MYND Domain-containing Protein Research Articles

Conditional inactivation of PDCD2 induces p53 activation and cell cycle arrest

ABSTRACTPDCD2 (programmed cell death domain 2) is a highly conserved, zinc finger MYND domain-containing protein essential for normal development in the fly, zebrafish and mouse. The molecular functions and cellular activities of PDCD2 remain unclear. In order to better understand the functions of PDCD2 in mammalian development, we have examined PDCD2 activity in mouse blastocyst embryos, as well as in mouse embryonic stem cells (ESCs) and embryonic fibroblasts (MEFs). We have studied mice bearing a targeted PDCD2 locus functioning as a null allele through a splicing gene trap, or as a conditional knockout, by deletion of exon2 containing the MYND domain. Tamoxifen-induced knockout of PDCD2 in MEFs, as well as in ESCs, leads to defects in progression from the G1 to the S phase of cell cycle, associated with increased levels of p53 protein and p53 target genes. G1 prolongation in ESCs was not associated with induction of differentiation. Loss of entry into S phase of the cell cycle and marked induction of nuclear p53 were also observed in PDCD2 knockout blastocysts. These results demonstrate a unique role for PDCD2 in regulating the cell cycle and p53 activation during early embryonic development of the mouse.

Structural insights into ERα methylation by histone methyltransferase SMYD2, a cellular event implicated in estrogen signaling regulation (LB267)

Estrogen receptor (ER) signaling plays a pivotal role in many developmental processes and has been implicated in numerous diseases including cancers. We recently showed that direct ERα methylation by the multi‐specificity histone lysine methyltransferase, SET and MYND domain‐containing protein 2 (SMYD2), regulates estrogen signaling through repressing ERα‐dependent transactivation. However, the mechanism controlling the specificity of the SMYD2‐ERα interaction and the structural basis of SMYD2 substrate binding diversity are unknown. Here we present the crystal structure of SMYD2 in complex with a target lysine (Lys266)‐containing ERα peptide. The structure reveals that ERα binds SMYD2 in a U‐shaped conformation with the binding specificity determined mainly by residues C‐terminal to the target lysine. The structure also reveals numerous intrapeptide contacts that ensure shape complementarity between the substrate and the active site of the enzyme, thereby likely serving as an additional structural determinant of substrate specificity. In addition, comparison of the SMYD2‐ERα and SMYD2‐p53 structures provides the first structural insight into the diverse nature of SMYD2 substrate recognition and suggests that the broad specificity of SMYD2 is achieved by multiple molecular mechanisms such as distinct peptide binding modes and the intrinsic dynamics of peptide ligands. Strikingly, a novel potentially SMYD2‐specific polyethylene glycol (PEG) binding site is identified in the C‐terminal domain, implicating possible functions in additional substrate binding or protein‐protein interactions. Our study thus provides the structural basis for the SMYD2‐mediated ERα methylation, and the resulting knowledge of SMYD2 substrate specificity and target‐binding diversity could have important implications in selective drug design against a wide range of ERα‐related diseases.Grant Funding Source: Supported by Leukemia Research Foundation, Aplastic Anemia & MDS International Foundation and American Heart Association.

Increased progesterone receptor A expression in labouring human myometrium is associated with decreased promoter occupancy by the histone demethylase JARID1A

Progesterone regulates female reproductive function predominantly through two nuclear progesterone receptors (PRs), PR-A and PR-B. During human parturition myometrial PR expression is altered to favour PR-A, which activates pro-labour genes. We have previously identified histone H3 lysine 4 trimethylation (H3K4me3) as an activator of myometrial PR-A expression at labour. To further elucidate the mechanisms regulating PR isoform expression in the human uterus at labour, we have (i) determined the methylation profile of the cytosine-guanine dinucleotides (CpG) island in the promoter region of the PR gene and (ii) identified the histone-modifying enzymes that target the H3K4me3 mark at the PR promoters in term and preterm human myometrial tissues obtained before and after labour onset. Bisulphite sequencing showed that despite overall low levels of PR CpG island methylation, there was a significant decrease in methylated CpGs with labour in both preterm (P < 0.05) and term (P < 0.01) groups downstream of the PR-B transcription start site. This methylation change was not associated with altered PR-B expression, but may contribute to the increase in PR-A expression with labour. Chromatin immunoprecipitation revealed that the histone methyltransferase, SET and MYND domain-containing protein 3 (SMYD3), bound to the PR gene at significantly higher levels at the PR-A promoter compared with the PR-B promoter (P < 0.010), with no labour-associated changes observed. The H3K4 demethylase, Jumonji AT-rich interactive domain 1A (JARID1A), also bound to the PR-A, but not to the PR-B promoter prior to term labour, and decreased significantly at the onset of labour (P = 0.014), providing a mechanism for the previously reported increase in H3K4me3 level and PR-A expression with labour. Our studies suggest that epigenetic changes mediated by JARID1A, SMYD3 and DNA methylation may be responsible, at least in part, for the functional progesterone withdrawal that precipitates human labour.

