The mechanism of sevoflurane post-treatment alleviating hypoxic-ischemic encephalopathy by affecting histone methyltransferase G9a in rats.

Hypoxic-ischemic encephalopathy (HIE) is recognized as the main cause of neonatal death, and efficient treatment strategies remain limited. This study aims to investigate the mechanism of sevoflurane (SF) post-treatment in alleviating HIE in rats. The HIE rat model and oxygen-glucose deprivation (OGD) cell model were established, and adeno-associated virus (AAV)-histone-lysine N-methyltransferase EHMT2 (G9a) was transfected after SF treatment. The learning and memory ability and the levels of nerve growth factor (NGF)/brain-derived neurotrophic factor (BDNF) were evaluated and determined. The levels of G9a/histone H3 lysine 9 dimethylation (H3K9me2) and the enrichment level of H3K9me2 in the promoter region of BDNF gene were analyzed. After SF post-treatment, the neurons in cerebral cortex, the learning and memory skills and the contents of NGF/BDNF were increased, while the apoptosis and G9a/H3K9me2 levels were reduced. After overexpression of G9a in vitro/vivo, the enrichment levels of H3K9me2 in the promoter region of BDNF were increased, the levels of BDNF were decreased, the neurons were damaged and the learning and memory abilities of HIE rats were impaired. The conclusion is that SF post-treatment can promote the expression of BDNF by inhibiting H3K9me2 on the BDNF gene promoter and inhibiting G9a, thus alleviating HIE in rats.

SetD7 (Set7/9) is a novel target of PPARγ that promotes the adaptive pancreatic ß-cell glycemic response.

Loss of functional pancreatic ß-cell mass leads to type 2 diabetes (T2D), attributable to modified ß-cell-dependent adaptive gene expression patterns. SetD7 is a histone methyltransferase enriched in pancreatic islets that mono- and dimethylates histone-3-lysine-4 (H3K4), promoting euchromatin modifications, and also maintains the regulation of key ß-cell function and survival genes. However, the transcriptional regulation of this important epigenetic modifier is unresolved. Here we identified the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPARγ) as a major transcriptional regulator of SetD7 and provide evidence for direct binding and functionality of PPARγ in the SetD7 promoter region. Furthermore, constitutive shRNA-mediated PPARγ knockdown in INS-1 ß-cells or pancreas-specific PPARγ deletion in mice led to downregulation of SetD7 expression as well as its nuclear enrichment. The relevance of the SetD7-PPARγ interaction in ß-cell adaptation was tested in normoglycemic 60% partial pancreatectomy (Px) and hyperglycemic 90% Px rat models. Whereas a synergistic increase in islet PPARγ and SetD7 expression was observed upon glycemic adaptation post-60% Px, in hyperglycemic 90% Px rats, islet PPARγ, and PPARγ targets SetD7 and Pdx1 were downregulated. PPARγ agonist pioglitazone treatment in 90% Px rats partially restored glucose homeostasis and ß-cell mass and enhanced expression of SetD7 and Pdx1. Collectively, these data provide evidence that the SetD7-PPARγ interaction serves as an important element of the adaptive ß-cell response.

Histone Mark Profiling in Pediatric Astrocytomas Reveals Prognostic Significance of H3K9 Trimethylation and Histone Methyltransferase SUV39H1.

Alterations in global histone methylation regulate gene expression and participate in cancer onset and progression. The profile of histone methylation marks in pediatric astrocytomas is currently understudied with limited data on their distribution among grades. The global expression patterns of repressive histone marks H3K9me3, H3K27me3, and H4K20me3 and active H3K4me3 and H3K36me3 along with their writers SUV39H1, SETDB1, EZH2, MLL2, and SETD2 were investigated in 46 pediatric astrocytomas and normal brain tissues. Associations between histone marks and modifying enzymes with clinicopathological characteristics and disease-specific survival were studied along with their functional impact in proliferation and migration of pediatric astrocytoma cell lines using selective inhibitors in vitro. Upregulation of histone methyltransferase gene expression and deregulation of histone code were detected in astrocytomas compared to normal brain tissues, with higher levels of SUV39H1, SETDB1, and SETD2 as well as H4K20me3 and H3K4me3 histone marks. Pilocytic astrocytomas exhibited lower MLL2 levels compared to diffusely infiltrating tumors indicating a differential pattern of epigenetic regulator expression between the two types of astrocytic neoplasms. Moreover, higher H3K9me3, H3K36me3, and SETDB1 expression was detected in grade IIΙ/IV compared to grade II astrocytomas. In univariate analysis, elevated H3K9me3 and MLL2 and diminished SUV39H1 expression adversely affected survival. Upon multivariate survival analysis, only SUV39H1 expression was revealed as an independent prognostic factor of adverse significance. Treatment of pediatric astrocytoma cell lines with SUV39H1 inhibitor reduced proliferation and cell migration. Our data implicate H3K9me3 and SUV39H1 in the pathobiology of pediatric astrocytomas, with SUV39H1 yielding prognostic information independent of other clinicopathologic variables.

