ROLE OF THE SMYD3 METHYLASE IN EMBRYONIC STEM CELL DIFFERENTIATION AND ZEBRAFISH DEVELOPMENT

Abstract

SET and MYND domain containing protein 3 (SMYD3) is a methyltransferase that methylates lysine 5 of histone 4 (H4K5) and a group of non-histone proteins. Furthermore, in order to regulate downstream genes, SMYD3 contacts RNA polymerase II and promotes the recruitment of positive coactivator 4 (PC4) and BRD4 at the proximal promoter and coding regions of target genes. SMYD3 is overexpressed in several cancers, such as liver, breast and colorectal carcinomas, in which it promotes proliferation, migration and invasion of tumor cells. in order to promote cancer progression, SMYD3 activates the transcription of several cell cycle regulators, Myc and Ctnnb1 oncogenes, components of the IL6-Jak-Stat3 cascade and Epithelial Mesenchymal Transition (EMT) markers. Recently, a novel role for SMYD3 in regulating homologous recombination (HR) repair and maintaining genome stability has been reported. SMYD3 plays a cytoplasmic role by methylating non-histone proteins. Indeed, SMYD3 methylates MAP3K2, vascular endothelial growth factor receptor 1 (VEGFR1), v-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1), and Human Epidermal Growth Factor Receptor 2 (HER2). Few evidences highlighted a possible role of SMYD3 in regulating developmental processes. In zebrafish, SMYD3 is required for correct maturation of cardiomyocytes and for trunk morphogenesis. However, the SMYD3-mediated molecular mechanisms, its functional role in development and its interplay with other proteins involved in development are still elusive. Aim of the present PhD project was to better understand SMYD3 role throughout development, by using mESCs and zebrafish as model systems. After knocking down SMYD3 in mESCs, we observed an upregulation of several mesendodermal markers at different time points of differentiation. In parallel, an increase in mesendodermal markers was detectable in smyd3 knocked down zebrafish embryos. Since SMYD3 is involved in cardiomyocyte maturation in zebrafish, we knocked down SMYD3 and prompted mESCs to differentiate towards cardiomyocytes by forming embryoid bodies (EBs). As a result, we observed an upregulation of early and late cardiovascular markers in SMYD3 depleted EBs. These data were corroborated by an upregulation of the endothelial marker kdrl in smyd3 morphants. Taken together, our results suggest that SMYD3 negatively modulates mesendodermal commitment during mESC and zebrafish development. As a second aim, we wanted to investigate the molecular mechanisms underlying SMYD3-mediated regulation of EMT. We assessed this aim by providing a link between TGFβ/SMAD signaling pathway, which is an important regulator of EMT during tumor progression, and SMYD3. Indeed, we demonstrated that SMYD3 directly interacts with SMAD3. Furthermore, the ability of SMYD3 inhibitor BCI-121 to impair cancer cell migration has been proved in zebrafish as a xenograft cancer model.