SMYD3 and its biological functions.
SET and MYND domain containg-3 is a member of the lysine methyltransferase family proteins, which requires S-adenosyl methionine (1) as a cofactor to methylate its substrates (2). Methylation of specific lysine residues serves as a post-translational epigenetic modification (PTM) that controls the expression of genes. It can function either by directly influence the organization of chromatin through altering histone-DNA and histone-histone interactions, or by cooperating with effector proteins which are referred to as readers of PTMs (3).
SMYD3 contains a SET domain that is divided into two segments by a MYND domain, followed by a cysteine-rich post-SET domain, and an extended C-terminal domain (CTD) (4). The MYND domain is a zinc finger motif that is important for protein-protein interaction (5). The SET domain is a conserved catalytic unit for lysine methylation that is found in nearly all protein lysine methyltransferases (6), and deletion of either the NHSC or GEEL motifs within the SET domain abolished its activity (7).
The CTD domain could form a cap to bind substrates effectively and selectively (8).
Genetic mouse models for SMYD3 depletion provide hints for investigating its functions. Smyd3 knockout mice develop normally and are fertile (9,10). However, Smyd3 knockout mouse embryos show defects in the growth in vitro and a reduction in
the number of viable offspring, which may be due to the suppression of the pluripotency genes, such as Oct4, Nanog and Sox2 (11). In addition, using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, the abrogating SMYD3 catalytic activity inhibits tumor development in response to oncogenic Ras (10).
SMYD3 is highly expressed in colorectal carcinomas, hepatocellular carcinomas, pancreatic cancer, prostate cancer, and breast cancer (7,12,13). Its high expression correlates with an aberrant pattern of histone modifications which causes abnormal gene expression (Appendix Fig. 1). SMYD3 regulates gene transcription through methylating histone substrates, including H3K4me2/3 (14), H4K20me2/3 (4), H4K5me1/2/3 (9) and H2A.ZK101 (15). For example, SMYD3 methylates H2A.Z to activate cyclin A1 expression and drive cancer proliferation (15), and H3K4 to upregulate MMP9 (16) and hTERT expression (17). Moreover, SMYD3 modifies non-histone proteins VEGFR and MAP3K2 to promote metastasis (18) and Ras/Raf/MEK/ERK signaling (10) in cancer development, respectively. SMYD3 mainly locates in the cytoplasm at G0/G1 phases and moves to the nucleus at S/G2 phases (19). Therefore, the function of SMYD3 may depend on its location.
The involvement of SMYD3 in human pathology is not restricted to cancer.
SMYD3 controls the proper development of skeletal muscle via the regulation of c-met and myostatin (20), a critical negative regulator of cell differentiation and muscle mass (21). SMYD3-depleted cell culture and the animal model show decreased expression of c-met and myostatin, resulting in protection against glucocorticoid-induced muscle atrophy. Notably, SMYD3 are more abundantly expressed in skeletal muscle compared with other tissues (19), which enables it an attractive target for the treatment of muscle disease with increased specificity.
DNA double-strand breaks repair pathways.
Cells inevitably encounter the challenge of chromosomal double-strand breaks (DSBs) during their lifetime. The oxidative byproducts of the normal metabolic process and exogenous factors such as chemical agents or ionizing radiation (IR) constantly
threaten the integrity of our genome. Unrepaired or misrepaired DNA lesions can lead to genome instability, which is a hallmark of cancer (22,23). Two major pathways, homologous recombination and non-homologous end joining (NHEJ), are responsible for repairing these breaks.
HR occurs predominantly at S and G2 phases when a sister chromatid is accessible (24). The repair is initiated by a resection process, which includes MRE11-RAD50-NBS1 (MRN) end sensing complex, CtIP endonuclease (25), EXO1 exonuclease (26), and BLM helicase (27), to remove oligonucleotides from each side of the DSB and expose single-stranded DNA (ssDNA) tails for forming RAD51-ssDNA filaments with the help of BRCA2 (28). Working in concert with RAD51-ssDNA filaments, RAD54B, a DNA-dependent ATPase, drives the search for a homologous template and strand invasion (29,30), which leads to accurate repair. Classical NHEJ (C-NHEJ) occurs throughout the cell cycle but predominately at G1 phase. During C-NHEJ, cells utilize Ku70/Ku80 heterodimer and DNA-dependent protein kinase (DNA-PK) to recognize and ligate DSB ends via little (less than ten base pairs) or no homology between the joined ends, which is, therefore, an error-prone pathway (31,32).
Besides, alternative end-joining pathways, such as microhomology-mediated end joining (MMEJ), do not use Ku- and DNA-PK. Initial resection produces relatively longer stretches of microhomology (5-25 base pairs), and subsequent flap trimming and end-joining often create the final mutagenic MMEJ repair products (33).
DSB repair is facilitated through chromatin modifications to open the compact barriers and improve the accessibility of repair proteins (34). For example, the rapid phosphorylation of H2A.X at S139 in mammals (forming γH2A.X) by ATM within minutes at DSB sites is considered as a major hallmark of DSB recognition (35,36).
γH2A.X further interacts with the mammalian repair mediator MDC1. MDC1 recruits RNF8 (37) and RNF168 (38) to catalyze K63-ubiquitilation on H2A and H2A.X to recruit BRCA1 and 53BP1 for HR and NHEJ, respectively (37,39-41). While the posttranslational modifications of proteins in DSB repair have been broadly studied (42,43), the evidence of transcriptional regulation of DSB repair proteins is comparatively scarce.
SMYD3 is involved in homologous recombination.
Previous studies have focused on the ability of how SMYD3 stimulates cell proliferation and metastasis. Here, we identify a new role of SMYD3 in regulating HR repair. After IR treatment, SMYD3-depleted cells showed a delay in the removal of γH2A.X foci, and are more vulnerable to the IR stress. The ratio of DNA breaks and the gross chromosomal rearrangement are increased in SMYD3-deficient cells post IR treatment. Moreover, inhibition of SMYD3 directly blunts HR efficiency by downregulating the expression of HR-related genes. Additionally, SMYD3 knockdown leads to decreased methylation of H3K4 and recruitment of RNA polymerase II (RNAPII) at the target gene promoters. These data reveal that SMYD3 maintains genome stability by ensuring normal expression levels of HR repair proteins.