SUV39H2 epigenetic silencing controls fate conversion of epidermal stem and progenitor cells.

Epigenetic histone trimethylation on lysine 9 (H3K9me3) represents a major molecular signal for genome stability and gene silencing conserved from worms to man. However, the functional role of the H3K9 trimethylases SUV39H1/2 in mammalian tissue homeostasis remains largely unknown. Here, we use a spontaneous dog model with monogenic inheritance of a recessive SUV39H2 loss-of-function variant and impaired differentiation in the epidermis, a self-renewing tissue fueled by stem and progenitor cell proliferation and differentiation. Our results demonstrate that SUV39H2 maintains the stem and progenitor cell pool by restricting fate conversion through H3K9me3 repressive marks on gene promoters encoding components of the Wnt/p63/adhesion axis. When SUV39H2 function is lost, repression is relieved, and enhanced Wnt activity causes progenitor cells to prematurely exit the cell cycle, a process mimicked by pharmacological Wnt activation in primary canine, human, and mouse keratinocytes. As a consequence, the stem cell growth potential of cultured SUV39H2-deficient canine keratinocytes is exhausted while epidermal differentiation and genome stability are compromised. Collectively, our data identify SUV39H2 and potentially also SUV39H1 as major gatekeepers in the delicate balance of progenitor fate conversion through H3K9me3 rate-limiting road blocks in basal layer keratinocytes.

Expression of G9a in Auditory Cortex Is Downregulated in a Rat Model of Age-Related Hearing Loss.

G9a is essential for dendritic plasticity and is associated with neurological disorders. The possible relationship between age-related hearing loss and G9a expression in the auditory cortex has not been fully explored. This study aimed to understand the expression patterns of G9a-mediated histone methylations in the auditory cortex during aging. Using immunofluorescence and western blotting, we demonstrated that a significant reduction in G9a expression observed in the auditory cortex of 24-month-old rats compared to 3-month-old rats, was associated with remarkable hearing threshold elevation and hair cell loss. Correspondingly, histone H3 lysine 9 (H3K9) mono- and dimethylation (marked by H3K9me1 and H3K9me2, respectively), which were regulated by G9a activity, also evidently decreased during aging. These findings, which merit further investigation, suggest a possible association between G9a-mediated histone methylations and central age-related hearing disorders.

High glucose inhibits vascular endothelial Keap1/Nrf2/ARE signal pathway via downregulation of monomethyltransferase SET8 expression.

Hyperglycemia-mediated reactive oxygen species (ROS) accumulation plays an important role in hyperglycemia-induced endothelial injury. Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway inhibition participates in hyperglycemia-induced ROS accumulation. Our previous study indicated that SET8 overexpression inhibits high glucose-mediated ROS accumulation in human umbilical vein endothelial cells (HUVECs). In the present study, we hypothesize that SET8 may play a major role in high glucose-induced ROS accumulation via modulation of Keap1/Nrf2/ARE pathway. Our data indicated that high glucose mediated cell viability reduction, ROS accumulation, and Nrf2/ARE signal pathway inhibition via upregulation of Keap1 expression in HUVECs. Moreover, high glucose inhibited the expressions of SET8 and H4K20me1 (a downstream target of SET8). SET8 overexpression improved high glucose-mediated Keap1/Nrf2/ARE pathway inhibition and endothelial oxidation. Consistently, the effects of sh-SET8 were similar to that of high glucose treatment and were reversed by si-Keap1. A mechanistic study found that H4K20me1 was enriched at the Keap1 promoter region. SET8 overexpression attenuated Keap1 promoter activity and its expression, while mutant SET8 R259G did not affect Keap1 promoter activity and expression. The results of this study demonstrated that SET8 negatively regulates Keap1 expression, thus participating in high glucose-mediated Nrf2/ARE signal pathway inhibition and oxidative injury in HUVECs.

