Mol. Cells 2015; 38(6): 580-586
Published online May 22, 2015
https://doi.org/10.14348/molcells.2015.0053
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: tlhuh@knu.ac.kr (TLH); mj.kim.lucky@gmail.com (MJK)
While increasing evidence indicates the important function of histone methylation during development, how this process influences cardiac development in vertebrates has not been explored. Here, we elucidate the functions of two histone H3 lysine 4 (H3K4) methylation enzymes, SMYD3 and SETD7, during zebrafish heart morphogenesis using gene expression profiling by whole mount
Keywords heart morphogenesis, histone methyltransferase, SETD7, SMYD3, zebrafish
During vertebrate development, heart is formed by a highly stereotypic and organized process, and the expression of genes in these processes need be tightly regulated in a spatiotemporal manner (Barnett et al., 2012; Wang, 2012). Recent reports suggest that epigenetic regulation modulates genetic program during differentiation and development of various organs including heart and somitic lineages (Wamstad et al., 2012). For instance, modifications of histone substantially alter the accessibility and structure of chromatin, therefore modifying transcriptional activity of specific gene clusters. Histones are known to undergo specific modification such as methylation and acetylation: while methylation and demethylation of histone are coordinated by histone methyltransferases (HMTs) and demethylase (HDMs), acetylation of histone is modulated by acetyltransferases (HAT) and deacetylases (HDAC) (Bhaumik et al., 2007; Helin and Dhanak, 2013). Due to their significance in regulating genetic program during development and diseases, recent efforts were directed to identify specific factors that mediate this process (Arrowsmith et al., 2012; Cayuso Mas et al., 2011; Kim et al., 2012a; 2012b). In case of methylation, histone H3 lysine 4 residue (H3K4) is sequentially modified through mono-, di-, and tri-methylation status, each of which step is mediated by specific enzymes. MLLs (Mll1?5), SETDB1, and SETDB2 have broad roles for H3K4 methylation, and can methylate all type of H3K4, such as non-, mono-, di-methylated H3K4 (Black et al., 2012; Sims and Reinberg, 2004). In contrast, SMYD3 methylate mono- and di-methylated H3K4, and SETD7 can only catalase the non-methylated H3K4 to transit to mono-methylated H3K4 (Hamamoto et al., 2004; Wang et al., 2001).
SET and MYND domain-containing proteins (SMYDs), involving SMYD1?5, are high conserved protein family across plant, fungi, and animal as well as some protozoa, and have two functional protein domains, SET and MYND domains (Del Rizzo and Trievel, 2011; Dillon et al., 2005). Especially, SET domain is important for histone lysine methylation activity and MYND domain can mediate the protein-protein interaction and bind to DNA motifs. SMYD3 was originally reported as histone lysine methyltransferase (HMT), which methlylates the mono-(H3K4me1) and di-methylated lysine 4 residue (H3K4me2) to generate H3K4me3, of histone H3, but SMYD3 also can bind to 5′-CCCTCC-3′ motif on promoter region of Nkx2.8 gene to induce its expression in cancer cells (Hamamoto et al., 2004). Recent researches showed the global decrease of histone H4 lysine 5 (H4K5) methylation in SMYD3 knock-down condition and structural preference for histone H4 lysine 20 (H4K20) of SMYD3, suggesting the substrate diversity of SMYD3 (Foreman et al., 2011; Van Aller et al., 2012). It is known that SMYD3 is highly expressed in a various cancers, including liver, prostate, rectal, and breast carcinomas, and stimulates the oncogenic activities, such as cell proliferation, adhesion and migration, of tumor cells (Frank et al., 2006; Hamamoto et al., 2006; Silva et al., 2008). In addition, SMYD3 is essential for myogenic differentiation through MyoD regulation (Proserpio et al., 2013). Actually, the deficient zebrafish embryos displayed the developmental cardiac and somite abnormalities, illuminating its functions in cardiac and somite muscle formation (Fujii et al., 2011).
