PHF2 regulates sarcomeric gene transcription in myogenesis.

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.

14-3-3 binding motif phosphorylation disrupts Hdac4-organized condensates to stimulate cardiac reprogramming.

Cell fate conversion is associated with extensive post-translational modifications (PTMs) and architectural changes of sub-organelles, yet how these events are interconnected remains unknown. We report here the identification of a phosphorylation code in 14-3-3 binding motifs (PC14-3-3) that greatly stimulates induced cardiomyocyte (iCM) formation from fibroblasts. PC14-3-3 is identified in pivotal functional proteins for iCM reprogramming, including transcription factors and chromatin modifiers. Akt1 kinase and protein phosphatase 2A are the key writer and key eraser of the PC14-3-3 code, respectively. PC14-3-3 activation induces iCM formation with the presence of only Tbx5. In contrast, PC14-3-3 inhibition by mutagenesis or inhibitor-mediated code removal abolishes reprogramming. We discover that key PC14-3-3-embedded factors, such as histone deacetylase 4 (Hdac4), Mef2c, and Foxo1, form Hdac4-organized inhibitory nuclear condensates. PC14-3-3 activation disrupts Hdac4 condensates to promote cardiac gene expression. Our study suggests that sub-organelle dynamics regulated by a PTM code could be a general mechanism for stimulating cell reprogramming.

MEF2C regulates NK cell effector functions through control of lipid metabolism.

Natural killer (NK) cells are a critical first line of defense against viral infection. Rare mutations in a small subset of transcription factors can result in decreased NK cell numbers and function in humans, with an associated increased susceptibility to viral infection. However, our understanding of the specific transcription factors governing mature human NK cell function is limited. Here we use a non-viral CRISPR-Cas9 knockout screen targeting genes encoding 31 transcription factors differentially expressed during human NK cell development. We identify myocyte enhancer factor 2C (MEF2C) as a master regulator of human NK cell functionality ex vivo. MEF2C-haploinsufficient patients and mice displayed defects in NK cell development and effector function, with an increased susceptibility to viral infection. Mechanistically, MEF2C was required for an interleukin (IL)-2- and IL-15-mediated increase in lipid content through regulation of sterol regulatory element-binding protein (SREBP) pathways. Supplementation with oleic acid restored MEF2C-deficient and MEF2C-haploinsufficient patient NK cell cytotoxic function. Therefore, MEF2C is a critical orchestrator of NK cell antiviral immunity by regulating SREBP-mediated lipid metabolism.

Folding of Class IIa HDAC Derived Peptides into α-helices Upon Binding to Myocyte Enhancer Factor-2 in Complex with DNA.

Interaction of transcription factor myocyte enhancer factor-2 (MEF2) family members with class IIa histone deacetylases (HDACs) has been implicated in a wide variety of diseases. Though considerable knowledge on this topic has been accumulated over the years, a high resolution and detailed analysis of the binding mode of multiple class IIa HDAC derived peptides with MEF2D is still lacking. To fulfil this gap, we report here the crystal structure of MEF2D in complex with double strand DNA and four different class IIa HDAC derived peptides, namely HDAC4, HDAC5, HDAC7 and HDAC9. All class IIa HDAC derived peptides form extended amphipathic α-helix structures that fit snugly in the hydrophobic groove of MEF2D domain. Binding mode of class IIa HDAC derived peptides to MEF2D is very similar and occur primarily through nonpolar interactions mediated by highly conserved branched hydrophobic amino acids. Further studies revealed that class IIa HDAC derived peptides are unstructured in solution and appear to adopt a folded α-helix structure only upon binding to MEF2D. Comparison of our peptide-protein complexes with previously characterized structures of MEF2 bound to different co-activators and co-repressors, highlighted both differences and similarities, and revealed the adaptability of MEF2 in protein-protein interactions. The elucidation of the three-dimensional structure of MEF2D in complex with multiple class IIa HDAC derived peptides provide not only a better understanding of the molecular basis of their interactions but also have implications for the development of novel antagonist.

Identification of two MEF2s and their role in inhibiting the transcription of the mstn2a gene in the yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782).