Evolution and structure of clinically relevant gene fusions in multiple myeloma.

Multiple myeloma is a plasma cell blood cancer with frequent chromosomal translocations leading to gene fusions. To determine the clinical relevance of fusion events, we detect gene fusions from a cohort of 742 patients from the Multiple Myeloma Research Foundation CoMMpass Study. Patients with multiple clinic visits enable us to track tumor and fusion evolution, and cases with matching peripheral blood and bone marrow samples allow us to evaluate the concordance of fusion calls in patients with high tumor burden. We examine the joint upregulation of WHSC1 and FGFR3 in samples with t(4;14)-related fusions, and we illustrate a method for detecting fusions from single cell RNA-seq. We report fusions at MYC and a neighboring gene, PVT1, which are related to MYC translocations and associated with divergent progression-free survival patterns. Finally, we find that 4% of patients may be eligible for targeted fusion therapies, including three with an NTRK1 fusion.

VHL-HIF-2α axis-induced SMYD3 upregulation drives renal cell carcinoma progression via direct trans-activation of EGFR.

It has been well established that the von Hippel-Lindau/hypoxia-inducible factor α (VHL-HIFα) axis and epidermal growth factor receptor (EGFR) signaling pathway play a critical role in the pathogenesis and progression of renal cell carcinoma (RCC). However, few studies have addressed the relationship between the two oncogenic drivers in RCC. SET and MYND domain-containing protein 3 (SMYD3) is a histone methyltransferase involved in gene transcription and oncogenesis, but its expression and function in RCC remain unclear. In the present study, we found that SMYD3 expression was significantly elevated in RCC tumors and correlated with advanced tumor stage, histological and nuclear grade, and shorter survival. Depletion of SMYD3 inhibited RCC cell proliferation, colony numbers, and xenograft tumor formation, while promoted apoptosis. Mechanistically, SMYD3 cooperates with SP1 to transcriptionally promote EGFR expression, amplifying its downstream signaling activity. TCGA data analyses revealed a significantly increased SMYD3 expression in primary RCC tumors carrying the loss-of-function VHL mutations. We further showed that HIF-2α can directly bind to the SMYD3 promoter and subsequently induced SMYD3 transcription and expression. Taken together, we identify the VHL/HIF-2α/SMYD3 signaling cascade-mediated EGFR hyperactivity through which SMYD3 promotes RCC progression. Our study suggests that SMYD3 is a potential therapeutic target and prognostic factor in RCC.

SETD2 deficiency accelerates MDS-associated leukemogenesis via S100a9 in NHD13 mice and predicts poor prognosis in MDS.

SETD2, the histone H3 lysine 36 methyltransferase, previously identified by us, plays an important role in the pathogenesis of hematologic malignancies, but its role in myelodysplastic syndromes (MDSs) has been unclear. In this study, low expression of SETD2 correlated with shortened survival in patients with MDS, and the SETD2 levels in CD34+ bone marrow cells of those patients were increased by decitabine. We knocked out Setd2 in NUP98-HOXD13 (NHD13) transgenic mice, which phenocopies human MDS, and found that loss of Setd2 accelerated the transformation of MDS into acute myeloid leukemia (AML). Loss of Setd2 enhanced the ability of NHD13+ hematopoietic stem and progenitor cells (HSPCs) to self-renew, with increased symmetric self-renewal division and decreased differentiation and cell death. The growth of MDS-associated leukemia cells was inhibited though increasing the H3K36me3 level by using epigenetic modifying drugs. Furthermore, Setd2 deficiency upregulated hematopoietic stem cell signaling and downregulated myeloid differentiation pathways in the NHD13+ HSPCs. Our RNA-seq and chromatin immunoprecipitation-seq analysis indicated that S100a9, the S100 calcium-binding protein, is a target gene of Setd2 and that the addition of recombinant S100a9 weakens the effect of Setd2 deficiency in the NHD13+ HSPCs. In contrast, downregulation of S100a9 leads to decreases of its downstream targets, including Ikba and Jnk, which influence the self-renewal and differentiation of HSPCs. Therefore, our results demonstrated that SETD2 deficiency predicts poor prognosis in MDS and promotes the transformation of MDS into AML, which provides a potential therapeutic target for MDS-associated acute leukemia.

