| Abstract |
BACKGROUND: SMYD5 is a Su(Var)3-9, Enhancer-of-zeste and Trithorax (SET) and Myeloid, Nervy, and DEAF-1 (MYND) domain-containing histone methyltransferase, which was originally described as a component of the nuclear receptor corepressor (NCoR) complex catalyzing histone 4 lysine 20 trimethylation (H4K20me3).
RATIONALE: Based on its domain composition and on previously published data indicating its role as a methyltransferase, we sought to identify putative methylation substrates of SMYD5 in vitro. In addition, given that other members of the SMYD family, such as SMYD2 and SMYD3, have shown to play important roles in cancer promotion, including liver and colon cancer, we investigated the potential implication of SMYD5 in carcinogenesis using murine models and human samples.
APPROACH: We performed an in vitro methylation assay using the recombinant human SMYD5 protein with histone and non-histone substrates. In addition, we generated Smyd5-deficient mice, in which the Smyd5 gene was inactivated either in all tissues (Smyd5flox/MX-Cre) or specifically in the liver (Smyd5flox/Alfp-Cre and Smyd5flox/Alb-Cre). Moreover, we produced intestine-specific Smyd5-deficient mice (Smyd5flox/Villin-Cre). Analysis of SMYD5 expression in human hepatocellular carcinoma (HCC) and colon cancer was conducted using The Human Cancer Genome Atlas (TCGA) datasets.
RESULTS: We show that purified, baculovirus-expressed SMYD5 preferentially methylates histones 2A and 2B (H2A and H2B) in vitro.
All four SMYD5 mouse knockout (KO) strains generated developed normally and did not display any visible phenotype. However, when subjected to a protocol of chemically induced carcinogenesis by diethylnitrosamine (DEN) injection, whole-tissue and liver Smyd5-deficient mice exhibited delayed liver tumor growth compared to wild-type (WT) mice exposed to the same treatment. The mice were subjected to macroscopic and histological analysis, including stainings to evaluate hepatic DNA damage and proliferation, as well as expression profiling by RNA-sequencing (RNA-seq) and by quantitative polymerase chain reaction (qPCR). A number of cancer- and proliferation-related genes showed reduced expression in the liver of Smyd5-deficient mice compared to WT mice. Analysis of Smyd5flox/Villin-Cre mice subjected to chemical treatment inducing colon carcinogenesis did not indicate any obvious phenotypic differences compared to WT mice receiving the same treatment.
Consistent with the mice data, we found a significant correlation between SMYD5 expression and the incidence of hepatocellular cancer. SMYD5 expression in human liver tumor samples negatively correlated with overall patient survival and the probability of “tumor-free” survival after chemotherapy for a given time frame, but positively correlated with the probability of progressing to high-grade (G3/G4) tumors. Furthermore, based on mouse experiments and human colon cancer samples from TCGA, we concluded that SMYD5 does not seem to play a role in colon cancer, at least using the specific parameters of our analysis.
CONCLUSIONS: We found that SMYD5 presents histone methyltransferase activity in vitro, with in vivo data from SMYD5 KO mouse models and human cancer samples supporting its role in liver carcinogenesis.
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