Study Of Molecular Mechanism Of Histone Acetyltransferase P300 In The Expression Of Mucin 5AC

Background: Histone acetylation is an important pathogenesis of bronchial asthma.It is mediated by histone acetyltransferase(HAT)and histone deacetylases(HDAC).P300 is a typical histone acetyltransferase.One of the pathological features of bronchial asthma is airway mucus hypersecretion,and mucin 5AC(MUC5AC)is the main airway mucin.Nowadays,many studies have shown that the transcriptional regulation of the MUC5 AC gene is related to histone acetylation.We envisaged that the histone acetyltransferase P300 is involved in the transcriptional regulation of MUC5 AC.Objective: To clone the MUC5 AC gene promoter and analyze its transcriptional activity find the core promoter areas.To investigate the molecular mechanism of histone acetyltransferase P300 in the expression of mucin 5AC.Methods: 1.The 1348 bp DNA sequence at human MUC5 AC gene 5 end was amplified by PCR method and the product of PCR was sequenced.2.By promoter deletion analysis,5 deletion promoter segments with different lengths were amplified by PCR,the products were identified by DAN sequence,and the 6 promotor segments were inserted into p GL3-enhancer vectors.The core promoter region was identified using a series of 5’ deletion promoter plasmids in the luciferase reporter assays.3.The MUC5 AC promoter activity,m RNA expression and protein expression levels in A549 cells were observed by using plasmid P300 wt and P300 mut as well as P300 si RNA and si-control.Results: 1.It was shown that the 5 end region sequence reporter plasmids of MUC5 AC gene of different lengths were successfully constructed through the blast furnace and comparing DNA sequence comparison tools.2.The luciferase reporter assays of series 5’ deletion reporter plasmids revealed that this constructed promoter activity declined significantly from-935 to-583 bp,these results indicated that the core promoter region of MUC5 AC was located within the region-935/+48 relative to the TSS.3.Compared with P300 mut,P300wt could reduce MUC5 AC promoter activity,m RNA levels and protein levels expression,just as P300 si RNA led to a marked increase on MUC5 AC expression levels.(P<0.05 或 P<0.01).Conclusion: The reporter gene vectors of human MUC5 AC gene with promoter segment different length were constructed successfully.The core promoter of MUC5 AC gene was located between-935 bp and +48bp,P300 could reduce MUC5 AC promoter activity,m RNA levels and protein levels expression.