Generation of both an shRNA-resistant MEF2A over expression construct and a dominant negative construct in adenovirus for rescue and knockout experiments in muscle

The Myocyte Enhancer Factor-2, or MEF2, transcription factor family is necessary for the differentiation and regeneration of both skeletal and cardiac muscle tissue. The transcription factors in this family are responsible for the activation of many muscle specific growth factor-induced and differentiation genes. There are four individual isoforms of MEF2; MEF2A, -B, -C, and –D, and the roles of these individual transcription factors are not completely understood. Knockdowns of these individual isoforms revealed that a MEF2A knockdown mouse model displays severe myofibrillar defects in cardiac muscle. This knockdown also has shown that MEF2A is required for myogenesis in vitro, where the other 3 isoforms, -B, -C, and –D, are not necessary for this process. One method of knocking down MEF2A to study its roles further is through the use of short hairpin RNAs (shRNA). The purpose of my research was two-fold. First, in order to test the specificity of this shRNA method, an shRNA-resistant MEF2A over expression construct in an adenoviral vector was created to perform rescue experiments. Second, to compare individual MEF2 isoform knockouts to a complete knockout of the entire MEF2 family, a dominant negative construct was created in an adenoviral vector. In both cases, a pShuttle-CMV adenoviral vector was used. The results of this experiment can be used to further investigate the roles of MEF2A in both regeneration and differentiation of skeletal and cardiac muscle tissue

Thrombin Protease-activated Receptor-1 Signals through Gq- and G13-initiated MAPK Cascades Regulating c-Jun Expression to Induce Cell Transformation

Although the ability of G protein-coupled receptors to stimulate normal and aberrant cell growth has been intensely investigated, the precise nature of the molecular mechanisms underlying their transforming potential are still not fully understood. In this study, we have taken advantage of the potent mitogenic effect of thrombin and the focus-forming activity of one of its receptors, protease-activated receptor-1, to dissect how this receptor coupled to Gi, Gq/11, and G12/13 transduces signals from the membrane to the nucleus to initiate transcriptional events involved in cell transformation. Using endogenous and transfected thrombin receptors in NIH 3T3 cells, ectopic expression of muscarinic receptors coupled to Gq and Gi, and chimeric G protein subunits and murine fibroblasts deficient in Gq/11, and G12/13, we show here that, although coupling to Gi is sufficient to induce ERK activation, the ability to couple to Gq and/or G13 is necessary to induce c-jun expression and cell transformation. Furthermore, we show that Gq and G13 can initiate the activation of MAPK cascades, including JNK, p38, and ERK5, which in turn regulate the activity of transcription factors controlling expression from the c-jun promoter. We also present evidence that c-Jun and the kinases regulating its expression are integral components of the transforming pathway initiated by protease-activated receptor-1

Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module

Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins

RNA microarray analysis in prenatal mouse cochlea reveals novel IGF-I target genes: implication of MEF2 and FOXM1 transcription factors

Background: Insulin-like growth factor-I (IGF-I) provides pivotal cell survival and differentiation signals during inner ear development throughout evolution. Homozygous mutations of human IGF1 cause syndromic sensorineural deafness, decreased intrauterine and postnatal growth rates, and mental retardation. In the mouse, deficits in IGF-I result in profound hearing loss associated with reduced survival, differentiation and maturation of auditory neurons. Nevertheless, little is known about the molecular basis of IGF-I activity in hearing and deafness. Methodology/Principal Findings: A combination of quantitative RT-PCR, subcellular fractionation and Western blotting, along with in situ hybridization studies show IGF-I and its high affinity receptor to be strongly expressed in the embryonic and postnatal mouse cochlea. The expression of both proteins decreases after birth and in the cochlea of E18.5 embryonic Igf1(-/-) null mice, the balance of the main IGF related signalling pathways is altered, with lower activation of Akt and ERK1/2 and stronger activation of p38 kinase. By comparing the Igf1(-/-) and Igf1(+/+) transcriptomes in E18.5 mouse cochleae using RNA microchips and validating their results, we demonstrate the up-regulation of the FoxM1 transcription factor and the misexpression of the neural progenitor transcription factors Six6 and Mash1 associated with the loss of IGF-I. Parallel, in silico promoter analysis of the genes modulated in conjunction with the loss of IGF-I revealed the possible involvement of MEF2 in cochlear development. E18.5 Igf1(+/+) mouse auditory ganglion neurons showed intense MEF2A and MEF2D nuclear staining and MEF2A was also evident in the organ of Corti. At P15, MEF2A and MEF2D expression were shown in neurons and sensory cells. In the absence of IGF-I, nuclear levels of MEF2 were diminished, indicating less transcriptional MEF2 activity. By contrast, there was an increase in the nuclear accumulation of FoxM1 and a corresponding decrease in the nuclear cyclin-dependent kinase inhibitor p27(Kip1). Conclusions/Significance: We have defined the spatiotemporal expression of elements involved in IGF signalling during inner ear development and reveal novel regulatory mechanisms that are modulated by IGF-I in promoting sensory cell and neural survival and differentiation. These data will help us to understand the molecular bases of human sensorineural deafness associated to deficits in IGF-I

