RESUMO
Expression of the nuclear orphan receptor gene Nur77 in neuronal cells is induced by activity-dependent increases in intracellular Ca2+ ions. Ca2+ responsiveness of the Nur77 gene has been attributed to two distinct DNA regulatory regions that recruit the transcription factors cAMP response element binding protein (CREB) and myocyte enhancer factor-2 (MEF2). Here we used dominant interfering and constitutively active mutants of CREB and MEF2 proteins to assess their relative contribution to depolarization-induced Nur77 expression in undifferentiated PC12 cells and hippocampal neurons. We show that while CREB is necessary for Ca2+-activated Nur77 expression MEF2 functions to modulate CREB-dependent Nur77 expression by acting as a repressor in quiescent cells.
Assuntos
Regulação da Expressão Gênica/fisiologia , Fatores de Regulação Miogênica/metabolismo , Neurônios/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Cálcio/metabolismo , Células Cultivadas , Ciclosporina/farmacologia , Inibidores Enzimáticos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Fatores de Transcrição MEF2 , Camundongos , Mutação/fisiologia , Fatores de Regulação Miogênica/genética , Fatores de Transcrição NFATC/metabolismo , Neurônios/efeitos dos fármacos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Células PC12/efeitos dos fármacos , Células PC12/metabolismo , Cloreto de Potássio/farmacologia , Regiões Promotoras Genéticas/genética , Ratos , Fatores de Tempo , Transfecção/métodosRESUMO
Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure. In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes. In hypertrophy, H3K9me2 was reduced following a miR-217-mediated decrease in expression of the H3K9 dimethyltransferases EHMT1 and EHMT2 (EHMT1/2). miR-217-mediated, genetic, or pharmacological inactivation of EHMT1/2 was sufficient to promote pathological hypertrophy and fetal gene reexpression, while suppression of this pathway protected against pathological hypertrophy both in vitro and in mice. Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy. These results suggest that targeting miR-217 and EHMT1/2 to prevent H3K9 methylation loss is a viable therapeutic approach for the treatment of heart disease.
Assuntos
Cardiomegalia/enzimologia , Cardiomegalia/prevenção & controle , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Epigênese Genética , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Clone stability and in vitro phytoextraction capacity of vegetative clones of P. x canescens (2n = 4x = 38) including two transgenic clones (ggs11 and lgl6) were studied as in vitro leaf disc cultures. Presence of the gshI-transgene in the transformed clones was detected in PCR reactions using gshI-specific primers. Clone stability was determined by fAFLP (fluorescent amplified DNA fragment length polymorphism) analysis. In total, 682 AFLP fragments were identified generated by twelve selective primer pairs after EcoRI-MseI digestion. Four fragments generated by EcoAGT-MseCCC were different (99.4% genetic similarity) which proves an unexpectedly low bud mutation frequency in P. x canescens. For the study of phytoextraction capacity leaf discs (8 mm) were exposed to a concentration series of ZnSO4 (10(-1) to 10(-5) M) incubated for 21 days on aseptic tissue culture media WPM containing 1 microM Cu. Zn2+ caused phytotoxicity only at high concentrations (10(-1) to 10(-2) M). The transgenic poplar cyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showed elevated heavy metal (Cu) uptake as compared to the non-transformed clone. These results suggest that gshI-transgenic poplars may be suitable for phytoremediation of soils contaminated with zinc and copper.
Assuntos
Biodegradação Ambiental , Cobre/farmacocinética , Plantas Geneticamente Modificadas/metabolismo , Polimorfismo Genético , Populus/metabolismo , Sequência de Bases , Primers do DNA , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Amplificação de Genes , Folhas de Planta , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
The causative organism of crown rust in ryegrasses (Puccinia coronata f.sp. lolii) is an obligate biotroph that causes significant economic losses within the temperate grazing industries of dairy, meat, and wool production. This study reports on the development, transferability, and utility of gene-associated simple sequence repeat (SSR) molecular markers for crown rust. Analysis of 1,100 expressed sequence tag (EST) sequences from a urediniospore-derived cDNA library detected 55 SSR loci. The majority of EST-SSR arrays contained perfect trinucleotide repeats with consistently low repeat numbers, and the motifs (ACC)n and (CAT)n were most commonly represented. DNA extraction from single pustules, in conjunction with multiple displacement amplification, provided the basis for PCR-based screening to evaluate genetic marker performance. An example of the identification of intraspecific genetic diversity was obtained from the analysis of 16 P. coronata isolates originating from the United Kingdom, Australia, New Zealand, and Japan. A subset of 12 robust EST-SSR markers was informative for determination of pathogen diversity within and between these localities. It was also demonstrated that crown rust EST-SSR markers were capable of cross-amplification in closely related fungal taxa (Puccinia spp.) and filamentous fungi within the Ascomycota.