The prognostic impact of tumour NSD2 expression in advanced prostate cancer.

Purpose: To assess the prognostic significance of the nuclear receptor binding SET protein 2 (NSD2), a co-activator of the NFkB-pathway, on tumour progression in patients with advanced prostate cancer (PCa).Methods: We retrospectively assessed NSD2 expression in 53 patients with metastatic and castration-resistant PCa. Immunohistochemical staining for NSD2 was carried out on specimen obtained from palliative resection of the prostate. Univariable and multivariable analyses were performed to assess the association between NSD2 expression and PCa progression.Results: Of the 53 patients, 41 had castration-resistant PCa and 48 men had metastases at time of tissue acquisition. NSD2 expression was increased in tumour specimen from 42 patients (79.2%). In univariable Cox regression analyses, NSD2 expression was associated with PSA progression, progression on imaging and overall survival (p = 0.04, respectively). In multivariable analyses, NSD2 expression did not retain its association with these endpoints.Conclusions: NSD2 expression is abnormal in almost 80% of patients with advanced PCa. Expression levels of this epigenetic regulator are easily detected by immunohistochemistry while this biomarker exhibited prognostic value for PCa progression and death in univariable analysis. Further studies on NSD2 involvement in PCa proliferation, progression, metastasis and resistance mechanisms are needed.

LncRNA NEAT1 promotes cardiac hypertrophy through microRNA-19a-3p/SMYD2 axis.

OBJECTIVE: The role of NEAT1 in cancers has been demonstrated. But the role of NEAT1 in cardiac hypertrophy still remains unknown. This study aimed to elucidate the specific function of long non-coding RNA (lncRNA) NEAT1 in cardiac hypertrophy and its underlying mechanism. PATIENTS AND METHODS: In this study, the in vivo and in vitro cardiac hypertrophy models were constructed by transverse aortic coarctation (TAC) procedure in rats and phenylephrine (PE) induction in primary cardiomyocytes, respectively. The expression levels of NEAT1, microRNA-19a-3p, SMYD2, and cardiac hypertrophic markers were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Cardiac hypertrophy was evaluated as calculating the surface area of hypertrophic cardiomyocyte by fluorescein isothiocyanate (FITC)-Phalloidin staining. Luciferase Reporter Gene Assay was conducted to detect the binding of NEAT1, SMYD2, and microRNA-19a-3p. RESULTS: The results showed that NEAT1 and SMYD2 were highly expressed in myocardium of rats with cardiac hypertrophy and PE-induced primary cardiomyocytes, whereas microRNA-19a-3p was lowly expressed. Besides, NEAT1 overexpression markedly upregulated levels of the cardiac hypertrophic markers. Moreover, FITC-Phalloidin staining also revealed hypertrophic cardiomyocytes overexpressing NEAT1. On the contrary, microRNA-19a-3p overexpression reduced the cardiomyocyte surface area and downregulated the levels of the cardiac hypertrophic markers. As luciferase reporter gene assay demonstrated, NEAT1 and SMYD2 could bind to microRNA-19a-3p. Finally, the gain-of-function experiments were designed to verify whether NEAT1 exerted its functions in cardiac hypertrophy by modulating SMYD2 and microRNA-19a-3p. Furthermore, both microRNA-19a-3p overexpression or SMYD2 knockdown could inhibit and reduce the cardiomyocyte surface area, and downregulate the levels of the cardiac hypertrophic markers. CONCLUSIONS: In summary, NEAT1 promotes the occurrence and progression of cardiac hypertrophy by upregulating SMYD2 by binding to microRNA-19a-3p.

Generation of MuRF-GFP transgenic zebrafish models for investigating murf gene expression and protein localization in Smyd1b and Hsp90α1 knockdown embryos.

