Early growth response gene – 1 (Egr1) encodes a protein widely present in mammalian body, such as connective tissue, cardiac tissue, the liver, and the brain. As a transcription factor (TF), it is involved in processes that take place in the endocrine, digestive, immune, musculo-skeletal and central nervous systems, for instance, B cell maturation upon B cell receptor activation, tendon repair upon mechano-stimulation, and long-term spatial memory formation. In mammalian brains, EGR1 controls the responses to environmental stimuli such as chronic stress and physical contact. It also participates in processes such as long-term memory consolidation and synapse re-structuring. It plays a role in enacting responses and qualities of gene transcription cascades upon neuronal stimulation. Inside the epigenetic realm, EGR1 recruits Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) to remove DNA methylation at target loci. Due to its critical functions during brain development and upon neuronal activation, mis-regulation of EGR1 is associated with neuropsychological disorders such as post-traumatic stress disorder (PTSD) and schizophrenia (SCZ) in humans. In this study, we performed bioinformatics analysis with brain methylomes and predicted EGR1 may interact with myocyte enhancer factor 2C (MEF2C), which is known to be involved in many similar processes as EGR1, such as synapse architecture, cell migration, and learning and memory. EGR1 and MEF2C ChIP-seq data derived from mouse frontal cortex suggest these two proteins may regulate a common set of downstream genes. To begin, co-immunoprecipitation experiments were performed with HEK293T cells co-transfected with EGR1-FLAG and MEF2C-HA tagged constructs, allowing for specific interaction identification without endogenous protein expression interference. Furthermore, co-immunoprecipitation experiments performed with brain tissues additionally indicated the two proteins interact with each other endogenously. Altogether, this study provides protein-protein interaction evidence for EGR1 and MEF2C in cultured HEK293 cells and in the cortices of adult male mice. This information provides a foundation for future examinations of how these two TFs interact to initiate cascading events following neuronal stimulation. / Master of Science / Early growth response gene – 1 (EGR1) encodes a protein that can be found in animals such as fruit flies, mice, rats, and humans. In mammals, it is widely expressed in the cardiovascular, endocrine, digestive, immune, musculo-skeletal and central nervous systems (CNS). Within the CNS, EGR1 is known as an essential transcription factor involved in brain development. More specifically, EGR1 plays a role in how the early brain develops in response to environmental stimuli, formation of synapse architecture and certain types of memories. Many gene networks involved in growth and development rely on EGR1 to regulate functions such as synapse reformation after exposure to the environment. EGR1 is known to have numerous partners with whom it interacts to execute its functions. It is also involved in epigenetic regulation, which is a process by which genes are silenced or activated without changing DNA sequences in the genome. EGR1 may directly interact with TET1 to demethylate EGR1 target sites in the genome, and to increase gene transcription. In memory development, EGR1 plays a key role ensuring short-term auditory fear memory can be converted to long-term memory, and also ensures long-term spatial memory. In this study, our computational analyses suggest that EGR1 may interact with MEF2C. This work provides evidence of a protein-protein interaction of EGR1 and MEF2C in cultured cells and in the brain cortical areas of mice. Such an interaction may explain why these two genes regulate overlapped biological processes within the brain and sheds lights on how cascading events are initiated following neuronal stimulation.