Effects of hexabromocyclododecane (HBCD) on cardiac development in zebrafish embryos (Danio rerio): the mechanisms involved

六溴环十二烷（1,2,5,6,9,10-Hexabromocyclododecane，HBCD）是被运用最广泛的阻燃剂之一，在全球的用量仅次于四溴双酚A和多溴联苯醚。HBCD具有持久性、生物累积性和生物放大效应，因而被定义为持久性有机污染物（persistentorganicpollutants，POPs）。已有研究表明HBCD具有发育毒性、神经毒性、内分泌毒性和生殖毒性，但对心脏发育毒性的研究十分有限，尤其缺乏对心脏发育毒性机制的研究。本研究以斑马鱼和H9C2细胞系为实验模型，采用低浓度HBCD（0，2nM，20nM和200nM）对其进行暴露，应用生态毒理学、分子生物学以及生物信息学技术检测...Hexabromocyclododecane (HBCD) is the third most commonly used brominated flame retardants, following tetrabromobisphenol A and polybrominated diphenyl ethers. HBCD has a strong potential to be persistent, bioaccumulative and biomagnified, so it is listed into persistent organic pollutants. Studies have reported that HBCD can cause developmental, neural, endocrine and reproductive toxicities. Howev...学位：理学博士院系专业：生命科学学院_动物学学号：2162012015376

The relevance of depression-associated genes in neuronal in vitro systems.

Um neue Erkenntnisse über die Bedeutung der 15 „Depressionsgene“ im Gehirn für die Pathophysiologie der IFN-α-induzierten sowie der endogenen Depressionserkrankungen zu erlangen, wurde in dieser Dissertation zunächst ein in vitro-Zellkultursystem aus murinen hippocampalen und kortikalen Primärneuronen entwickelt. Durch Nachahmung einer Hepatitis-C-Virus-Therapie (HCV) durch Kostimulation mit murinem Interferon-alpha (mIFN-α) (1000 IU/ml) und dem TLR3-Agonisten Polyinosin:Polycytidylsäure (Poly(I:C)) (100 μg/ml) wurde eine starke Überexpression der „Depressionsgene“ Gch1, Disc1, Tor1b, Dynlt1 und Mef2a in primären Neuronen in vitro gezeigt. Stat1, Ube2L6, Rtp4 und Gbp1 wurden genau so wie in den in vitro Vorversuchen mit der hippocampalen HT22 Zelllinie höher als alle anderen „Depressionsgene“ exprimiert. Die schnelle Regulation dieser Gene lässt vermuten, dass diese als nützliche Marker für eine Depression geeignet sind. Die synergistische mIFN/Poly(I:C)-Wirkung förderte zugleich eine Überexpression der proinflammatorischen Zytokine Cxcl1, Cxcl10, Ccl5, Tnf, Il6 und Ifng auf molekularen und proteinbiochemischen Ebenen in hippocampalen und kortikalen Neuronen in vitro, die hauptsächlich einer STAT1-abhängigen transkriptionellen Antwort auf IFN-α unterliegen. Diese „Downstream“-Zytokine stellen einen möglichen Zusammenhang zwischen den Entzündungsprozessen im Gehirn und der Veränderung der Neurotransmitterbiosynthese durch eine synergetisch erhöhte Aktivität des Enzyms Indolamin-2,3-Dioxygenase (Ido1), des Serotonintransporters (Slc6a4) und -rezeptors (Htr1a) sowie durch eine gewebespezifische Veränderung der Expression des Dopamintransporters (Slc6a3) auf indirektem Weg in primären Neuronen in vitro dar. Da die transkriptionelle Antwort auf IFN-α in Neuronen vollständig von STAT1 abhängt, beeinflusst die in dieser Arbeit gefundene starke IFN/Poly(I:C)-vermittelte STAT1-Aktivierung in primären Neuronen die Induktion der proinflammatorischen Zytokine, die wiederum eine mögliche Überexpression von inflammatorischen Genen steuert. Durch einen gezielten STAT1-„Knockdown“ mittels RNAi- Technologie wurde eine signifikante Suppression der „Depressionsgene“ Gch1, Disc1, Mef2a in hippocampalen und kortikalen Neuronen in vitro nachgewiesen. Da erstens das Kandidatengen Gch1 in die Serotonin/Dopamin-Biosynthese involviert ist, zweitens Disc1 mit der Entstehung von Schizophrenie und Depression und drittens Mef2a mit der Hemmung des Neuritenwachstums und der kortikalen Entwicklung assoziiert ist, scheint die transiente STAT1-Suppression ein wirksames molekulares Target für die Hemmung der „Depressionsgen“-Funktion zu sein. Die STAT1-Suppression hat trotz anschließender mIFN/Poly(I:C)-Kostimulation ebenfalls eine signifikant hemmende Wirkung auf die Expression von Ccl5, Il6 und Cxcl10, der Ido1 und Slc6a4 in primären Neuronen. Somit spielt STAT1 eine wichtige Rolle im IFN-abhängigen Signalweg der Gehirnzellen und scheint ein Schlüsselmolekül in der Regulation der besonders wichtigen „Depressionsgene“, Neurotransmittermoleküle und der „Downstream“- Zytokine zu sein. Die gezielte STAT1-Suppression im Gehirn kann einem möglichen therapeutischen Ansatz zur Verbesserung/Minderung der Symptome einer exogenen/endogenen Depression dienen