Abstract 5290: MicroRNA-495-3p functions as tumor suppressor by targeting multiple oncogenic epigenetic effector molecules in gastric cancer.

Abstract Epigenetic effector molecules are involved in many biological processes by regulating post-translational histone modification and DNA methylation. Recent evidence has suggested that several epigenetic effectors are aberrantly over-expressed in gastric cancer (GC). However, underlying regulatory mechanisms of those molecules are not completely understood. In this study, we identified miR-495-3p which regulates multiple epigenetic effectors through a comprehensive miRNA profiling analysis with in silico target prediction in GC. The down-regulation of miR-495-3p was demonstrated in a subset of GC tissue, revealing inverse correlation with expression levels of epigenetic effectors that were predicted to be targeted by miR-495-3p. Ectopic overexpression of miR-495-3p in GC cells suppressed protein expression and 3’-UTR (untranslated region) reporter luciferase activity of multiple epigenetic effectors including histone deacetylase 2 (HDAC2), SET and MYND domain-containing protein 3 (SMYD3), DNA methyltransferase 1 (DNMT1) and metastasis-associated protein1 (MTA1). Moreover, miR-495-3p attenuated cancer cell proliferation accompanied with LC3B-II conversion, PARP cleavage, p16INK4A expression and down-regulation of CDK4/6 and cyclin D1, which were implicated in autophagic cell death, apoptosis and cell cycle regulation, respectively. In addition, ectopically over-expressed miR-495-3p inhibits cell migration via suppressing epithelial-mesenchymal transition (EMT)-related molecules, Snail and SLUG. Taken together, we suggest that a loss or down-regulation of endogenous miR-495-3p triggers aberrant overexpression of multiple oncogenic epigenetic effector molecules, and thereby miR-495-3p functions as a tumor suppressor in gastric tumorigenesis. Citation Format: Min Gyu Kim, Seung Jin Kim, Hyun Jin Bae, Ji Heon Noh, Jeong Kyu Kim, Jung Woo Eun, Kwang Hwa Jung, Young Gyoon Chang, Qingyu Shen, Won Sang Park, Jung Young Lee, Suk Woo Nam. MicroRNA-495-3p functions as tumor suppressor by targeting multiple oncogenic epigenetic effector molecules in gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5290. doi:10.1158/1538-7445.AM2013-5290 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 3145: Smyd3 catalyzes a novel methylation mark and regulates cancer cell proliferation

Abstract SET and MYND domain-containing protein 3 (Smyd3) is a lysine methyltransferase over-expressed in liver, breast and rectal carcinomas. We have shown that knockdown of Smyd3 has an anti-proliferative effect in cancer cell lines and that this effect can be rescued with wild type but not catalytically inactive Smyd3, directly linking Smyd3 catalytic activity to proliferation. Published reports indicate Smyd3 methylates lysine 4 of histone H3, lysine 20 of histone H4 and VEGFR1. However, activity testing of a peptide library and intact recombinant nucleosomes indicate that a unique methylation mark is catalyzed by Smyd3. LC-MS/MS analysis of in vitro reaction products have identified the location of this novel site of methylation. The existence of this novel mark and its dependence on Smyd3 activity was confirmed in cancer cells and mouse embryonic fibroblasts. We speculate that overexpression of Smyd3 causes aberrant methylation and plays an important role in the deregulation of gene expression leading to uncontrolled cellular proliferation in cancer making Smyd3 a promising target for anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3145. doi:1538-7445.AM2012-3145