CATALÀ:L'enzim carnitina palmitoïltransferasa1 (CPT1), localitzat a la membrana mitocondrial externa, constitueix el principal punt de control de l'entrada d'àcids grassos de cadena llarga (LCFA) al mitocondri i és clau en el manteniment d'àcids grassos circulants. Existeixen tres isotips descrits (CPT1A, CPT1B i CPT1C) que codifiquen per enzims amb diferents característiques cinètiques i patrons d'expressió.1.- Mecanismes de control transcripcional del gen CPT1B humà.El promotor humà de la CPT1B inclou un element de resposta a PPAR i un lloc d'unió a MEF-2. Hem investigat el paper d'aquests elements de resposta i la possible interacció entre PPARα i MEF2C en la regulació transcripcional d'aquest promotor. Dels resultats obtinguts podem concloure que la resposta del promotor a PPARα depèn: del context cel·lular, de l'element de resposta a MEF-2 i de la disposició espacial d'aquest respecte al PPRE. La combinació d'aquests elements cis en el promotor de la CPT1B indueix l'expressió màxima del gen en resposta a diferents senyals. La concurrència de senyals metabòlics i miogènics en aquest promotor genera una conformació transcripcional permissiva d'aquest promotor que porta a una activació sinèrgica del promotor en aquells teixits on es troben els corresponents factors de transcripció (MEF2C, PPARα/RXRα) i el substrat metabòlic de l'enzim, els àcids grassos que activen PPARα. En la mateixa una regió promotor una zona rica en GC capaç d'unir Sp1 ha resultat fonamental per l'expressió basal del gen i per la transactivació per PPARα però no per la de PPARδ. 2.- Relació estructura/funció de l'enzim CPT1 de porc.A nivell cinètic, les CPT1A mostren una alta afinitat per la carnitina i una baixa sensibilitat al malonil-CoA mentre que les CPT1B presenten característiques contràries. Una excepció a aquesta relació és la CPT1A de porc (PLCPT1) que es comporta com una quimera natural entre els isotips A i B, presentant afinitats pels substrats similars a les CPT1A i una IC50 pel malonil-CoA típica de les CPT1B. Utilitzant quimeres entre la CPT1A de rata i la CPT1A de porc hem demostrat que l'extrem C-terminal de les CPT1A es comporta com un únic domini que dicta la sensibilitat total a malonil-CoA de l'enzim. El grau de sensibilitat a l'inhibidor ve determinat per l'estructura adoptada per aquest domini. Utilitzant mutants delecionats hem mostrat que la sensibilitat a malonil-CoA també depèn de la interacció d'aquest únic domini carboxil amb els primers 18 aminoàcids de la proteïna. D'aquests resultats podem concloure que les CPt1A de rata i porc presenten diferent sensibilitats a malonil-CoA perquè els primers 18 aminoàcids dels enzims interaccionen diferent amb el domini C-terminal.Hem aïllat l'isotip muscular de la CPT1 de porc (PMCPT1), una proteïna de 772 aminoàcids molt similar a les CPT1B. Expressada en Pichia pastoris la CPt1B de porc ha resultat ser un enzim amb característiques cinètiques pròximes a les CPT1A. 3.- Efecte de C75 sobre el sistema CPT. Els enzims CPT1 i CPT2 són components del sistema llançadora CPT. Aquest sistema es troba finament regulat pels nivells de malonil-CoA, un inhibidor reversible de la CPT1. Per la seva capacitat d'inhibir la sintasa d'àcids grassos (FAS), el C75 és capaç d'incrementar els nivells intracel·lular de malonil-CoA intracel·lular. Paradoxalment també activa l'oxidació d'àcids grassos de cadena llarga. Per tal d'identificar la diana exacta del C75 en el sistema CPT vam analitzar l'activitat enzimàtica de CPT1A, CPT1B i CPT2 davant el tractament amb C75. Els resultats d'aquests experiments indiquen que el C75 actua sobre el sistema CPT activant CPT1A, CPT1B i CPT2 de manera independent de malonil-CoA. / The outer mitochondrial membrane enzyme carnitine palmitoyltransferase1 (CPTI) catalyzes the initial and regulatory step in the β-oxidation of long-chain fatty acids. There are three characterized isotypes: CPT1A, CPT1B and CPT1C. The human CPT1B promoter includes a functional PPAR responsive element and a myocite-specific site that binds MEF2C. We investigated the roles of these sites and the potential interaction between PPARα and MEF2C regulating this promoter. The combination of cis elements in the promoter of the CPT1B maximally induces the expression of this gene in response to a combination of signals. The concurrence of myogenic and metabolic signals generates a transcriptionally permissive conformation of the promoter that gives rise to a synergistic transcription of the gene in tissues containing the corresponding transcription factors and fatty acids that activate PPARα.Kinetic hallmarks of the CPT1A are high affinity for carnitine and low sensitivity to malonyl-CoA inhibition, while the opposite characteristics are intrinsic to the CPT1B isotype. Pig and rat CPT1A share common Km values for their substrates but differ in their sensitivity to malonyl-CoA inhibition. Using chimeras between rat CPT1A and pig CPT1A, we show that the C-terminal region behaves as a single domain, which dictates the overall malonyl-CoA sensitivity of this enzyme. Using deletion mutation analysis, we show that malonyl-CoA sensitivity also depends on the interaction of this single domain with the first 18 N-terminal amino acid residues. Pig and rat CPT1A have different malonyl-CoA sensitivity, because the first 18 N-terminal amino acids interact differently with the C-terminal domain. Pig CPT1B is a protein of 772 amino acids that shares extensive sequence identity with CPT1B. Expressed in Pichia pastoris, pig CPT1B shows kinetic characteristics similar to those of the CPT1A isotype. CPT1 and CPT2 enzymes are components of the CPT shuttle system. This system is tightly regulated by malonyl-CoA. Because of its ability to inhibit fatty acid synthase, C75 is able to increase malonyl-CoA intracellular levels. Paradoxically it also activates β-oxidation. To identify the exact target of C75 within the CPT system, we expressed CPT1 and CPT2 in Pichia pastoris. We show that C75 acts on recombinant CPT1A, CPT1B and CPT2.

The Hedgehog (HH) signalling pathway and its primary transducer, GLI2, regulate cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells. To further assess the role of HH signalling during mouse embryonic stem (mES) cell differentiation, we studied the effects of GLI2 overexpression during mES cell differentiation. GLI2 overexpression resulted in temporal enhancement of cardiac progenitor genes, Mef2c and Nkx2-5, along with enhancement of Tbx5, Myhc6, and Myhc7 in day 6 differentiating mES cells. Mass spectrometric analysis of proteins that immunoprecipitate with GLI2 determined that GLI2 forms a complex with BRG1 during mES cell differentiation. Furthermore, modulation of HH signalling during P19 EC cell differentiation followed by chromatin immunoprecipitation with an anti-BRG1 antibody determined that HH signalling regulates BRG1 enrichment on Mef2c. Therefore, HH signalling accelerates cardiac progenitor gene expression during mES cell differentiation potentially by recruiting a chromatin remodelling factor to at least one cardiac progenitor gene.

L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.