Muscle-specific RING-finger proteins (MuRFs) are E3 ubiquitin ligases that play important roles in protein quality control in skeletal and cardiac muscles. Here we characterized murf gene expression and protein localization in zebrafish embryos. We found that the zebrafish genome contains six murf genes, including murf1a, murf1b, murf2a, murf2b, murf3 and a murf2-like gene that are specifically expressed in skeletal and cardiac muscles of zebrafish embryos. To analyze the subcellular localization, we generated transgenic zebrafish models expressing MurF1a-GFP or MuRF2a-GFP fusion proteins. MuRF1a-GFP and MuRF2a-GFP showed distinct patterns of subcellular localization. MuRF1a-GFP displayed a striated pattern of localization in myofibers, whereas MuRF2a-GFP mainly exhibited a random pattern of punctate distribution. The MuRF1a-GFP signal appeared as small dots aligned along the M-lines of the sarcomeres in skeletal myofibers. To determine whether knockdown of smyd1b or hsp90α1 that increased myosin protein degradation could alter murf gene expression or MuRF protein localization, we knocked down smyd1b or hsp90α1 in wild type, Tg(ef1a:MurF1a-GFP) and Tg(ef1a:MuRF2a-GFP) transgenic zebrafish embryos. Knockdown of smyd1b or hsp90α1 had no effect on murf gene expression. However, the sarcomeric distribution of MuRF1a-GFP was abolished in the knockdown embryos. This was accompanied by an increased random punctate distribution of MuRF1a-GFP in muscle cells of zebrafish embryos. Collectively, these studies demonstrate that MuRFs are specifically expressed in developing muscles of zebrafish embryos. The M-line localization MuRF1a is altered by sarcomere disruption in smyd1b or hsp90α1 knockdown embryos.

CDK2 regulates the NRF1/Ehmt1 axis during meiotic prophase I.

Meiosis generates four genetically distinct haploid gametes over the course of two reductional cell divisions. Meiotic divisions are characterized by the coordinated deposition and removal of various epigenetic marks. Here we propose that nuclear respiratory factor 1 (NRF1) regulates transcription of euchromatic histone methyltransferase 1 (EHMT1) to ensure normal patterns of H3K9 methylation during meiotic prophase I. We demonstrate that cyclin-dependent kinase (CDK2) can bind to the promoters of a number of genes in male germ cells including that of Ehmt1 through interaction with the NRF1 transcription factor. Our data indicate that CDK2-mediated phosphorylation of NRF1 can occur at two distinct serine residues and negatively regulates NRF1 DNA binding activity in vitro. Furthermore, induced deletion of Cdk2 in spermatocytes results in increased expression of many NRF1 target genes including Ehmt1 We hypothesize that the regulation of NRF1 transcriptional activity by CDK2 may allow the modulation of Ehmt1 expression, therefore controlling the dynamic methylation of H3K9 during meiotic prophase.

Transcriptomic Analysis of Histone Methyltransferase Setd7 Knockdown and Phenethyl Isothiocyanate in Human Prostate Cancer Cells.

BACKGROUND/AIM: Transcriptomic analysis was performed to evaluate the differential gene expression profiles of Setd7 knockdown (KD) and the effects of phenethyl isothiocyanate (PEITC) in human prostate cancer (PCa) LNCaP cells. MATERIALS AND METHODS: RNA isolated from wild-type and Setd7-KD LNCaP cells in the presence or absence of PEITC was subjected to microarray analysis followed by Ingenuity® Pathway Analysis (IPA). RESULTS: Setd7 KD impacted a larger set of genes and caused a higher fold change compared to PEITC treatment. Several signaling pathways were altered particularly inflammation-related TNFR signaling and PTEN/PI3K/AKT signaling by Setd7 KD and PEITC. Interestingly, PEITC and Setd7 KD at a small subset of genes that could be potential molecular targets. CONCLUSION: This study offers new insights into the mechanisms of action of the epigenetic modifier Setd7 and the effects of PEITC treatment in PCa cells and enhances our understanding of the potential cancer preventive/treatment effects of isothiocyanate compounds such as PEITC in PCa.

Overexpression of SET and MYND Domain-Containing Protein 2 (SMYD2) Is Associated with Tumor Progression and Poor Prognosis in Patients with Papillary Thyroid Carcinoma.