SET domain containing protein 7 (SETD7), also termed as SET7/9, is another type of histone lysine methyltransferase (HMT) and only have SET domain for methyltransferase activity, but not MYND domain (Wang et al., 2001). It was initially discovered as a specific methyltransferase for only non-methylated histone H3 lysine 4 (H3K4) that converted to mono-methylated histone H3 lysine 4 (H3K4me1) (Wang et al., 2001). Subsequently, recent accumulating evidence suggests that non-histone proteins, such as Yap, DNMT1, E2F1, and STAT3, are also the substrates for SETD7 (Est?ve et al., 2009; Oudhoff et al., 2013; Pradhan et al., 2009; Yang et al., 2010). The mouse knock out allele of SETD7 is a viable and did not show any developmental and gross defect (Campaner et al., 2011; Lehnertz et al., 2011). In zebrafish developmental model, knock-down of
Here, we examined the function of
Zebrafish AB strain as wild-type control for all experiments, expect the experiments using
Control,
To make digoxigenin-labelled antisense ribo-probe, zebrafish
To generate the synthetic
To delineate functions of HMTs, such as
To further delineate the roles of HMTs in heart development, we carried out knock-down experiments of SMYD3 and SETD7. First, to determine the functional requirement of SMYD3 during zebrafish heart development, we knocked SMYD3 down with a morpholino (MO)-based gene targeting system. For this purpose, splicing blocking MO that binds to the junction of exon2 and intron2 of
Similarly, we examined the function of SETD7 during development. MOs targeting
During histone H3 lysine 4 (H3K4) methylation, SETD7 can only methylate non-methylated H3K4 to produce mono-methylated H3K4 (H3K4me1), which then is further methylated by SMYD3 to become H3K4me2 and H3K4me4 (Hamamoto et al., 2004; Wang et al., 2001). Individual roles of SMYD3 and SETD7 during cardiac and skeletal muscle development in zebrafish have been reported (Fujii et al., 2011; Tao et al., 2011). Given that SMYD3 and SETD7 successively methylate H3K4, we can assume that these two enzymes function synergistically during zebrafish development. To test this possibility, we examined whether co-injection of
Next, we delineated the synergistic roles of SMYD3 and SETD7 in zebrafish heart development. For this purpose, we used the heart-specific transgenic fish
To test whether the developing heart defects in the SMYD3-and SETD7-deficient embryos are caused by the expressional changes of early cardiac muscle genes, we determined the early expressions of
To investigate whether enforced expression of
Taken together, our results indicate that methylation can provide essential regulatory input during organ development in zebrafish. Since similar developmental defects were observed in embryos with either excessive or reduced level of SMDY3 and/or SETD7 activity, it appears that appropriate level of histone methylation is critical to ensure the formation of cardiac and skeletal muscle in zebrafish.
Heart morphogenesis in vertebrate development is a highly complicated event that achieved by correct cell specification, migration, proliferation, apoptosis, and transition as well as controlled beating of heart myocytes (Barnett et al., 2012). In this process, proper gene expressions for structure of cardiac chamber at early developmental stages are critical for late heart forming event. Our data presented here indicates that proper level of histone methylation, which is mediated by a series of enzymes commonly known as HMTs, including
During zebrafish development, the expression pattern of HMTs indicates that transcripts encoding these proteins may be maternally deposited. In addition, considering their ubiquitous expression, these proteins may provide essential functions in development by regulating histone methylation for all types of cells (Figs. 1 and 2). However, knock-down of
It was report that knocked-down
Interestingly, in skeletal muscle development, SETD7 directly interacts with MyoD to induce myogenin and MEF2 expression to generate differentiated myocytes (Tao et al., 2011). Therefore, it is tempting to speculate that SETD7 manipulation in zebrafish may lead to the alteration in expression of certain structural genes. However, we did not find any discernible change in the expression of
Here, we have shown the knock-down and overexpression phenotypes of SMYD3 and SETD7 in zebrafish development. The results suggest that H3K4 methyltransferases are crucial genetic regulators for normal heart development. These findings extend our current knowledge of the roles of HMTs and increase our understanding of the functional mechanism of HMTs in heart development.
Mol. Cells 2015; 38(6): 580-586
Published online June 30, 2015 https://doi.org/10.14348/molcells.2015.0053
Copyright © The Korean Society for Molecular and Cellular Biology.
