9418-y.PubMedPubMedCentralCrossRefGoogle Scholar
- Calpena E, Palau F, Espinos C, Galindo MI. Evolutionary history of the Smyd Gene family in Metazoans: a framework to identify the orthologs of human Smyd Genes in Drosophila and other animal species. PloS one. 2015;10:e0134106. doi: 10.1371/journal.pone.0134106.PubMedPubMedCentralCrossRefGoogle Scholar
- Jiang Y, Trescott L, Holcomb J, Zhang X, Brunzelle J, Sirinupong N, et al. Structural insights into estrogen receptor alpha methylation by histone methyltransferase SMYD2, a cellular event implicated in estrogen signaling regulation. J Mol Biol. 2014;426:3413–25. doi: 10.1016/j.jmb.2014.02.019.PubMedCrossRefGoogle Scholar
- Proserpio V, Fittipaldi R, Ryall JG, Sartorelli V, Caretti G. The methyltransferase SMYD3 mediates the recruitment of transcriptional cofactors at the myostatin and c-Met genes and regulates skeletal muscle atrophy. Genes Dev. 2013;27:1299–312. doi: 10.1101/gad.217240.113.PubMedPubMedCentralCrossRefGoogle Scholar