| Part I Effects of alcohol exposure on histone modification of cardiac development related nuclear transcription factors in mice during pregnancyObjectiveTo explore the effects of alcohol exposure on HATs activity and HDACs activity and histone acetylation of cardiac development related genes GATA4, MEF2C, NKX2.5 and TBX5 in mice during pregnancy.MethodsPregnant Kunming mice were randomly divided into blank control group and alcohol group on ED 8.5, and pregnant mice were gavaged with alcohol or saline. Fetal mouse hearts were collected for analysis on ED 14.5, ED 16.5, PND 0.5 and PND 7. (1) HATs and HDACs activities were tested by colorimetry. (2) The acetylation level of ac-H3 and H3AcK9 in myocardial tissues of fetal mice were tested by Western blot. (3) The acetylation level of ac-H3 and H3AcK9 on the promoter of cardiac nuclear transcription factors GATA4, MEF2C, NKX2.5 and TBX5 were analyzed using ChIP-Q-PCR. (4) Blood alcohol concentration of pregnant mice were tested by headspace gas chromatography, and alcohol related side effects were observed.Results(1) Alcohol concentration of blood was 147.5 mg/100 ml after 40 minutes by gavaging alcohol with 5 mg/kg, and the ratio of abortion, stillbirth and intestinal tympanites were about 15%. With the increase of alcohol dose, the blood alcohol concentration and the incidence of side effects caused by alcohol increased gradually.(2) The results of colorimetry showed that alcohol could increase significantly HATs activities in myocardial tissues on ED 14.5, ED 16.5 and PND 0.5 (P<0.05 vs blank control group). However, alcohol could not increase significantly HDACs activities in the same samples (P> 0.05 vs blank control group).(3) Western blot results showed that alcohol could increase significantly the acetylation level of ac-H3 and H3AcK9 in myocardial tissues of fetal and neonatal mice (P<0.05 vs blank control group).(4) The results of ChIP-Q-PCR showed that alcohol could increase significantly the acetylation level of ac-H3 and H3AcK9 on the promoter of cardiac nuclear transcription factors GATA4, MEF2C, NKX2.5 and TBX5 (P<0.05 vs blank control group).Conclusions(1) Alcohol could increase selectively HATs activities in myocardial tissues of fetal and neonatal mice during pregnancy, but not HDACs activities.(2) The acetylation level of ac-H3 and H3AcK9 were increased in myocardial tissues of fetal mice exposed to alcohol during pregnancy.(3) Alcohol exposure during pregnancy could lead to histone ac-H3 and H3AcK9 hyperacetylation on the promoter of cardiac nuclear transcription factors GATA4, MEF2C, NKX2.5 and TBX5.Part Ⅱ Histone acetylation regulation of histone acetylases on cardiac nuclear transcription factorsObjectiveTo investigate histone acetylation regulation of HATs on cardiac nuclear transcription factors GATA4, MEF2C, NKX2.5 and TBX5, and to explore the effects of histone hyperacetylation induced by alcohol exposure on transcription activities of cardiac nuclear transcription factors GATA4, MEF2C, NKX2.5 and TBX5.Methods(1) Normal fetal mice as the research objectives, specific primers were designed for recognizing the promoter before 1000 bp of GATA4, MEF2C, NKX2.5 and TBX5. Real-Time PCR amplification DNA fragment after ChIP by precipitating anti-p300 antibody, anti-CBP antibody, anti-PCAF antibody, anti-SRC 1 antibody, anti-GCN5 antibody with ChIP grade, to explore regulation of isoforms of HATs on GATA4, MEF2C, NKX2.5 and TBX5.(2) Fetal and neonatal mice on ED 14.5, ED 16.5, PND 0.5 and PND 7 were randomly divided into blank control group and alcohol group. Specific primers were designed for recognizing the promoter before 1000 bp of GATA4, MEF2C, NKX2.5 and TBX5. Real-Time PCR were used to test the effects of alcohol on the binding of HATs to the promoter ofGATA4, MEF2C, NKX2.5 and TBX5 after ChIP by anti-p300 antibody, anti-CBP antibody, anti-PCAF antibody, anti-SRC 1 antibody, anti-GCN5 antibody with ChIP grade.(3) Fetal and neonatal mice on ED 14.5, ED 16.5, PND 0.5 and PND 7 were randomly divided into blank control group and alcohol group. Specific primers were designed for recognizing CDS core coding regions of GATA4, MEF2C, NKX2.5 and TBX5. Quantitative RT-PCR were used to test the mRNA expression of GATA4, MEF2C, NKX2.5 and TBX5.Results(1) The results of ChIP-PCR showed that isoforms of HATs that include p300, CBP, PCAF, SRC1 and GCN5 all of them could bind to the promoter of cardiac nuclear transcription factors GATA4, MEF2C, NKX2.5 and TBX5.(2) The binding of p300-HAT, CBP-HAT, PCAF-HAT and SRC 1-HAT to the promoter of GATA4, MEF2C, NKX2.5 and TBX5 were increased significantly in the hearts of mice exposed to alcohol (P< 0.05 vs blank control group). However, the binding of GCN5-HAT to the promoter of GATA4, MEF2C, NKX2.5 and TBX5 have no significant increase in the hearts of mice exposed to alcohol (P>0.05 vs blank control group).