| Objective: To investigate the role of transcription cofactor p300 in regulating the repair pathway selection of non-homologous end junctions(NHEJ)of DNA double-strand breaks and the molecular mechanism of chromosomal translocation.Methods:(1)A fluorescence indicator reporting system(hereinafter referred to as NHEJ fluorescence reporting system)was constructed in HEK 293 T cells to indicate different intracellular NHEJ repair pathways(intra-NHEJ and inter-NHEJ).Green fluorescence indicated intra-NHEJ and red fluorescence indicated inter-NHEJ.The proportion of green fluorescent cells and red fluorescent cells was detected by flow cytometry.(2)RNA-sequencing was used to screen differentially expressed genes related to DNA damage repair in different fluorescent cells.Chromatin immunoprecipitation(Ch IP)technique was used to detect the differences of protein molecules recruited at DNA repair ends in different fluorescent cells.(3)Interference or overexpression of p300 or p300 HAT specific inhibitor C646 was applied to the fluorescence reporting system.Flow cytometry was used to detect the variations in the proportion of fluorescent cells with different color,different color cells were sorted out and proteins were extracted for further testing.Western blot was used to detect the protein expression of p300,MSH2,KU70 and XRCC4 in different fluorescent cells,and the acetylated histone levels at different sites were detected.The interaction between p300 and histone H3/H4 was detected by co-immunoprecipitation(Co-IP),and the activity of p300 HAT in different fluorescent cells was determined using a HAT detection kit.(4)Peripheral blood samples and aborted chorions of patients with recurrent spontaneous abortion were collected.According to the results of karyotype analysis,it was divided into normal karyotype group and structural chromosomal variation group.The protein expressions of p300,MSH2,KU70 and XRCC4 were detected by Western blot,and the acetylated histone levels at different sites were also detected.p300 HAT activity was determined using HAT assay kit.(5)Single nucleotide polymorphisms of EP300(p300),MSH2,KU70 and XRCC4 in cases of recurrent spontaneous abortion with structural chromosomal variation were detected by whole-exon sequencing(WES)and verified by Sanger sequencing.Peripheral blood mononuclear cells with different SNPs genotypes were isolated from the normal karyotype group,and the frequency of chromosomal translocation was detected by fluorescence in situ hybridization(FISH)after X-ray irradiation.Meanwhile,the expression of p300,p300 Ac,MSH2,KU70,XRCC4 and p300 HAT were detected.(6)The significant SNP locus in wild type cell line TEV-1 was edited by CRISPR technique,and the frequency of chromosomal translocation was detected by fluorescence in situ hybridization(FISH)after X-ray irradiation.The expression of p300,p300 Ac,MSH2,KU70,XRCC4 and p300 HAT activity in different locus cells were detected.(7)The expression of p300 protein,HAT activity and acetylated histone levels in chorionic tissue of recurrent spontaneous abortion were determined.Results:(1)The intracellular NHEJ fluorescence reporting system was successfully constructed.The fluorescence reporting system can indicate different NHEJ repair pathways through presenting different fluorescence accurately.(2)Differentially expressed genes were EP300,MSH2,KU70 and XRCC4 in fluorescent cells with different colors.KU70 and XRCC4 were recruited near DNA repair sites in fluorescent cells with different colors.(3)The interference,overexpression of p300 or p300 HAT specific inhibitor C646 in fluorescence reporting system showed that the total number of NHEJ repair events decreased accompanied by the proportion of inter-NHEJ repair increased.The protein level of KU70 in red fluorescent cells was significant higher than that in green fluorescent cells,while the XRCC4 is the opposite(P < 0.05).Further detection of p300 HAT activity and histone acetylation at different sites showed that p300 HAT activity,H3K18 Ac,H3K56Ac and H4K16 Ac protein levels in red fluorescent cells were lower than those in green fluorescent cells(P <0.05).Co-immunoprecipitation(Co-IP)confirmed the interaction between p300 and histone H3/H4.(4)The protein levels of KU70,XRCC4,H3K18 Ac,H3K56Ac and H4K16 Ac in peripheral blood of recurrent spontaneous abortion group with structure chromosome abnormal were lower than those of normal karyotype group(P < 0.05).The p300 Ac protein level and total HAT activity were significantly lower in structure chromosome abnormal group than that in normal karyotype group(P < 0.05).(5)The frequency of EP300 rs20551 and XRCC6 rs132788 in recurrent spontaneous abortion with structural abnormalities(mainly chromosomal translocation)was significantly higher than that in normal control group(P < 0.05).The chromosome translocation frequency in peripheral blood mononuclear cells of spontaneous abortion with rs20551(AG)or rs132788(GT)was significantly higher than that in wild-type group(P < 0.05)after X-ray irradiation.Meanwhile,p300 Ac protein level and HAT activity were significantly decreased in rs20551 /rs132788(AG/GG)group(P < 0.05).(6)After the locus of rs20551 was changed from A to G by CRISPR,the p300 HAT activity,p300 Ac,H3K18Ac,H3K56 Ac and H4K16 Ac protein levels were significantly decreased(P < 0.05).(7)The activity of p300 HAT,protein levels of p300,H3K18 Ac,H3K56Ac,H4K12 Ac and H4K16 Ac in chorionic tissues of recurrent spontaneous abortion with chromosomal structural variation were lower than that in normal karyotype group and control group(P < 0.05,P <0.05 respectively).Conclusions:(1)p300 interacts with histone H3 and H4,acetylates the histone H3K18,H3K56 and H4K16 sites through its HAT activity,affects the transcription of XRCC6(KU70)and XRCC4 genes,promotes NHEJ repair occurs in the error-prone pathway,causes the formation of chromosomal translocation.(2)The status of rs20551 locus in EP300 can affect the HAT activity of p300.(3)EP300 rs20551(or reduced p300 HAT activity)might be a risk factor to recurrent spontaneous abortion with structural chromosomal variation. |