High expression of trimethylated histone H3 lysine 4 is associated with poor prognosis in hepatocellular carcinoma

Tumor-associated epigenetic alterations including DNA methylation and histone modifications are important determinants in the initiation and progression of hepatocellular cancer (HCC) and represent promising biomarkers and therapeutic targets. Locus-specific trimethylation of histone H3 lysine 4 (H3K4me) is a well-known modification linked to the enhanced transcriptional expression of many genes activated in HCC. Our aim was to assess the cellular expression pattern of H3K4me3 in HCC and its association with clinicopathologic variables and outcome. Expression of H3K4me3 and the histone methyltransferase (HMT) SET and MYND domain-containing protein 3 (SMYD3) was studied by Western blotting and immunohistochemistry in cell lines and tumor tissue microarray from a well-characterized series of HCC patients (n = 168). The optimal cut-point of H3K4me3 expression for prognosis was determined by the X-tile program. The prognostic significance was evaluated using Kaplan-Meier survival estimates and log-rank tests. Tumor tissue microarray from another independent HCC patients cohort (n = 147) was used for validation studies. Expression of H3K4me3 and SMYD3 were enhanced in HCC cell lines. In tumor specimens, enhanced expression of H3K4me3 was correlated with reduced overall survival (P < .0001), especially in early-stage HCC patients (TNM I/II). Furthermore, both univariate and multivariate analyses revealed that H3K4me3 level was a significant and independent predictor of poor survival (hazard ratio, 3.592; 95% confidence interval, 2.302-5.605). In addition, H3K4m3 expression was positively correlated with SMYD3 expression in both testing and validation cohorts (P < .0001). In conclusion, H3K4me3 level defines unrecognized subsets of HCC patients with distinct epigenetic phenotype and clinical outcome and can thus be a novel predictor for poor prognosis of HCC patients, especially at TNM I/II stage.

The role of NeuroD1 in the upregulation of SMYD3 by HBx.

183 Background: SET and MYND Domain-Containing Protein 3 (SMYD3) is frequently overexpressed in hepatocellular carcinoma (HCC) exhibiting increased malignant phenotypes. It has also been known that the hepatitis B virus x protein (HBx) is strongly associated with HCC development and progression. Although overexpression of both proteins is related to HCC, the relationship between the two has not been well studied. Methods: Immunohistochemical staining was used to detect the expression of HBx and SMYD3 in HCC tumor tissues. HBx gene transfection, RNAi, and histone methyltransferase(H3-K4) activity assay were performed to reveal the transcrpitionally activation of HBx on functional SMYD3 gene expression. Chromatin immunoprecipitation (ChIP), Co-immunoprecipitation (Co-IP), Electrophoretic mobility shift assay (EMSA) were applied to investigate the underlying mechanism. Dual-luciferase reporter assay was used to search for the HBx responsive cis-element of SMYD3 gene. Results: Immunohistochemistry identified the positive correlation between HBx and SMYD3 expression in 42 HCC tissues. Up-regulation of HBx on SMYD3 expression was validated through experiments involving overexpression or knock-down of HBx in different HCC cell lines. And up-regulated SMYD3 is functionally active as histone methyltransferase. Next we found that HBx transcriptionally regulated SMYD3 gene expression by interacting with RNA polymerase IIand altering its binding site to a proximal promoter region(SD2) from a distant promoter region(SD6) of SMYD3. Truncated and mutant reporter assays revealed that the cis-element mapped in -178~-203bp in SMYD3 promotor is responsive for HBx-transactivation. And this 25bp cis-element contains a E-box 3 unit, which is a binding site for the transcriptional factor Neurogenic differentiation 1(NeuroD1). EMSA and Chip showed that HBx increased NeuroD1 binding to SMYD3 proximal promotor, however transcient expression of antisense NeuroD1 abolished HBx-induced SMYD3 expression. Conclusions: HBx transcriptionally up-regulates SMYD3 and that this process is mediated by NeuroD1 through binding to the E-box 3 site of SMYD3 promotor.

