Research On The Mechanism Of Gene Regulation Of Map2 After Induction Of AtRA

Research on the mechanism of gene regulation of map2 after induction of atRAThe development of mammalian nervous system is one of the most complicated events in vivo. The development and re-construction of the cellular framework, the fibrous structural network of the cells and their signal transmission functions are efficiently and accurately controlled and regulated. MAPs (Microtubule-Associated Proteins, MAPs) play important roles in the development of neuronal cells. As a major member of MAPs, MAP2 is important in the assembly of microtubes, the growth of processes and the communication between microtubes and other components of the neuron. Moreover, abnormality in its expression and/or modification of the MAP2 often implicated in the occurrence of neuro-degenerative diseases. It has been known that the appropriate targeting of the microtubule-associated proteins in neurodegenerative diseases is capable of either relieve its symptoms or block its occurrence. Here, mouse embryonal caracinoma p19 cells were adopted as a model to study the effects and mechanisms of atRA (all trans retinoid acid) induced differentiation of the cells.We have first found that the cell aggregation and the gradual elongation of its processes in atRA treated p19 cells are accompanied by an elevated level of MAP2. The roles and mechanisms of chromatin remodeling factors and transcription factors in the regulation of map2 gene were thus studied in detail.1. The regulation of RA and p53 on the promoter activity of map2 gene in p19 cellsEffects of RA on the promoter activity of map2 geneReporter plasmid pGL3-map2-1.3k containing an 1.3kb fragment of map2 promoter was co-transfected into p19 cells with a control plasmid of pRL-TK to study the promoter activity of the map2 gene. atRA was added into medium at the 6th hour after transfection. Results showed that RA increased the promoter activity of map2 gene with the intensity getting higher as the treatment extended. Effects of p53 protein on the promoter activity of map2 geneWild type or mutant p53 expression plasmid was individually cotransfected into p19 cells with the above mentioned reporter plasmid and the control plasmid. Results showed that p53 increased the promoter activity of map2 gene, whereas, mutant-p53 played an opposite role on the gene. Moreover, RA could strengthen the enhancing effect of p53 protein on the map2 promoter activity but not mutant-p53.As shown by immunofluorescence assays, p53 protein translocated from cytoplasm to the nucleus more efficiently in RA treated cells, which suggested that RA may indirectly regulate the expression of map2 via the p53 pathway.2. Effect of Histone acetyl transferases PCAF and p300 on the promoter activity of map2 gene in RA induced p19 cellsThe expression plasmid of PCAF or p300 was individually cotransfected the p19 cells as mentioned above and the promoter activity of map2 was detected by Dual-Luciferase(?) Reporter Assay System. PCAF showed an increasing activity on the promoter activity of map2 as the treatment of RA extended. In contrast, p300, either with or without HAT domain, showed limited effect on the gene even under RA treatment.3. The binding of chromatin modifiers to the promoter of map2 in RA treated p19 cellsIn chromatin immunoprecipitation (ChIP) assays, it was found after RA treatment, acetylated H3K9 was the first indicator for an open conformation at the promoter region of map2 gene, which was followed by a～6 folds increase of p300. PCAF was found consistently exhibited at the map2 gene and was induced by RA to elevate gradually and reach a peak at day 4 when p19 cells were induced to show morphologically differentiation phenotype. Accordingly, RNA Polymerase II was recruited to the promoter on day 4 suggesting possible transcription of map2 gene initiated. Meanwhile, the acetylated p53 was slightly enhanced on day 2-4 suggesting a role of p53 acetylation in the early stage of differentiation via map2 gene activation.Summarizing the above, we suggest that The treatment of RA in p19 cells promoted PCAF to acetylate p53, the acetylated p53 might bind to its specific binding site on map2 promoter,then recruit components of HAT family to map2 promoter region to remodel the chromatin around the map2 transcription initiation site. So the expression of map2 gene was promoted.This study provides novel clues on targets of drug development that could eventually benefit clinic treatments for neurodegenerative diseases.