Myocyte-specific enhancer binding factor 2 (MEF2), which belongs to the MADS superfamily, is a pivotal and conserved transcription factor that combines with the E-box motif to control the expression of muscle genes. Myostatin (mstn), a muscle growth inhibitor, is a vital member of the TGF-ß superfamily. Currently, an understanding of the mechanisms of A. latus mstn (Almstn) transcriptional regulation mediated by MEF2 in fish muscle development is lacking. In the present study, two AlMEF2s (AlMEF2A and AlMEF2B) and Almstn2a were characterized from Acanthopagrus latus. AlMEF2A and AlMEF2B had 456 and 315 amino acid (aa) residues, respectively. Two typical regions, a MADS-box, MEF2, and transcriptionally activated (TAD) domains, are present in both AlMEF2s. The expression profiles of the two AlMEF2 genes were similar. The AlMEF2 genes were mainly expressed in the brain, white muscle, and liver, while Almstn2a expression was higher in the brain than in other tissues. Moreover, the expression trends of AlMEF2s and Almstn2a were significantly changed after starvation and refeeding in the five groups. Additionally, truncation experiments showed that -987 to +168 and -105 to +168 were core promoters of Almstn2a that responded to AlMEF2A and AlMEF2B, respectively. The point mutation experiment confirmed that Almstn2a transcription relies on the mutation binding sites 1 or 5 (M1/5) and mutation binding sites 4 or 5 (M4/5) for AlMEF2A and AlMEF2B regulation, respectively. The electrophoretic mobile shift assay (EMSA) further verified that M1 (-527 to -512) was a pivotal site where AlMEF2A acted on the Almstn2a gene. Furthermore, a siRNA interference gene expression experiment showed that reduced levels of AlMEF2A or AlMEF2B could prominently increase Almstn2a transcription. These results provide new information about the regulation of Almstn2a transcriptional activity by AlMEF2s and a theoretical basis for the regulatory mechanisms involved in muscle development in fish.

METTL3 promotes microglial inflammation via MEF2C in spinal cord injury.

Spinal cord injury (SCI) is a significant contributor to disability in contemporary society, resulting in substantial psychological and economic burdens for patients and their family. Microglia-mediated inflammation is an important factor affecting the nerve repair of SCI patients. N6-methyladenosine (m6A) is a prevalent epigenetic modification in mammals, which shows a strong association with inflammation. However, the mechanism of m6A modification regulating microglia-mediated inflammation is still unclear. Here, we observed that METTL3, a m6A methylase, was increased in SCI mice and lipopolysaccharide (LPS)-exposed BV2 cells. Knockdown of METTL3 inhibited the increased expression of iNOS and IL-1ß induced by LPS in vitro. Subsequently, MEF2C, myocyte-specific enhancer factor 2C, was decreased in SCI mice and LPS-exposed BV2 cells. Knockdown of MEF2C promoted the expression of iNOS and IL-1ß. Sequence analysis showed that there were multiple highly confident m6A modification sites on the MEF2C mRNA. METTL3 antibody could pull down a higher level of MEF2C mRNA than the IgG in RNA binding protein immunoprecipitation assay. Knockdown of METTL3 promoted MEF2C protein expression and MEF2C mRNA expression, accompanied by a reduced m6A modification level on the MEF2C mRNA. Knockdown of MEF2C inhibited the anti-inflammatory effect of METTL3 siRNA. Our results suggest that METTL3 promotes microglia inflammation via regulating MEF2C mRNA m6A modification induced by SCI and LPS treatment.

Structural insights into the HDAC4-MEF2A-DNA complex and its implication in long-range transcriptional regulation.

Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7 and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation.

Deciphering the roles of subcellular distribution and interactions involving the MEF2 binding region, the ankyrin repeat binding motif and the catalytic site of HDAC4 in Drosophila neuronal morphogenesis.

