6533b82dfe1ef96bd1291159

RESEARCH PRODUCT

Transcriptional regulation of egr-1 gene in murine cells. Towards the understanding of the role of chromatin.

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description

Eukaryotic gene expression is a highly regulated process that has to ensure the right cellular response to any type of stimulus. Nevertheless, chromatin structure, despite the fact of being dinamic, imposes certain contraints to this regulation. To solve this restrictions, cells have organized the recruitment of chromatin modifying enzymes and ATP-dependent chromatin remodelling enzymes. The first class of enzymes are responsible for the covalent modifications of histones which alter the global charge of the histones and also serve to recruit specific co-factors to the chromatin. The second group of enzymes uses the energy from ATP hydrolisis to modify the interactions DNA-histones, therefore increasing the nucleosomal DNA accesibility to the transcriptional machinery. The understanding of the mechanisms that the cell uses to regulate expression of an immediate-early gene such as egr-1, are very important, not only for the basic molecular knowledge but also for the understanding of different pathologies. The coordinated action of the signalling pathways, the union of transcriptional factors and enzymatic complexes that act over the chromatin is crucial for the right interpretation of the genetic code, which is specific for each gene under study. In the case of egr-1, treating MLP29 cells with TPA activates expression after 5 min of treatment, has a peak around 15 and 30 min, and decreases subsequently to be undetectable at 180min after the treatment. This induction takes place through the MAP and p38 kinase cascades which also act phosphorylating the promoter transcriptional factors ELK1 and CREB. Chromatin immunoprecipitation experiments (ChIP), suggest that egr-1 is a gene "ready to be expressed", as the RNAPol II and the SRF, ELK1, CREB and SP1 transcriptional factors are already present in the basal state of the gene. During the induction process SP1 leaves the promoter and EGR-1, NAB1 and NAB2 are recruited. In the same manner, ChIP experiments have detected the presence of two HDAC complexes implicated in egr-1 repression (mSIN3A and HDAC3-NcoR) and the presence of two HAT complexes (CBP, with a possible role as maintaining the basal acetylation levels and GCN5 which putative role would favour the induction process). The presence of these enzymes has been correlated with a general increase in the levels of acetylation of histone H3 and H4 by immunoprecipitating against specific histone modifications. Additionally, experiments of Micrococcal nuclease protection have suggested the existence of three positioned nucleosomes in egr-1 promoter and also the movement of the more proximal to the transcription start during the induction process. That nucleosomal mobility has been related to the presence of the ATPase BRM and BRG1 in the promoter of the gene and also with specific increases in the acetylation levels of the three nucleosomes as revealed by mononuclesomal ChIP experiments. Moreover, to study the biological effect of the lack of EGR1, it has been designed a siRNA sequence that is able to decrease egr-1 expression up to a 70%, and which biological activity has been shown by a decrease in nab2 (EGR1 target gene) expression of 40%. Lastly, the interconnection between these results has allowed us to hypothesize a model for the epigenetic regulation of egr-1 gene in murine cells treated with TPA.