ABSTRACT
Objective: To clone AbCYP80F1 gene and its promoter, analyze the Cis-acting elements related to control signals and transcription factors in AbCYP80F1 gene promoter and study the effect of AbCYP80F1 overexpression on scopolamine accumulation in Atropa belladonna. Methods: CYP80F1cDNA from Hyoscyamus niger was used as query sequence to do blastn search in A. belladonna transcriptome database to retrieve homologous unigenes. Full-length of AbCYP80F1 cDNA and gene promoter were acquired by RACE and hiTAIL-PCR, respectively. Agrobacterium rhizogenes-dipping method was adopted to induce AbCYP80F1- overexpressed A. belladonna hairy roots, and scopolamine content in hairy roots was detected by HPLC. Results: A full-length of 1 675 bp of AbCYP80F1 gene was obtained and the deduced protein had a closed homology with CYP80F1 from Anisodus luridus. A length of 2 000 bp promoter contained Cis-acting elements related to light, anaerobic induction, auxin, MeJA, gibberellins and low temperature, as well as recognition sites of transcription factor of MYB, AP2/ERF, WRKY and bHLH. Overexpression of AbCYP80F1 gene increased the scopolamine content in hairy roots of A. belladonna by an average of 1.8 times. Conclusion: AbCYP80F1 is a secondary root-specific gene in scopolamine biosynthetic pathway and its overexpression can enhance scopolamine accumulation. Expression of AbCYP80F1 may be regulated by WRKY and bHLH transcription factors.
ABSTRACT
A família de fatores de transcrição NFAT (Fator Nuclear de Células T ativadas) tem diferentes funções regulatórias no ciclo celular, apoptose, diferenciação celular e angiogênese. O proto-oncogene c-myc, que está envolvido em todos estes mecanismos, é reprimido em alguns modelos, em resposta à Ciclosporina A, que inibe a ativação das proteínas NFAT. Dados anteriores de nosso laboratório mostraram que os linfócitos de camundongos NFAT1-/- sensibilizados com ovalbumina, apresentaram níveis aumentados de c-MYC quando comparados com os linfócitos dos camundongos NFAT1+/+. Desta forma, o objetivo deste trabalho foi avaliar se o NFAT1 regula diretamente a expressão de c-MYC. Este estudo mostrou que linfócitos T de camundongos NFAT1-/- naives superexpressam c-MYC em relação aos linfócitos NFAT1+/+, através de ensaios de PCR em tempo real. Uma análise de bioinformática encontrou sete supostos sítios de ligação para NFAT no promotor de c-myc, conservados em humanos e camundongos. Três desses sítios foram confirmados por um ensaio de mudança de mobilidade eletroforética, incluindo o sítio proximal, que é regulado por NFAT2. Além disso, um ensaio de imunoprecipitação de cromatina com linfócitos murinos demonstrou que o NFAT1 se liga diretamente ao promotor de c-myc in vivo. Estes resultados sugerem que o NFAT1 tem um importante papel na regulação do promotor de c-myc, aparentemente regulando negativamente sua expressão.
The Nuclear Factor of Activated T Cells (NFAT) family of transcription factors has different regulatory functions in the cell cycle, apoptosis, cell differentiation and angiogenesis. The c-myc proto-oncogene, which is also involved in those mechanisms, is repressed, in some models, in response to Cyclosporine A that inhibits NFAT activation. Previous data of our laboratory found that lymphocytes from NFAT1-/- mice sensitized with ovalbumin presented higher levels of c-MYC mRNA when compared with the NFAT1+/+. Hence, the aim of this work was to evaluate whether the NFAT1 directly regulates the c-MYC expression. This study showed that T lymphocytes from NFAT1-/- naive mice overexpress c-MYC mRNA when compared with the NFAT1+/+ mice assessed by Real Time PCR. A bioinformatic analysis found seven putative NFAT binding sites in the c-myc promoter, conserved in human and mouse. Three of them were confirmed by an Eletrophoretic Mobility Shift Assay, including the proximal site, which is upregulated by NFAT2. Additionally, a Chromatin Immunoprecipitation Assay with mouse T lymphocytes demonstrated that NFAT1 directly binds to c-myc promoter in vivo. These findings suggest that NFAT1 plays an important role in the regulation of c-myc promoter apparently through the inhibition of this expression.
Subject(s)
Male , Female , Humans , Cyclosporine , Gene Expression Regulation , Genes, myc , NFATC Transcription Factors , Proto-OncogenesABSTRACT
To identify regulatory molecules which play key roles in the development of obesity, we investigated the transcriptional profiles in 3T3-L1 cells at early stage of differentiation and analyzed the promoter sequences of differentially regulated genes. One hundred and sixty-one (161) genes were found to have significant changes in expression at the 2nd day following treatment with differentiation cocktail. Among them, 86 transcripts were up-regulated and 75 transcripts were down-regulated. The 161 transcripts were classified into 10 categories according to their functional roles; cytoskeleton, cell adhesion, immune, defense response, metabolism, protein modification, protein metabolism, regulation of transcription, signal transduction and transporter. To identify transcription factors likely involved in regulating these differentially expressed genes, we analyzed the promoter sequences of up- or -down regulated genes for the presence of transcription factor binding sites (TFBSs). Based on coincidence of regulatory sites, we have identified candidate transcription factors (TFs), which include those previously known to be involved in adipogenesis (CREB, OCT-1 and c-Myc). Among them, c-Myc was also identified by our microarray data. Our approach to take advantage of the resource of the human genome sequences and the results from our microarray experiments should be validated by further studies of promoter occupancy and TF perturbation.