BACKGROUND: Dysregulation of nucleocytoplasmic shuttling of histone deacetylase 4 (HDAC4) is associated with several neurodevelopmental and neurodegenerative disorders. Consequently, understanding the roles of nuclear and cytoplasmic HDAC4 along with the mechanisms that regulate nuclear entry and exit is an area of concerted effort. Efficient nuclear entry is dependent on binding of the transcription factor MEF2, as mutations in the MEF2 binding region result in cytoplasmic accumulation of HDAC4. It is well established that nuclear exit and cytoplasmic retention are dependent on 14-3-3-binding, and mutations that affect binding are widely used to induce nuclear accumulation of HDAC4. While regulation of HDAC4 shuttling is clearly important, there is a gap in understanding of how the nuclear and cytoplasmic distribution of HDAC4 impacts its function. Furthermore, it is unclear whether other features of the protein including the catalytic site, the MEF2-binding region and/or the ankyrin repeat binding motif influence the distribution and/or activity of HDAC4 in neurons. Since HDAC4 functions are conserved in Drosophila, and increased nuclear accumulation of HDAC4 also results in impaired neurodevelopment, we used Drosophila as a genetic model for investigation of HDAC4 function. RESULTS: Here we have generated a series of mutants for functional dissection of HDAC4 via in-depth examination of the resulting subcellular distribution and nuclear aggregation, and correlate these with developmental phenotypes resulting from their expression in well-established models of neuronal morphogenesis of the Drosophila mushroom body and eye. We found that in the mushroom body, forced sequestration of HDAC4 in the nucleus or the cytoplasm resulted in defects in axon morphogenesis. The actions of HDAC4 that resulted in impaired development were dependent on the MEF2 binding region, modulated by the ankyrin repeat binding motif, and largely independent of an intact catalytic site. In contrast, disruption to eye development was largely independent of MEF2 binding but mutation of the catalytic site significantly reduced the phenotype, indicating that HDAC4 acts in a neuronal-subtype-specific manner. CONCLUSIONS: We found that the impairments to mushroom body and eye development resulting from nuclear accumulation of HDAC4 were exacerbated by mutation of the ankyrin repeat binding motif, whereas there was a differing requirement for the MEF2 binding site and an intact catalytic site. It will be of importance to determine the binding partners of HDAC4 in nuclear aggregates and in the cytoplasm of these tissues to further understand its mechanisms of action.

The transcription factor ZEB2 drives the formation of age-associated B cells.

Age-associated B cells (ABCs) accumulate during infection, aging, and autoimmunity, contributing to lupus pathogenesis. In this study, we screened for transcription factors driving ABC formation and found that zinc finger E-box binding homeobox 2 (ZEB2) is required for human and mouse ABC differentiation in vitro. ABCs are reduced in ZEB2 haploinsufficient individuals and in mice lacking Zeb2 in B cells. In mice with toll-like receptor 7 (TLR7)-driven lupus, ZEB2 is essential for ABC formation and autoimmune pathology. ZEB2 binds to +20-kb myocyte enhancer factor 2b (Mef2b)'s intronic enhancer, repressing MEF2B-mediated germinal center B cell differentiation and promoting ABC formation. ZEB2 also targets genes important for ABC specification and function, including Itgax. ZEB2-driven ABC differentiation requires JAK-STAT (Janus kinase-signal transducer and activator of transcription), and treatment with JAK1/3 inhibitor reduces ABC accumulation in autoimmune mice and patients. Thus, ZEB2 emerges as a driver of B cell autoimmunity.

LncRNA CASC2 Alleviates Renal Interstitial Inflammation and Fibrosis through MEF2C Downregulation-Induced Hinderance of M1 Macrophage Polarization.