BACKGROUND SET and MYND domain-containing protein 2 (SMYD2), which is identified as a protein-lysine methyltransferase, plays a crucial role in the progression of some tumors such as bladder carcinoma. However, the clinical significance of SMYD2 in patients with papillary thyroid carcinoma (PTC) has not been elucidated. In the present study, we aimed to investigate the expression and role of SMYD2 in human PTC. MATERIAL AND METHODS Clinicopathological analysis was performed in 107 patients with PTC. Expression of SMYD2 was determined by immunohistochemistry staining, quantitative RT-PCR, or Western blotting in PTC tissues, adjacent normal tissues, and PTC cells (K1 and B-CPAP). The prognostic value of SMYD2 in PTC patients was assessed by univariate and multivariate analysis. Clinical outcomes were evaluated by Kaplan-Meier log-rank tests. Cell proliferation was examined in PTC cells following overexpression or knockdown of SMYD2. RESULTS SMYD2 was highly expressed in PTC tissues compared to adjacent thyroid tissues. Additionally, high expression of SMYD2 was significantly related to tumor size, lymph node metastasis, and TNM stage. Moreover, SMYD2 was identified as an independent prognosis factor by multivariate analysis. Using 2 PTC cell lines, K1 and B-CPAP, we demonstrated that high expression of SMYD2 can promote tumor cell proliferation. CONCLUSIONS SMYD2 expression was upregulated in PTC tissues and significantly related to the poorer prognosis of PTC patients. Our studies suggested the potential role of SMYD2 as a new therapeutic target and prognostic biomarker in human PTC.

Paediatric patients with acute leukaemia and KMT2A (MLL) rearrangement show a distinctive expression pattern of histone deacetylases.

Histone deacetylase inhibitors (HDACi) had emerged as promising drugs in leukaemia, but their toxicity due to lack of specificity limited their use. Therefore, there is a need to elucidate the role of HDACs in specific settings. The study of HDAC expression in childhood leukaemia could help to choose more specific HDACi for selected candidates in a personalized approach. We analysed HDAC1-11, SIRT1, SIRT7, MEF2C and MEF2D mRNA expression in 211 paediatric patients diagnosed with acute leukaemia. There was a global overexpression of HDACs, while specific HDACs correlated with clinical and biological features, and some even predicted outcome. Thus, some HDAC and MEF2C profiles probably reflected the lineage and the maturation of the blasts and some profiles identified specific oncogenic pathways active in the leukaemic cells. Specifically, we identified a distinctive signature for patients with KMT2A (MLL) rearrangement, with high HDAC9 and MEF2D expression, regardless of age, KMT2A partner and lineage. Moreover, we observed an adverse prognostic value of HDAC9 overexpression, regardless of KMT2A rearrangement. Our results provide useful knowledge on the complex picture of HDAC expression in childhood leukaemia and support the directed use of specific HDACi to selected paediatric patients with acute leukaemia.

Hijacking a key chromatin modulator creates epigenetic vulnerability for MYC-driven cancer.

While the genomic binding of MYC protein correlates with active epigenetic marks on chromatin, it remains largely unclear how major epigenetic mechanisms functionally impact the tumorigenic potential of MYC. Here, we show that, compared with the catalytic subunits, the core subunits, including DPY30, of the major H3K4 methyltransferase complexes were frequently amplified in human cancers and selectively upregulated in Burkitt lymphoma. We show that DPY30 promoted the expression of endogenous MYC and was also functionally important for efficient binding of MYC to its genomic targets by regulating chromatin accessibility. Dpy30 heterozygosity did not affect normal animal physiology including lifespan, but significantly suppressed Myc-driven lymphomagenesis, as cells failed to combat oncogene-triggered apoptosis as a result of insufficient epigenetic modulation and expression of a subset of antiapoptotic genes. Dpy30 reduction also greatly impeded MYC-dependent cellular transformation, without affecting normal cell growth. These results suggest that MYC hijacks a major epigenetic pathway - H3K4 methylation - to facilitate its molecular activity in target binding and to coordinate its oncogenic program for efficient tumorigenesis, meanwhile creating "epigenetic vulnerability." DPY30 and the H3K4 methylation pathway are thus potential epigenetic targets for treating certain MYC-driven cancers.

DNA methylation levels and expression patterns of Smyd1a and Smyd1b genes during Metamorphosis of the Japanese Flounder (Paralichthys olivaceus).