(3) The mRNA expression of cardiac nuclear transcription factors GATA4, MEF2C, NKX2.5 and TBX5 in alcohol group were increased compared with blank control group, and the mRNA expression of GATA4 and MEF2C in alcohol group were higher than that in blank control group on ED 14.5 and ED 16.5 (P< 0.05). There was a significant difference in NKX2.5 mRNA expression between alcohol group and blank control group on ED 14.5, ED 16.5 and PND 0.5(P<0.05); the mRNA expression of TBX5 in alcohol group was higher than that in blank control group on ED 14.5 (P< 0.05).Conclusions(1) Isoforms of HATs that p300, CBP, PCAF, SRC1 and GCN5 all could bind to the promoter of cardiac nuclear transcription factors GATA4, MEF2C, NKX2.5 and TBX5 under physiological conditions, and regulate histone acetylation modification of transcription factors that above mentioned.(2) Binding of p300, CBP, PCAF and SRC1, but not GCN5, were increased on the promoter of GATA4, MEF2C, NKX2.5 and TBX5 relative to the saline treated group.(3) Alcohol exposure could increase significantly the transcription activities of cardiac nuclear transcription factors GATA4, MEF2C, NKX2.5 and TBX5 in the hearts of fetal mice.Part Ⅲ Anacardic acid inhibits histone acetylation to down regulate overexpression of cardiac development related genes induced by alcohol during pregnancyObjectiveTo explore the effects of HATs inhibitor anacardic acid inhibits histone acetylation to downregulate overexpression of cardiac nuclear transcription factors GATA4, TBX5 and cardiac development related genes α-MHC, Cx43, cTnT and α-actin induced by alcohol during pregnancy.MethodsPregnant Kunming mice on ED 8.5 were randomly divided into six group i.e. blank control group, blank control+DMSO group, alcohol+ DMSO group, alcohol+DMSO+anacardic acid group, blank control+ DMSO+anacardic acid group. Pregnant mice were gavaged with alcohol (5 mg/kg-d) or saline for one week, and pregnant mice exposed to alcohol were administered with anacardic acid (5 mg/kg·d) that dissolve in sterile DMSO by intraperitoneal injection; and the hearts of fetal mice were collected on ED 14.5 to assay as follows:(1) ChIP trials were conducted by use of anti-p300 antibody, anti-PCAF antibody, anti-SRC 1 antibody and anti-H3AcK9 antibody, and specific primers were designed for recognizing the promoter of GATA4, MEF2C, NKX2.5 and TBX5. DNA fragments of precipitation by antibodies that above mentioned were amplified by Real-Time PCR to assay the acetylation level of H3AcK9 and the binding of p300, PCAF and SRC1 to the promoter of GATA4 and TBX5.(2) The mRNA expression of cardiac nuclear transcription factors GATA4, TBX5 and cardiac development related genes α-MHC, Cx43, cTnT and a-actin were assayed by quantitative RT-PCR.(3) The protein expression of cardiac development related genes a-MHC, Cx43, cTnT and a-actin and the acetylation level of H3AcK9 were assayed by Western blot.Results(1) Western blot results showed that the acetylation level of H3AcK9 in myocardial tissues of fetal mice exposed to alcohol was higher than that the myocardial tissues of fetal mice treated with saline (P< 0.05 vs blank control group), and ChIP-Q-PCR results showed that the acetylation level of H3AcK9 on the promoter of cardiac nuclear transcription factors GATA4 and TBX5 in myocardial tissues of fetal mice exposed to alcohol was increased significantly (P< 0.05 vs blank control group), and anacardic acid could inhibit obviously hyperacetylation of H3AcK9 caused by alcohol exposure in the hearts of fetal mice during pregnancy.(2) The results of ChIP-Q-PCR showed that the binding of p300, PCAF and SRC1 to the promoter of cardiac nuclear transcription factors GATA4 and TBX5 in alcohol group was higher than that in blank control group (P<0.05); and anacrdic acid could decrease obviously the binding of p300 and PCAF to the promoter of cardiac nuclear transcription factors GATA4 and TBX5 (P< 0.05 vs alcohol group), but not SRC1 (P> 0.05 vs alcohol group).(3) Quantitative RT-PCR results showed that alcohol exposure could upregulate significantly the mRNA expression of cardiac nuclear transcription factors GATA4 and TBX5 and cardiac development related genes a-MHC, Cx43 and cTnT (P< 0.05 vs blank control group). However, alcohol exposure could not increase obviously the mRNA expression of a-actin in the same samples (P> 0.05 vs blank control group). While anacardic acid could downregulate obviously the mRNA overexpression of GATA4, TBX5, a-MHC, Cx43 and cTnT induced by alcohol exposure during pregnancy (P< 0.05 vs alcohol group).(4) Western blot results showed that alcohol exposure could increase obviously the expression of a-MHC, Cx43 and cTnT at the level of protein (P<0.05 vs blank control group). However, alcohol exposure could not increase obviously the expression of a-actin at the level of protein (P> 0.05 vs blank control group). While anacardic acid could downregulate significantly the over-expression of α-MHC, Cx43 and cTnT caused by alcohol exposure in the level of protein (P< 0.05 vs alcohol group).Conclusions(1) Anacardic acid inhibits the binding of p300 and PCAF to the promoter of cardiac nuclear transcription factors GATA4 and TBX5, and decreases the acetylation of H3AcK9 in the promoter region, and eventually downregulates the transcription activities of GATA4 and TBX5.(2) Anacardic acid could inhibit the over-expression of cardiac development related genes a-MHC, Cx43 and cTnT at the level of transcription and translation, but not affect the expression of a-actin at the level of transcription and protein. |
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