INTRODUCTION: Long noncoding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) alleviates the progression of diabetic nephropathy by inhibiting inflammation and fibrosis. This study investigated how CASC2 impacts renal interstitial fibrosis (RIF) through regulating M1 macrophage (M1) polarization. METHOD: Nine-week-old mice underwent unilateral ureteral obstruction (UUO) establishment. Macrophages were induced toward M1 polarization using lipopolysaccharide (LPS) in vitro and cocultured with fibroblasts to examine how M1 polarization influences RIF. LnCeCell predicted that CASC2 interacted with myocyte enhancer factor 2 C (MEF2C), which was validated by dual-luciferase reporter assay. CASC2/MEF2C overexpression was achieved by lentivirus-expressing lncRNA CASC2 injection in vivo or CASC2 and MEF2C transfection in vitro. Renal injury was evaluated through biochemical analysis and hematoxylin-eosin/Masson staining. Macrophage infiltration and M1 polarization in the kidney and/or macrophages were detected by immunofluorescence, flow cytometry, and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR). Expressions of CASC2, MEF2C, and markers related to inflammation/M1/fibrosis in the kidney/macrophages/fibroblasts were analyzed by qRT-PCR, fluorescence in situ hybridization, enzyme-linked immunosorbent assay, and/or Western blot. RESULT: In the kidneys of mice, CASC2 was downregulated and macrophage infiltration was promoted time-dependently from days 3 to 14 post-UUO induction; CASC2 overexpression alleviated renal histological abnormalities, hindered macrophage infiltration and M1 polarization, downregulated renal function markers serum creatinine and blood urea nitrogen and inflammation/M1/fibrosis-related makers, and offset UUO-induced MEF2C upregulation. LncRNA CASC2 overexpression inhibited fibroblast fibrosis and M1 polarization in cocultured fibroblasts with LPS-activated macrophages. Also, CASC2 bound to MEF2C and inhibited its expression in LPS-activated macrophages. Furthermore, MEF2C reversed the inhibitory effects of lncRNA CASC2 overexpression. CONCLUSION: CASC2 alleviates RIF by inhibiting M1 polarization through directly downregulating MEF2C expression. CASC2 might represent a promising value of future investigations on treatment for RIF.

Brusatol enhances MEF2A expression to inhibit RCC progression through the Wnt signalling pathway in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most aggressive subtype of kidney tumour with a poor prognosis and an increasing incidence rate worldwide. Brusatol, an essential active ingredient derived from Brucea javanica, exhibits potent antitumour properties. Our study aims to explore a novel treatment strategy for RCC patients. We predicted 37 molecular targets of brusatol based on the structure of brusatol, and MEF2A (Myocyte Enhancer Factor 2A) was selected as our object through bioinformatic analyses. We employed various experimental techniques, including RT-PCR, western blot, CCK8, colony formation, immunofluorescence, wound healing, flow cytometry, Transwell assays and xenograft mouse models, to investigate the impact of MEF2A on RCC. MEF2A expression was found to be reduced in patients with RCC, indicating a close correlation with MEF2A deubiquitylation. Additionally, the protective effects of brusatol on MEF2A were observed. The overexpression of MEF2A inhibits RCC cell proliferation, invasion and migration. In xenograft mice, MEF2A overexpression in RCC cells led to reduced tumour size compared to the control group. The underlying mechanism involves the inhibition of RCC cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) through the modulation of Wnt/ß-catenin signalling. Altogether, we found that MEF2A overexpression inhibits RCC progression by Wnt/ß-catenin signalling, providing novel insight into diagnosis, treatment and prognosis for RCC patients.

A Ubiquitin-Dependent Switch on MEF2D Senses Pro-Metastatic Niche Signals to Facilitate Intrahepatic Metastasis of Liver Cancer.

Effective treatment for metastasis, a leading cause of cancer-associated death, is still lacking. To seed on a distal organ, disseminated cancer cells (DCCs) must adapt to the local tissue microenvironment. However, it remains elusive how DCCs respond the pro-metastatic niche signals. Here, systemic motif-enrichment identified myocyte enhancer factor 2D (MEF2D) as a critical sensor of niche signals to regulate DCCs adhesion and colonization, leading to intrahepatic metastasis and recurrence of liver cancer. In this context, MEF2D transactivates Itgb1 (coding ß1-integrin) and Itgb4 (coding ß4-integrin) to execute temporally unique functions, where ITGB1 recognizes extracellular matrix for early seeding, and ITGB4 acts as a novel sensor of neutrophil extracellular traps-DNA (NETs-DNA) for subsequent chemotaxis and colonization. In turn, an integrin-FAK circuit promotes a phosphorylation-dependent USP14-orchastrated deubiquitination switch to stabilize MEF2D via circumventing degradation by the E3-ubiquitin-ligase MDM2. Clinically, the USP14(pS432)-MEF2D-ITGB1/4 feedback loop is often hyper-active and indicative of inferior outcomes in human malignancies, while its blockade abrogated intrahepatic metastasis of DCCs. Together, DCCs exploit a deubiquitination-dependent switch on MEF2D to integrate niche signals in the liver mesenchyme, thereby amplifying the pro-metastatic integrin-FAK signaling. Disruption of this feedback loop is clinically applicable with fast-track potential to block microenvironmental cues driving metastasis.