Biochemical Characterization of Human SET and MYND Domain-Containing Protein 2 Methyltransferase

SET and MYND domain-containing protein 2 (SMYD2) is a protein lysine methyltransferase that catalyzes the transfer of methyl groups from S-adenosylmethionine (AdoMet) to acceptor lysine residues on histones and other proteins. To understand the kinetic mechanism and the function of individual domains, human SMYD2 was overexpressed, purified, and characterized. Substrate specificity and product analysis studies established SMYD2 as a monomethyltransferase that prefers nonmethylated p53 peptide substrate. Steady-state kinetic and product inhibition studies showed that SMYD2 operates via a rapid equilibrium random Bi Bi mechanism at a rate of 0.048 ± 0.001 s(-1), with K(M)s for AdoMet and the p53 peptide of 0.031 ± 0.01 μM and 0.68 ± 0.22 μM, respectively. Metal analyses revealed that SMYD2 contains three tightly bound zinc ions that are important for maintaining the structural integrity and catalytic activity of SMYD2. Catalytic activity was also shown to be dependent on the GxG motif in the S-sequence of the split SET domain, as a G18A/G20A double mutant and a sequence deletion within the conserved motif impaired AdoMet binding and significantly decreased enzymatic activity. The functional importance of other SMYD2 domains including the MYND domain, the cysteine-rich post-SET domain, and the C-terminal domain (CTD), were also investigated. Taken together, these results demonstrated the functional importance of distinct domains in the SMYD family of proteins and further advanced our understanding of the catalytic mechanism of this family.

Hepatitis C virus core upregulates the methylation status of the RASSF1A promoter through regulation of SMYD3 in hilar cholangiocarcinoma cells

Increasing evidence has been accumulated indicating the important role of epigenetic regulation in tumor genesis. Previously, we observed that the transfection of hepatitis C virus core (HCVc) protein led to malignant transformation in normal biliary cells, and that tumor suppressor gene RASSF1A was downregulated in many hilar cholangiocarcinoma patients by hypermethylation in the promoter region. In the present study, we found SET and MYND domain-containing protein 3 (SMYD3), a novel histone methyltransferase, was overexpressed in cholangiocarcinoma patients especially in those with HCV infection. Transfection of HCVc into hilar cholangiocarcinoma cell lines QBC939 and FRH0201 could upregulate the expression of SMYD3 and promote cell growth, which was consistent with the results of our clinical research. This phenomenon indicated that SMYD3 was related to the epigenetic regulation of cholangiocarcinoma genesis with HCV infection. Overexpression of SMYD3 could inhibit RASSF1A expression, whereas inhibition of SMYD3 by siRNA improved its expression. Methylation-specific polymerase chain reaction (MS-PCR) results showed the methylation status of RASSF1A promoter was regulated by SMYD3. In conclusion, HCVc could upregulate the methylation status of the RASSF1A promoter through regulation of SMYD3, and histone methylation may affect the DNA methylation of downstream gene by an unknown mechanism.

Effects of SMYD3 over-expression on cell cycle acceleration and cell proliferation in MDA-MB-231 human breast cancer cells

SET and MYND domain-containing protein 3 (SMYD3) is a histone methyltransferase that plays an important role in transcriptional regulation in human carcinogenesis. It can specifically methylate histone H3 at lysine 4 and activate the transcription of a set of downstream genes, including several oncogenes (e.g., N-myc, CrkL, Wnt10b, RIZ and hTERT) and genes involved in the control of cell cycle (e.g., CyclinG1 and CDK2) and signal transduction (e.g., STAT1, MAP3K11 and PIK3CB). To determine the effects of SMYD3 over-expression on cell proliferation, we transfected SMYD3 into MDA-MB-231 cells and found that these cells showed several transformed phenotypes as demonstrated by colony growth in soft agar. Besides, we show here that down-regulation of SMYD3 could induce G1-phase cell cycle arrest, indicating the potent induction of apoptosis by SMYD3 knockdown. These results suggest the regulatory mechanisms of SMYD3 on the acceleration of cell cycle and facilitate the development of strategies that may inhibit the progression of cell cycle in breast cancer cells.