Jun-Dae Kim1,4,5, Eunmi Kim1,5, Soonil Koun1,5, Hyung-Jin Ham1, Myungchull Rhee2, Myoung-Jin Kim1,*, and Tae-Lin Huh1,3,*
1School of Life Science and Biotechnology (BK 21 plus program), Kyungpook National University, Daegu 702-701, Korea, 2Department of Biological Sciences, College of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea, 3Korea Basic Science Institute Daegu Center, Daegu 702-701, Korea, 4Present address: Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA
Correspondence to:*Correspondence: tlhuh@knu.ac.kr (TLH); mj.kim.lucky@gmail.com (MJK)
While increasing evidence indicates the important function of histone methylation during development, how this process influences cardiac development in vertebrates has not been explored. Here, we elucidate the functions of two histone H3 lysine 4 (H3K4) methylation enzymes, SMYD3 and SETD7, during zebrafish heart morphogenesis using gene expression profiling by whole mount
Keywords: heart morphogenesis, histone methyltransferase, SETD7, SMYD3, zebrafish
During vertebrate development, heart is formed by a highly stereotypic and organized process, and the expression of genes in these processes need be tightly regulated in a spatiotemporal manner (Barnett et al., 2012; Wang, 2012). Recent reports suggest that epigenetic regulation modulates genetic program during differentiation and development of various organs including heart and somitic lineages (Wamstad et al., 2012). For instance, modifications of histone substantially alter the accessibility and structure of chromatin, therefore modifying transcriptional activity of specific gene clusters. Histones are known to undergo specific modification such as methylation and acetylation: while methylation and demethylation of histone are coordinated by histone methyltransferases (HMTs) and demethylase (HDMs), acetylation of histone is modulated by acetyltransferases (HAT) and deacetylases (HDAC) (Bhaumik et al., 2007; Helin and Dhanak, 2013). Due to their significance in regulating genetic program during development and diseases, recent efforts were directed to identify specific factors that mediate this process (Arrowsmith et al., 2012; Cayuso Mas et al., 2011; Kim et al., 2012a; 2012b). In case of methylation, histone H3 lysine 4 residue (H3K4) is sequentially modified through mono-, di-, and tri-methylation status, each of which step is mediated by specific enzymes. MLLs (Mll1?5), SETDB1, and SETDB2 have broad roles for H3K4 methylation, and can methylate all type of H3K4, such as non-, mono-, di-methylated H3K4 (Black et al., 2012; Sims and Reinberg, 2004). In contrast, SMYD3 methylate mono- and di-methylated H3K4, and SETD7 can only catalase the non-methylated H3K4 to transit to mono-methylated H3K4 (Hamamoto et al., 2004; Wang et al., 2001).
SET and MYND domain-containing proteins (SMYDs), involving SMYD1?5, are high conserved protein family across plant, fungi, and animal as well as some protozoa, and have two functional protein domains, SET and MYND domains (Del Rizzo and Trievel, 2011; Dillon et al., 2005). Especially, SET domain is important for histone lysine methylation activity and MYND domain can mediate the protein-protein interaction and bind to DNA motifs. SMYD3 was originally reported as histone lysine methyltransferase (HMT), which methlylates the mono-(H3K4me1) and di-methylated lysine 4 residue (H3K4me2) to generate H3K4me3, of histone H3, but SMYD3 also can bind to 5′-CCCTCC-3′ motif on promoter region of Nkx2.8 gene to induce its expression in cancer cells (Hamamoto et al., 2004). Recent researches showed the global decrease of histone H4 lysine 5 (H4K5) methylation in SMYD3 knock-down condition and structural preference for histone H4 lysine 20 (H4K20) of SMYD3, suggesting the substrate diversity of SMYD3 (Foreman et al., 2011; Van Aller et al., 2012). It is known that SMYD3 is highly expressed in a various cancers, including liver, prostate, rectal, and breast carcinomas, and stimulates the oncogenic activities, such as cell proliferation, adhesion and migration, of tumor cells (Frank et al., 2006; Hamamoto et al., 2006; Silva et al., 2008). In addition, SMYD3 is essential for myogenic differentiation through MyoD regulation (Proserpio et al., 2013). Actually, the deficient zebrafish embryos displayed the developmental cardiac and somite abnormalities, illuminating its functions in cardiac and somite muscle formation (Fujii et al., 2011).