MEF2C and miR-194-5p: New Players in Triple Negative Breast Cancer Tumorigenesis.

Among breast cancer (BC) subtypes, the most aggressive is triple negative BC (TNBC), which is prone to metastasis. We previously found that microRNA (miR)-194-5p is downregulated at the early stages of TNBC brain metastasis development. Additionally, the transcription factor myocyte enhancer factor 2 (MEF2)C, a bioinformatically predicted miR-194-5p target, was increasingly expressed throughout TNBC brain metastasis formation and disease severity. However, the contributions of these two players to malignant cells' features remain undetermined. This study aimed at disclosing the role of miR-194-5p and MEF2C in TNBC tumorigenesis. The transfection of 4T1 cells with a silencer for MEF2C or with a pre-miRNA for miR-194-5p was employed to study TNBC cells' phenotypic alterations regarding epithelial and mesenchymal markers, as well as migratory capability alterations. MEF2C-silenced cells presented a decline in both vimentin and cytokeratin expression, whereas the overexpression of miR-194-5p promoted an increase in cytokeratin and a reduction in vimentin, reflecting the acquisition of an epithelial phenotype. Both treatments reduced TNBC cells' migration. These results suggest that MEF2C may determine TNBC cells' invasive properties by partially determining the occurrence of epithelial-mesenchymal transition, while the overexpression of miR-194-5p promotes a decline in TNBC cells' aggressive behavior and reinforces this miRNA's role as a tumor suppressor in TNBC.

ERK5 Cooperates With MEF2C to Regulate Nr4a1 Transcription in MA-10 and MLTC-1 Leydig Cells.

Leydig cells produce hormones required for the development and maintenance of sex characteristics and fertility in males. MEF2 transcription factors are important regulators of Leydig cell gene expression and steroidogenesis. ERK5 is an atypical member of the MAP kinase family that modulates transcription factor activity, either by direct phosphorylation or by acting as a transcriptional coactivator. While MEF2 and ERK5 are known to cooperate transcriptionally, the presence and role of ERK5 in Leydig cells remained unknown. Our goal was to determine whether ERK5 is present in Leydig cells and whether it cooperates with MEF2 to regulate gene expression. We found that ERK5 is present in Leydig cells in testicular tissue and immortalized cell lines. ERK5 knockdown in human chorionic gonadotrophin-treated MA-10 Leydig cells reduced steroidogenesis and decreased Star and Nr4a1 expression. Luciferase assays using a synthetic reporter plasmid containing 3 MEF2 elements revealed that ERK5 enhances MEF2-dependent promoter activation. Although ERK5 did not cooperate with MEF2 on the Star promoter in Leydig cell lines, we found that ERK5 and MEF2C do cooperate on the Nr4a1 promoter, which contains 2 adjacent MEF2 elements. Mutation of each MEF2 element in a short version of the Nr4a1 promoter significantly decreased the ERK5/MEF2C cooperation, indicating that both MEF2 elements need to be intact. The ERK5/MEF2C cooperation did not require phosphorylation of MEF2C on Ser387. Taken together, our data identify ERK5 as a new regulator of MEF2 activity in Leydig cells and provide potential new insights into mechanisms that regulate Leydig cell gene expression and function.

Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

The current study intended to delve into the mechanisms of dexmedetomidine (Dex) in regulating myocardial pyroptosis against myocardial ischemia/reperfusion injury (MIRI). The rat MIRI models were induced by ligation/release of the coronary artery in vivo and Langendorff perfusion ex vivo. Hemodynamic parameters, infarction sizes, and histopathological changes were assessed to understand the effects of Dex on MIRI. We explored the mechanisms through functional experiments on an H9c2 cell hypoxia/reoxygenation (H/R) model. Cell viability and apoptosis were evaluated using cell counting kit 8 (CCK-8) and AV/PI dual staining respectively. The expressions of miR-665 and MEF2D mRNA were detected by qRT-PCR. Western blot was employed to determine the expression levels of pyroptosis- and signaling pathway- related proteins. The interplays between miR-665 and MEF2D were validated by Dual-luciferase reporter assays. Our findings indicated that Dex preconditioning dramatically attenuated hemodynamic derangements, infarct size, and histopathological damage in rats undergoing MIRI. Dex markedly augmented cell viability, while suppressing cell apoptosis and expressions of NLRP3, cleaved-caspase-1, ASC, GSDMD, IL-1ß, and IL-18 in H9c2 cells subjected to H/R injury. MiR-665 was significantly upregulated, MEF2D and Nrf2 downregulated following H/R, whereas Dex preconditioning reversed these changes. MEF2D was validated to be a target gene of miR-665. Overexpression of miR-665 decreased the expression of MEF2D and blunted the protective effects of Dex in H9c2 cells. Moreover, the functional rescue experiment further verified that Dex regulated MEF2D/Nrf2 pathway via miR-665. In conclusion, Dex mitigates MIRI through inhibiting pyroptosis via regulating miR-665/MEF2D/Nrf2 axis.

Effect of Bovine MEF2A Gene Expression on Proliferation and Apoptosis of Myoblast Cells.

Myocyte enhancer factor 2A (MEF2A) is a member of the myocyte enhancer factor 2 family. MEF2A is widely distributed in various tissues and organs and participates in various physiological processes. This study aimed to investigate the effect of MEF2A expression on the proliferation and apoptosis of bovine myoblasts. CCK8, ELISA, cell cycle, and apoptosis analyses were conducted to assess cell status. In addition, the mRNA expression levels of genes associated with bovine myoblast proliferation and apoptosis were evaluated using RT-qPCR. The results showed that the upregulation of MEF2A mRNA promoted the proliferation rate of myoblasts, shortened the cycle process, and increased the anti-apoptotic rate. Furthermore, the RT-qPCR results showed that the upregulation of MEF2A mRNA significantly increased the cell proliferation factors MyoD1 and IGF1, cell cycle factors CDK2 and CCNA2, and the apoptotic factors Bcl2 and BAD (p < 0.01). These results show that the MEF2A gene can positively regulate myoblast proliferation and anti-apoptosis, providing a basis for the analysis of the regulatory mechanism of the MEF2A gene on bovine growth and development.

The MEF2 homolog of Penaeus vannamei is essential for maintaining the WSSV latent infection.

White spot syndrome virus (WSSV) is a lethal shrimp pathogen that has a latent infection cycle. The latent virus can easily turn into an acute infection when the culture environment changes, leading to widespread shrimp mortality. However, the mechanism of WSSV latent infection is poorly understood. Bioinformatic analysis revealed that the promoters of WSSV latency-related genes (i.e., wsv151, wsv366, wsv403, and wsv427) contained putative myocyte enhancer factor 2 (MEF2) binding sites. This suggested that the transcription factor MEF2 may be involved in WSSV latent infection. To further investigate this, a MEF2 homolog (PvMEF2) was cloned from Penaeus vannamei and its role in WSSV latent infection was explored. The results showed that knockdown of PvMEF2 led to an increase in the copy number of WSSV, indicating reactivation of WSSV from a latent infection. It was further demonstrated that suppression of PvMEF2 significantly decreased expression of the viral latency-related genes in WSSV-latent shrimp, while overexpression of PvMEF2 in Drosophila S2 cells activated the promoter activity of the viral latency-related gene. Additionally, we demonstrated that silencing of PvMEF2 was able to upregulate the expression of pro-apoptosis genes, thereby promoting cell apoptosis during latent infection. Collectively, the present data suggest that PvMEF2 could promote the expression of virus latency-related genes and enhance cell survival to maintain WSSV latent infection. This finding would contribute to a better understanding of the maintenance mechanism of WSSV latent infection.

Comprehensive analysis and identification of the circ_0084615/miR-451a/MEF2D axis in benzo(a)pyrene exposed tumor cells in hepato-carcinogenesis.