Novobiocin decreases SMYD3 expression and inhibits the migration of MDA‐MB‐231 human breast cancer cells

SET and MYND domain-containing protein 3 (SMYD3) is a histone methyltransferase that plays an important role in transcriptional regulation in human carcinogenesis, and heat-shock protein HSP90A has been shown to increase the activity of SMYD3. We previously reported that overexpression of SMYD3 stimulated the migration of cells. In this study, we further found that novobiocin, a HSP90 inhibitor, could decrease the expression of SMYD3 and dose dependently inhibit the proliferation and migration of MDA-MB-231 human breast cancer cells. As a control, the short hairpin RNA (shRNA) targeting SMYD3 gene also showed similar effects with novobicin. This study is the first to show that novobiocin can inhibit the migration of breast cancer cells and such event may involve the downregulation of SMYD3. These findings might throw light on the development of novel therapeutic approaches to human cancers, and lend further understanding to the potential role of SMYD3 in human carcinogenesis.

Alternative Splicing of PDCD2 Regulates Hematopoietic Stem Cell Specification, and Drives Multilineage Leukemia Development.

Abstract 258PDCD2 (Programmed cell death domain 2) is a highly conserved zinc finger MYND domain-containing nuclear protein expressed in a variety of tissues. Our group has demonstrated that in Drosophila, PDCD2 mutants display massive lymph gland hyperplasia phenotype suggestive of hematopoietic hyperproliferation concomitant with a block in differentiation. The human PDCD2 gene is located on chromosome 6q27 and encodes two alternative transcripts with multiple splice variants. We analyzed primary human leukemias and multiple tumor cell lines and detected a PDCD2 splice variant whose expression is associated exclusively with malignant but not normal hematopoietic samples. To study the role of alternative PDCD2 splicing in hematopoiesis in a biologically relevant vertebrate, we isolated the zebrafish PDCD2 and found that it is expressed in early embryonic stages, and its expression latter on colocalized with the hemangioblast and hematopoietic stem cell (HSC) markers cMyb, Flk1 and RUNX1. Promoter analysis demonstrated multiple binding sites for hematopoietic and lymphoid transcription factors controlling embryonic and adult hematopoiesis. We next generated transgenic zebrafish expressing EGFP under the control of the PDCD2 promoter. EGFP expression in zebrafish recapitulates PDCD2 expression pattern in HSC. Morpholino-mediated PDCD2 knockdown resulted in developmental defects with marked decrease in the number of embryonic blood cells and circulatory failure at 4 dpf. Morpholino studies using transgenic zebrafish with labeled endothelial and HSC progenitors demonstrated that the effects of PDCD2 knockdown are restricted to HSC but not endothelial progenitors. Concordant with this observation, we examined the expression of several markers for early hematopoiesis including SCL, RUNX1 and cMyb using in situ hybridization (ISH). PDCD2 knockdown resulted in a severe reduction in RUNX1+ cells suggesting that HSC formation in these embryos is compromised. Our ISH analysis was extended to examine the effects of PDCD2 loss on lineage-committed hematopoiesis with Gata1, Mpx, L-plastin, Ikaros, Tcr-α, Lck, and IgM hematopoietic lineage-specific probes. Utilizing a competitive RT-PCR assay, we found that in zebrafish leukemias, similar to human leukemias, a unique (tumor–specific) PDCD2 isoform is upregulated. To investigate the tumor suppressor and/or oncogenic function of these PDCD2 isoforms, we have generated multiple transgenic zebrafish expressing the different PDCD2 splice variants. Overexpression of PDCD2 variants derived from the alternatively spliced PDCD2 mRNAs resulted in an overt leukemic phenotype in 30% of the transgenic fish. Preliminary examination of the affected fish suggests that PDCD2 overexpression has the potential to drive leukemogenesis in either the myeloid, lymphoid, or erythroid lineages in individual cases. Moreover, we observed a bias toward erythroleukemia (M6) in the case of one isoform. Our data demonstrate a spectrum of leukemia arising from PDCD2 dysregulation. In light of our analysis of the normal PDCD2 function in hematopoiesis, we conclude that the alternative isoforms of PDCD2 play an important role in regulating normal HSC functions, and deviation from this role is a powerful promoter of leukemogenesis. Disclosures:Steward:UMDNJ: Patents & Royalties. Schaar:UMDNJ: Patents & Royalties. Sabaawy:UMDNJ: Patents & Royalties.