Japanese flounder (Paralichthys olivaceus) undergoes metamorphosis by changing its body from the larval to the juvenile form, and this process involves muscle development. Smyd1, a histone methyltransferase, plays a role in the skeletal muscle. In the present study, the Smyd1a and Smyd1b expression patterns and their 5' UTR and exon 1 DNA methylation levels were analyzed during metamorphosis of the Japanese flounder. Sample were analyzed 21 days post-hatching (dph) (with no migration of right eye; M1stage), 28 dph (during migration of right eye; M2 stage), and 35 dph (after migration of right eye; M3 stage). The results show that Smyd1a expression was highest in the M2 stage and then decreased, whereas Smyd1b expression continued to rise during the three stages. Methylation levels of CpG sites at positions -2318 and -2217 of the Smyd1a P region (-2462 to -2181 region of the 5' UTR), and the CpG sites at positions -351, -330, -284, -190, and - 92 of the Smyd1b promoter, with both regions containing putative transcription factor binding sites, showed significant differences in the three stages (p < 0.05). Interestingly, the methylation levels of these CpG sites were negatively correlated with mRNA expression. We inferred that binding of the predicted transcription factors might be affected by methylation of the CpG sites and thus modulate gene expression. Taken together, our results suggest that DNA methylation in the Smyd1a and Smyd1b genes participates in the regulation of metamorphosis, and epigenetics may provide clues for further studies of the mechanisms of metamorphosis in the Japanese flounder.

Upregulation of histone-lysine methyltransferases plays a causal role in hexavalent chromium-induced cancer stem cell-like property and cell transformation.

While hexavalent chromium [Cr(VI)] is generally considered as a genotoxic environmental carcinogen, studies showed that Cr(VI) exposure also causes epigenetic changes. However, whether Cr(VI)-caused epigenetic dysregulations plays an important role in Cr(VI) carcinogenicity remain largely unknown. The aim of this study was to determine if chronic low dose Cr(VI) exposure causes epigenetic changes, the underlying mechanism and whether chronic low dose Cr(VI) exposure-caused epigenetic dysregulation contributes causally to Cr(VI)-induced cancer stem cell (CSC)-like property and cell transformation. Two immortalized human bronchial epithelial cell lines (BEAS-2B and 16HBE) were exposed to 0.25 µM of K2Cr2O7 for 20 and 40 weeks to induce cell transformation, respectively. Cr(VI)-induced epigenetic changes were examined in Cr(VI)-transformed cells and Cr(VI) exposure-caused human lung cancer tissues. Pharmacological inhibitors and gene knockdown experiments were used to determine the role of epigenetic dysregulation in Cr(VI) carcinogenicity. We found that chronic Cr(VI) exposure causes epigenetic dysregulation as evidenced by the increased levels of histone H3 repressive methylation marks (H3K9me2 and H3K27me3) and the related histone-lysing methyltransferases (HMTases). Pharmacological inhibition or knockdown of HMTases reduces H3 repressive methylation marks and malignant phenotypes of Cr(VI)-transformed cells. Moreover, knockdown of HMTases in parental cells significantly reduces chronic Cr(VI) exposure-induced CSC-like property and cell transformation. Further mechanistic study revealed that knockdown of HMTases decreases Cr(VI) exposure-caused DNA damage. Our findings indicate that chronic Cr(VI) exposure increases H3 repressive methylation marks by increasing the related HMTases expression; and that increased expression of HMTases plays a causal role in Cr(VI)-induced CSC-like property and cell transformation.

Prdm13 is required for Ebf3+ amacrine cell formation in the retina.

Amacrine interneurons play a critical role in the processing of visual signals within the retina. They are highly diverse, representing 30 or more distinct subtypes. Little is known about how amacrine subtypes acquire their unique gene expression and morphological features. We characterized the gene expression pattern of the zinc-finger transcription factor Prdm13 in the mouse. Consistent with a developmental role, Prdm13 was expressed by Ptf1a+ amacrine and horizontal precursors. Over time, Prdm13 expression diverged from the transiently expressed Ptf1a and marked just a subset of amacrine cells in the adult retina. While heterogeneous, we show that most of these Prdm13+ amacrine cells express the transcription factor Ebf3 and the calcium binding protein calretinin. Loss of Prdm13 did not affect the number of amacrine cells formed during development. However, we observed a modest loss of amacrine cells and increased apoptosis that correlated with the onset timing of Ebf3 expression. Adult Prdm13 loss-of-function mice had 25% fewer amacrine cells, altered calretinin expression, and a lack of Ebf3+ amacrines. Forcing Prdm13 expression in retinal progenitor cells did not significantly increase amacrine cell formation, Ebf3 or calretinin expression, and appeared detrimental to the survival of photoreceptors. Our data show that Prdm13 is not required for amacrine fate as a class, but is essential for the formation of Ebf3+ amacrine cell subtypes. Rather than driving subtype identity, Prdm13 may act by restricting competing fate programs to maintain identity and survival.