Hepatocellular carcinoma (HCC) is caused by genetic and epigenetic alterations, as well as abnormal lifestyle and dietary habits, including contaminated food intake. Benzo(a)pyrene (B[a]P), derived from deep-fried meats, is regarded as the main dietary factor for tumorigenesis in epidemiological investigations. Although various studies have illustrated the adverse effects of B[a]P in malignancy through cell and animal models, the correlation between B[a]P exposure and clinical data remain to be explored. In the present study, we analyzed and identified novel B[a]P-associated circular RNA (circRNA) from microarray databases of liver tumor cells and HCC patient samples. Considering that circRNA regulates mRNA as a miRNA sponge, molecular circRNA-miRNA-mRNA interactions based on the stimulation of B[a]P exposure were predicted and established. Furthermore, up-regulated circ_0084615 in B[a]P-treated tumor cells was verified as a miRNA sponge via fluorescence in situ hybridization (FISH) assays, and the repression between circ_0084615 and target miR-451a exhibited a contrasting effect on hepatocarcinogenesis. Therefore, we performed integrated bioinformatics analysis and molecular experiments to establish the circ_0084615/miR-451a/MEF2D pathway, which provided a better understanding of the adverse effects of fried food preference on human health.

mef2ca and mef2cb Double Mutant Zebrafish Show Altered Craniofacial Phenotype and Motor Behaviour.

The transcription factor MEF2C is crucial in neuronal, cardiac, bone and cartilage molecular processes, as well as for craniofacial development. MEF2C was associated with the human disease MRD20, whose patients show abnormal neuronal and craniofacial development. Zebrafish mef2ca;mef2cb double mutants were analysed for abnormalities in craniofacial and behaviour development through phenotypic analysis. Quantitative PCR was performed to investigate the expression levels of neuronal marker genes in mutant larvae. The motor behaviour was analysed by the swimming activity of 6 dpf larvae. We found that mef2ca;mef2cb double mutants display several abnormal phenotypes during early development, including those already described in zebrafish carrying mutations in each paralog, but also (i) a severe craniofacial phenotype (comprising both cartilaginous and dermal bone structures), (ii) developmental arrest due to the disruption of cardiac oedema and (iii) clear alterations in behaviour. We demonstrate that the defects observed in zebrafish mef2ca;mef2cb double mutants are similar to those previously described in MEF2C-null mice and MRD20 patients, confirming the usefulness of these mutant lines as a model for studies concerning MRD20 disease, the identification of new therapeutic targets and screening for possible rescue strategies.

LncRNA TCONS_00323213 Promotes Myogenic Differentiation by Interacting with PKNOX2 to Upregulate MyoG in Porcine Satellite Cells.

Myogenic differentiation is a complex biological process that is regulated by multiple factors, among which long noncoding RNAs (lncRNAs) play an essential role. However, in-depth studies on the regulatory mechanisms of long noncoding RNAs (lncRNAs) in myogenic differentiation are limited. In this study, we characterized the role of the novel lncRNA TCONS_00323213, which is upregulated during porcine skeletal muscle satellite cell (PSC) differentiation in myogenesis. We found that TCONS_00323213 affected the proliferation and differentiation of PSC in vitro. We performed quantitative polymerase chain reaction (qPCR), 5-ethynyl-20-deoxyuridine (EdU), western blotting, immunofluorescence staining, pull-down assays, and cleavage under targets and tagmentation (CUT and Tag) assays to clarify the effects and action mechanisms of TCONS_00323213. LncRNA TCONS_00323213 inhibited myoblast proliferation based on analyses of cell survival rates during PSC proliferation. Functional analyses revealed that TCONS_00323213 promotes cell differentiation and enhances myogenin (MyoG), myosin heavy chain (MyHC), and myocyte enhancer factor 2 (MEF2C) during myoblast differentiation. As determined by pull-down and RNA immunoprecipitation (RIP) assays, the lncRNA TCONS_00323213 interacted with PBX/Knotted Homeobox 2 (PKNOX2). CUT and Tag assays showed that PKNOX2 was significantly enriched on the MyoG promoter after lncRNA TCONS_00323213 knockdown. Our findings demonstrate that the interaction between lncRNA TCONS_00323213 and PKNOX2 relieves the inhibitory effect of PKNOX2 on the MyoG promoter, increases its expression, and promotes PSC differentiation. This novel role of lncRNA TCONS_00323213 sheds light on the molecular mechanisms by which lncRNAs regulate porcine myogenesis.