SET domain containing protein 7 (SETD7), also termed as SET7/9, is another type of histone lysine methyltransferase (HMT) and only have SET domain for methyltransferase activity, but not MYND domain (Wang et al., 2001). It was initially discovered as a specific methyltransferase for only non-methylated histone H3 lysine 4 (H3K4) that converted to mono-methylated histone H3 lysine 4 (H3K4me1) (Wang et al., 2001). Subsequently, recent accumulating evidence suggests that non-histone proteins, such as Yap, DNMT1, E2F1, and STAT3, are also the substrates for SETD7 (Est?ve et al., 2009; Oudhoff et al., 2013; Pradhan et al., 2009; Yang et al., 2010). The mouse knock out allele of SETD7 is a viable and did not show any developmental and gross defect (Campaner et al., 2011; Lehnertz et al., 2011). In zebrafish developmental model, knock-down of
Here, we examined the function of
Zebrafish AB strain as wild-type control for all experiments, expect the experiments using
Control,
To make digoxigenin-labelled antisense ribo-probe, zebrafish
To generate the synthetic
To delineate functions of HMTs, such as
To further delineate the roles of HMTs in heart development, we carried out knock-down experiments of SMYD3 and SETD7. First, to determine the functional requirement of SMYD3 during zebrafish heart development, we knocked SMYD3 down with a morpholino (MO)-based gene targeting system. For this purpose, splicing blocking MO that binds to the junction of exon2 and intron2 of
Similarly, we examined the function of SETD7 during development. MOs targeting
During histone H3 lysine 4 (H3K4) methylation, SETD7 can only methylate non-methylated H3K4 to produce mono-methylated H3K4 (H3K4me1), which then is further methylated by SMYD3 to become H3K4me2 and H3K4me4 (Hamamoto et al., 2004; Wang et al., 2001). Individual roles of SMYD3 and SETD7 during cardiac and skeletal muscle development in zebrafish have been reported (Fujii et al., 2011; Tao et al., 2011). Given that SMYD3 and SETD7 successively methylate H3K4, we can assume that these two enzymes function synergistically during zebrafish development. To test this possibility, we examined whether co-injection of
Next, we delineated the synergistic roles of SMYD3 and SETD7 in zebrafish heart development. For this purpose, we used the heart-specific transgenic fish
To test whether the developing heart defects in the SMYD3-and SETD7-deficient embryos are caused by the expressional changes of early cardiac muscle genes, we determined the early expressions of
To investigate whether enforced expression of
Taken together, our results indicate that methylation can provide essential regulatory input during organ development in zebrafish. Since similar developmental defects were observed in embryos with either excessive or reduced level of SMDY3 and/or SETD7 activity, it appears that appropriate level of histone methylation is critical to ensure the formation of cardiac and skeletal muscle in zebrafish.
Heart morphogenesis in vertebrate development is a highly complicated event that achieved by correct cell specification, migration, proliferation, apoptosis, and transition as well as controlled beating of heart myocytes (Barnett et al., 2012). In this process, proper gene expressions for structure of cardiac chamber at early developmental stages are critical for late heart forming event. Our data presented here indicates that proper level of histone methylation, which is mediated by a series of enzymes commonly known as HMTs, including
During zebrafish development, the expression pattern of HMTs indicates that transcripts encoding these proteins may be maternally deposited. In addition, considering their ubiquitous expression, these proteins may provide essential functions in development by regulating histone methylation for all types of cells (Figs. 1 and 2). However, knock-down of
It was report that knocked-down
Interestingly, in skeletal muscle development, SETD7 directly interacts with MyoD to induce myogenin and MEF2 expression to generate differentiated myocytes (Tao et al., 2011). Therefore, it is tempting to speculate that SETD7 manipulation in zebrafish may lead to the alteration in expression of certain structural genes. However, we did not find any discernible change in the expression of
Here, we have shown the knock-down and overexpression phenotypes of SMYD3 and SETD7 in zebrafish development. The results suggest that H3K4 methyltransferases are crucial genetic regulators for normal heart development. These findings extend our current knowledge of the roles of HMTs and increase our understanding of the functional mechanism of HMTs in heart development.
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