Identification of miR-379/miR-656 (C14MC) cluster downregulation and associated epigenetic and transcription regulatory mechanism in oligodendrogliomas

Introduction Although role of individual microRNAs (miRNAs) in the pathogenesis of gliomas has been well studied, their role as a clustered remains unexplored in gliomas. Methods In this study, we performed the expression analysis of miR-379/miR-656 miRNA-cluster (C14MC) in oligodendrogliomas (ODGs) and also investigated the mechanism underlying modulation of this cluster. Results We identified significant downregulation of majority of the miRNAs from this cluster in ODGs. Further data from The Cancer Genome Atlas (TCGA) also confirmed the global downregulation of C14MC. Furthermore, we observed that its regulation is maintained by transcription factor MEF2. In addition, epigenetic machinery involving DNA and histone-methylation are also involved in its regulation, which is acting independently or in synergy. The post- transcriptionally regulatory network of this cluster showed enrichment of key cancer-related biological processes such as cell adhesion and migration. Also, there was enrichment of several cancer related pathways viz PIK3 signaling pathway and glioma pathways. Survival analysis demonstrated association of C14MC (miR-487b and miR-409-3p) with poor progression free survival in ODGs. Conclusion Our work demonstrates tumor-suppressive role of C14MC and its role in pathogenesis of ODGs and therefore could be relevant for the development of new therapeutic strategies

Control of asymmetric Hopfield networks and application to cancer attractors

The asymmetric Hopfield model is used to simulate signaling dynamics in gene/transcription factor networks. The model allows for a direct mapping of a gene expression pattern into attractor states. We analyze different control strategies aiming at disrupting attractor patterns using selective local fields representing therapeutic interventions. The control strategies are based on the identification of signaling $bottlenecks$, which are single nodes or strongly connected clusters of nodes that have a large impact on the signaling. We provide a theorem with bounds on the minimum number of nodes that guarantee controllability of bottlenecks consisting of strongly connected components. The control strategies are applied to the identification of sets of proteins that, when inhibited, selectively disrupt the signaling of cancer cells while preserving the signaling of normal cells. We use an experimentally validated non-specific network and a specific B cell interactome reconstructed from gene expression data to model cancer signaling in lung and B cells, respectively. This model could help in the rational design of novel robust therapeutic interventions based on our increasing knowledge of complex gene signaling networks

Smad7:β-catenin Complex Regulates Myogenic Gene Transcription

Recent reports indicate that Smad7 promotes skeletal muscle differentiation and growth. We previously documented a non-canonical role of nuclear Smad7 during myogenesis, independent of its role in TGF-β signaling. Here further characterization of the myogenic function of Smad7 revealed β-catenin as a Smad7 interacting protein. Biochemical analysis identified a Smad7 interaction domain (SID) between aa575 and aa683 of β-catenin. Reporter gene analysis and chromatin immunoprecipitation demonstrated that Smad7 and β-catenin are cooperatively recruited to the extensively characterized ckm promoter proximal region to facilitate its muscle restricted transcriptional activation in myogenic cells. Depletion of endogenous Smad7 and β-catenin in muscle cells reduced ckm promoter activity indicating their role during myogenesis. Deletion of the β-catenin SID substantially reduced the effect of Smad7 on the ckm promoter and exogenous expression of SID abolished β-catenin function, indicating that SID functions as a trans dominant-negative regulator of β-catenin activity. β-catenin interaction with the Mediator kinase complex through its Med12 subunit led us to identify MED13 as an additional Smad7-binding partner. Collectively, these studies document a novel function of a Smad7-MED12/13-β-catenin complex at the ckm locus, indicating a key role of this complex in the program of myogenic gene expression underlying skeletal muscle development and regeneration.York University Librarie