Effects of SMYD3 overexpression on transformation, serum dependence, and apoptosis sensitivity in NIH3T3 cells

The SET and MYND domain-containing protein 3 (SMYD3) gene was found to encode a novel histone methyltransferase involved in human cancer cells. It could specifically methylate histone H3 at lysine 4 and activate the transcription of a set of downstream genes, including of several oncogenes (e.g., N-Myc, CrkL, Wnt10b, RIZ, and hTERT) and genes involved in the control of cell cycle (e.g., Cyclin G1 and CDK2) and signal transduction (e.g., STAT1, MAP3K11, and PIK3CB). To determine the effects of SMYD3 overexpression on cell transformation, serum dependence and apoptosis sensitivity, we expressed SMYD3 in NIH3T3 cells, and these cells showed several transformed phenotypes as demonstrated by foci formation and colony growth in soft agar. Besides, these transfectants also showed increased serum dependence and depression of sensitivity to apoptosis induced by dexamethasone. These findings lend further understanding to the role of SMYD3 in the genesis of human cancers and might throw light on the development of novel therapeutic approaches to human cancers.

Overexpression of SMYD2 relates to tumor cell proliferation and malignant outcome of esophageal squamous cell carcinoma

Although we have identified two putative targets, ATF3 and CENPF, for a frequently gained/amplified region around 1q32-q41 in esophageal squamous cell carcinoma (ESCC), it is possible that other amplification targets remain to be identified. In this study, we tested whether SET and MYND domain-containing protein 2 (SMYD2), located between those two genes and encoding a lysine methyltransferase for histone H3K36 and p53K370 that regulates transcription and inhibits transactivation activity, respectively, acts as a cancer-promoting gene through activation/overexpression in ESCC. Frequent overexpression of SMYD2 messenger RNA and protein was observed in KYSE150 cells with remarkable amplification at 1q32-41.1 and other ESCC cell lines (11/43 lines, 25.6%). Overexpression of SMYD2 protein was frequently detected in primary tumor samples of ESCC (117/153 cases, 76.5%) as well and significantly correlated with gender, venous invasion, the pT category in the tumor-lymph node-metastases classification and status of recurrence. Patients with SMYD2-overexpressing tumors had a worse overall rate of survival than those with non-expressing tumors, and SMYD2 positivity was independently associated with a worse outcome in the multivariate analysis. Knockdown of SMYD2 expression inhibited and ectopic overexpression of SMYD2 promoted the proliferation of ESCC cells in a TP53 mutation-independent but SMYD2 expression-dependent manner. These findings suggest that SMYD2 plays an important role in tumor cell proliferation through its activation/overexpression and highlight its usefulness as a prognosticator and potential therapeutic target in ESCC.

SET and MYND domain-containing protein 3 decreases sensitivity to dexamethasone and stimulates cell adhesion and migration in NIH3T3 cells

SET and MYND domain-containing protein 3 decreases sensitivity to dexamethasone and stimulates cell adhesion and migration in NIH3T3 cells

SET and MYND domain-containing protein 3 decreases sensitivity to dexamethasone and stimulates cell adhesion and migration in NIH3T3 cells

SET and MYND domain-containing protein 3 (SMYD3) is a novel histone methyltransferase that plays an important role in transcriptional regulation in human carcinogenesis. It activates the transcription of a set of downstream genes. Of these downstream genes, there are several oncogenes and genes associated with cell adhesion (including those of N-Myc, CrkL, Wnt10b, L-selectin, CD31 and galectin-4), which have been shown to have effects on cell viability, adhesion, migration and metastasis by many studies. To determine whether SMYD3 has such functions, in this study, we examined these types of biological activities in mouse fibroblast NIH3T3 cells by stably transfecting the human SMYD3 gene. The SMYD3-gene-transfected cells showed an increased proliferation rate and became more resistant to cell death induced by dexamethasone. Furthermore, the SMYD3-transfected cells also exhibited increased rates of cell adhesion to both type IV collagen and endothelial cells, and enhanced cell migration ability in both two-dimensional and three-dimensional assays. This study is the first to show that the overexpression of the SMYD3 gene affects cell viability, adhesion and migration, indicating that SMYD3 may be a promising new target of therapeutic intervention for the treatment of cancers or other pathological processes associated with cell adhesion and migration.

