| PART I ACETYLATION MEDIATED BY P300 REGULATES THE EXPRESSION OF GATA4Objective: GATA4 is a particularly important cardiogenic transcription factor and serves as a potent driver of cardiogenesis.Recent progress in the field has made it clear that histone acetylation can influence gene expression.Our previous research had revealed that hypo-acetylation could repress GATA4 expression in cardiocytes,however the underlying mechanism by which this occurred was still unclear.To reveal the mechanism of histone acetylation involved in the regulation of GATA4 transcription,we concentrated on P300,one of the important histone acetyltransferases associated with cardiogenesis.Methods:(1)Lentivirus containing p300 siRNA and empty plasmid were transfected to cardiac progenitor cells,respectively;(2)Efficiency of lentiviral transfection was detected by flow cytometry;(3)Real-time quantitive PCR were used to detect the expression of P300,GATA4 and Tbx5 mRNA;(4)CHIP-qPCR were used to detect the global acetylation of H3 and the acetylation of H3K4,H3K9,and H3K27 near GATA4 and Tbx5 promoters,and binding level of histone acetylases in the GATA4 promoter.Results:(1)The transfection efficiency in negative control and interference group was 62.6% and 73.7% after 48 h of interference,respectively;(2)The expression of P300 mRNA was significantly lower than that in the negative control group(P<0.05);(3)The expression of GATA4 mRNA was significantly lower than that in the negative control group(P<0.05),while the expression of Tbx5 mRNA was not changed as compared with negative control group(P>0.05);(4)The global acetylation of H3 and the acetylation of H3K4,H3K9,and H3K27 in GATA4 promoter was significantly lower than that in the negative control group(P<0.05),the global acetylation of H3 and the acetylation of H3K4 and H3K27 in Tbx5 promoter was significantly lower than that in the negative control group(P<0.05),while the acetylation of H3K9 Tbx5 promoter was significantly higher than that in the negative control group(P<0.05);(5)The binding levels of GATA4 histone protein modifier P300 to GATA4 promoter was significantly lower than that in the negative control group(P<0.05),while the binding levels of GATA4 histone protein modifier CBP and PCAF to GATA4 promoter were significantly higher than those in the negative control group(P<0.05).Conclusions:(1)Histone acetylation mediated by P300 plays an important role in regulation of GATA4 expression in cardiocytes.(2)Acetylation of H3K4,H3K9,and H3K27 might regulate the expression of GATA4.PART II DEACETYLATION MEDIATED BY hdac1 REGULATES THE EXPRESSION OF CARDIAC TROPONIN IObjective: The previous part confirmed that histone deacetylation involved in the regulation of cTnI gene expression,and HDAC1 may play an important role in this process.To reveal the mechanism of HDAC1 regulating cTnI,we down regulate HDAC1 and detect its effect on histone acetylation of cTnI promoter and cTnI gene transcription.Methods:(1)Myocardial primary cell was separated from new born mice heart and transfected with adenovirus containing HDAC1 siRNA and empty plasmid,respectively,for 48 hours;(2)Real-time quantitive PCR were used to detect the expression of HDAC1 mRNA to reveal the efficiency of adenoviral transfection;(3)CHIP-qPCR were used to detect binding level of HDAC1 to GATA element of cTnI promoter;(4)Real-time quantitive PCR and Western blot were used to detect the expression of cTnI mRNA and protein,respectively;(5)CHIP-qPCR were used to detect the global acetylation of H3 and the acetylation of H3K4,H3K9,and H3K27 near GATA element of cT nI promoter and binding level of GATA4 to GATA element of cTnI promoter.Results:(1)The expression of HDAC1 mRNA was lower than that in the negative control group(P<0.05),when MOI was equal to 80,theefficiency of adenoviral transfection was highest;(2)The binding levels of HDAC1 to GATA element of cTnI promoter was significantly lower than that in the negative control group(P<0.05);(3)The expression of cTnI mR NA and protein was significantly higher than that in the negative control group(P<0.05);(4)The global acetylation of H3 and the acetylation of H3K4 and H3K9 near GATA element of cTnI promoter was significantly higher than that in the negative control group(P<0.05),while the acetylation of H3K27 near GATA element of cTnI promoter was not changed as compared with negative control group(P>0.05);(5)The binding levels of GATA4 to GATA element of cTnI promoter was significantly higher than that in the negative control group(P<0.05);Conclusions:(1)Histone deacetylase mediated by HDAC1 has an essential role in cTnI transcription.(2)Acetylation of H3K4 and H3K9 might regulate the expression of cTnI.(3)Histone acetylation can recruit GATA4 binding to GATA element of cTnI promoter to elevate cTnI transcriptionPART III ACETYLATION REGULATES THE EXPRESSION OF CARDIAC TROPONIN I AND PROTECTS CARDIAC DYSTONIC DISFUNCTIONObjective: We previously identified that histone acetylation plays an important role in cTnI expression,however the underlying mechanism by which this occurred was still unclear.To reveal the mechanism of histone deacetylation involved in the regulation of cTnI transcription and diastolic dysfunction,we reduced histone deacetylation by one of histone deacetylation inhibitor,vorinostat.Methods:(1)Fifteen-month-old aging mice were employed and subcutaneous injected with 1μl/g normal saline,DMSO,and vorinostat(50μg/μl/g)for 7 days,respectively;(2)High resolution echocardiography was used to detected cardiac function;(3)Real-time quantitive PCR and Western blot were used to detect the expression of cTnI mRNA and protein,respectively;(4)CHIP-qPCR were used to detect binding level of HDAC1 to GATA element of c TnI promoter,the global acetylation of H3 and the acetylation of H3K4,H3K9,and H3K27 near GATA element of cTnI promoter,and binding level of GATA4 to GATA element of cTnI promoter.Results:(1)The E/A ratio in vorinostat group was higher than that in the negative control group(P<0.05),while the HR,IVSd,PWd,LVEDD,IVSs,PWs,LVESD,FS and EF were not changed as compared with controlgroup(P>0.05);(2)The expression of cTnI mRNA and protein in vorinostat group was higher than that in the negative control group(P<0.05);(3)The binding levels of HDAC1 to GATA element of cTnI promoter was significantly lower than that in the negative control group(P<0.05);(4)The global acetylation of H3 and the acetylation of H3K4 and H3K9 near GATA element of cTnI promoter was significantly higher than that in the negative control group(P<0.05),while the acetylation of H3K27 near GATA element of cTnI promoter was not changed as compared with negative control group(P>0.05);(5)The binding levels of GATA4 to GATA element of cTnI promoter was significantly higher than that in the negative control group(P<0.05);Conclusions:(1)Acetylation of H3K4 and H3K9 might recruit GATA4 binding to GATA element of cTnI promoter to elevate cTnI.(2)Histone acetylase can recruit GATA4 binding to GATA element of cTnI promoter and elevate cTnI to improve cardiac diastolic function... |
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