The Telomerase Reverse Transcriptase (hTERT) Gene Is a Direct Target of the Histone Methyltransferase SMYD3

Recent evidence has accumulated that the dynamic histone methylation mediated by histone methyltransferases and demethylases plays key roles in regulation of chromatin structure and transcription. In the present study, we show that SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase implicated in oncogenesis, directly trans-activates the telomerase reverse transcriptase (hTERT) gene that is essential for cellular immortalization and transformation. SMYD3 occupies its binding motifs on the hTERT promoter and is required for maintenance of histone H3-K4 trimethylation, thereby contributing to inducible and constitutive hTERT expression in normal and malignant human cells. Knocking down SMYD3 in tumor cells abolished trimethylation of H3-K4, attenuated the occupancy by the trans-activators c-MYC and Sp1, and led to diminished histone H3 acetylation in the hTERT promoter region, which was coupled with down-regulation of hTERT mRNA and telomerase activity. These results suggest that SMYD3-mediated trimethylation of H3-K4 functions as a licensing element for subsequent transcription factor binding to the hTERT promoter. The present findings provide significant insights into regulatory mechanisms of hTERT/telomerase expression; moreover, identification of the hTERT gene as a direct target of SMYD3 contributes to a better understanding of SMYD3-mediated cellular transformation.

Muscle‐specific expression of the smyd1 gene is controlled by its 5.3‐kb promoter and 5′‐flanking sequence in zebrafish embryos

Zebrafish SmyD1 is a SET and MYND domain-containing protein that plays an important role in myofiber maturation and muscle contraction. SmyD1 is required for myofibril organization and sarcomere assembly during myofiber maturation. Whole-mount in situ hybridization revealed that smyd1 mRNAs are specifically expressed in skeletal and cardiac muscles in zebrafish embryos. However, it is unknown if smyd1 is expressed in other striated muscles, such as cranial and fin muscles, and moreover, the regulatory elements required for its muscle-specific expression. We report here the analyses of smyd1 expression using smyd1-gfp transgenic zebrafish. smyd1-gfp transgenic zebrafish were generated using the 5.3-kb smyd1 promoter and its 5'-flanking sequence. GFP expression was found in the skeletal and cardiac muscles of smyd1-gfp transgenic embryos. GFP expression appeared stronger in slow muscles than fast muscles in transgenic zebrafish larvae. In addition, GFP expression was also detected in cranial and fin muscles of smyd1-gfp transgenic zebrafish larvae. In situ hybridization confirmed smyd1 mRNA expression in these tissues, suggesting that the expression of the smyd1-gfp transgene recapitulated that of the endogenous smyd1 gene. Deletion analysis revealed that the 0.5-kb sequence in the proximal promoter of smyd1 was essential for its muscle specificity. Together, these data indicate that smyd1 is specifically expressed in most, if not all, striated muscles, and the muscle specificity is controlled by the 5.3-kb promoter and flanking sequences.

BS69, a Specific Adaptor in the Latent Membrane Protein 1-Mediated c-Jun N-Terminal Kinase Pathway

We previously demonstrated that the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) potently activates the cellular c-Jun N-terminal kinase (JNK) pathway by sequentially engaging an unknown adaptor, TRAF6, TAB1/TAK1, and JNKKs. We now show that BS69, a MYND domain-containing cellular protein, is the missing adaptor that bridges LMP1 and TRAF6, as the MYND domain and a separate region of BS69 bind to the carboxyl termini of LMP1 and TRAF6, respectively. While LMP1 promotes the interaction between BS69 and TRAF6, the complex formation between LMP1 and TRAF6 is BS69 dependent. A fraction of LMP1 and BS69 is constitutively colocalized in the membrane lipid rafts. Importantly, knockdown of BS69 by small interfering RNAs specifically inhibits JNK activation by LMP1 but not tumor necrosis factor alpha. Although overexpression of either BS69 or a mutant LMP1 without the cytoplasmic carboxyl tail is not sufficient to activate JNK, interestingly, when BS69 is covalently linked to the mutant LMP1, the chimeric protein restores the ability to activate JNK. This indicates that the recruitment and aggregation of BS69 is a prerequisite for JNK activation by LMP1.