Epigenetic Regulation Mechanism Of Apoptosis And Nucleolar Structure

Apoptosis is a suicidal behavior made by cells in response to internal and external environmental signals in order to maintain homeostasis.Apoptosis is found to be involved in multiple processes including normal development of organisms,tissue self-renewal,and it also play an important part in the pathogenesis of various diseases such as cancer and nervous system diseases due to the mutation of major apoptosis genes.DNA methylation plays a crucial role in numerous biological processes and carcinogenesis.5-azacytidine(5-AC)is a DNA methylation inhibitor that has been used clinically to treat myelodysplastic syndromes,leukemia and other diseases.Our previous study has found that 5-AC can specifically inhibit the apoptotic process,but the molecular pathways involved need further exploration.Nucleolus is a nuclear organelle responsible for ribosome synthesis.Recent research and development of nucleolar proteomics have shown that it plays an important role in a variety of cellular activities.The structure of the nucleolus is a manifestation of its normal biological function.Apoptotic cells exhibit condensed chromatin and broken nucleoli.Studies have shown that histone H3 lysine 9 dimethylation(H3K9me2)is an indispensable factor for nucleolus to maintain its structural integrity,however the underlying molecular mechanism about regulation of nucleolar structure remains to be further explored.The main results are as follows.1.DNA methylation inhibitor 5-AC specifically inhibits apoptosis of NIH-3T3 cells induced by tumor necrosis factor(TNF-?)and cycloheximide(CHX)by affecting the anti-apoptotic protein BCL-XLTreatment with 5-AC causes a decrease in the whole methylation level of the cancer cells,including the methylation level of a promoter which belongs to a specific apoptosisrelated gene,thereby causing apoptosis of cancer cells.Due to the diversity of intracellular molecular environments,different cell types show varied responses to 5-AC treatment.Our previous study demonstrated that the mouse embryonic fibroblast cell line NIH-3T3 is highly tolerant to 5-AC and that genomic methylation levels in NIH-3T3 cells rises during TNF-? and CHX-induced apoptosis,and 50 ?mol/l of 5-AC pretreatment for 3 h resulted in genomic DNA hypomethylation and significantly blocked the apoptotic process.To detect whether DNA methylation was involved in the NIH-3T3 apoptosis,three otherDNA methylation inhibitors,5-aza-2'-deoxycytidine(5-d AC),6-thioguanine(6-TG),and RG108 was used.Dot blot analysis showed that 5-d AC,6-TG and RG108 reduced the overall genomic methylation levels of NIH-3T3 cells to almost normal levels which are just comparable to 5-AC,however,results of flow cytometry(FCM)detecting cells co-staining with Annexin-V-FITC and PI showed these three DNA methylation inhibitors could not inhibit the NIH-3T3 cells apoptosis process induced by TNF-? and CHX as compared with 5-AC.In addition,transient knockdown of DNA methyltransferase DNMT1 by using small interfering RNA(siRNA)reduced genomic methylation level to a degree lower than the control.However,FCM analysis of the apoptotic rate showed that the knockdown of DNMT1 hardly affected apoptosis of the NIH-3T3 cells.Taken together,our results suggested that 5-AC specifically inhibit the NIH-3T3 apoptosis process as induced by TNF-? and CHX via a genomic DNA methylation-independent pathway.Subsequently,western blot was conducted to analyze the changes in the BCL-2 family proteins during the apoptosis process;the results indicated that both anti-apoptotic proteins BCL-2 and BCL-XL,and proapoptotic protein BAX were downregulated.Furthermore,FCM analysis indicated that siRNA-mediated knockdown of the antiapoptotic protein BCL-XL would abolish the function of inhibiting apoptosis by 5-AC.On the other hand,knockdown of anti-apoptotic BCL-2 or pro-apoptotic BAX protein did not interfere with the inhibition function of 5-AC.These data indicated that 5-AC exerts an inhibitory effect on TNF-? and CHX-induced apoptosis of NIH-3T3 by affecting the anti-apoptotic protein BCL-XL.2.Decreased H3K9me2 level at the rDNA site leads to an increase in rDNA transcription initiation and disorganized nucleoliIn this study,I analyzed the effect of three rRNA polymerase I transcription inhibitors,ActD,BMH21 or CX5461,on human lung cancer A549 cells for 24 hours.Immunofluorescence(IF)staining of the nucleolar marker fibrillarin(FC)exhibited a significant increase in the number of interphase nuclei containing more than three nucleoli.Real-time quantitative PCR(q RT-PCR)was performed using the 5'-end primer of the rDNA gene coding region and showed a conspicuous increase in the rDNA transcriptional initiation as indicated by an increase in the number of 45 S pre-rRNAs(45S pre).A similar result was observed in human cervical carcinoma HeLa cells.Chromatin immunoprecipitation(ChIP)assay indicated that the level of heterochromatin marker H3K9me2 at the rDNA site of HeLa cells reduced dramatically.HeLa cells were treated with ActD for different times,including 10 min,20 min,30 min and 1 h.The results of IF staining displayed that the number of interphase nuclei containing more than three nucleoli started to increase after treatment for 20 min,and q RT-PCR revealed that rDNA transcription initiation level also increased after treatment for 20 min.However,ChIP results demonstrated that the level of H3K9me2 at the rDNA locus was significantly reduced after 10 min of ActD treatment,indicating that the change of H3K9me2 precedes in the mechanism resulting for increasing number of nucleoli and rDNA transcription initiation,and therefore suggesting a close link between initiation of the transcription of rDNA and the nucleolar disruption.BIX-01294(BIX)is capable of specifically inhibiting the activity of histone lysine methyltransferase G9 a and reducing the genomic level of H3K9me2.In the present study,ChIP experiments on BIX-treated HeLa cells exhibited reduction in the overall level of H3K9me2 at the rDNA region.IF staining of BIX-treated HeLa cells showed an increase in the number of interphase nucleoli,and q RT-PCR exhibited that the expression of the transcriptional initiation product 45 S pre of rDNA also increased significantly.These findings suggested that H3K9me2 at the rDNA locus of HeLa cells regulates rDNA transcription initiation and the integrity of nucleolar structure and organization.3.The accumulation of R-loop at the rDNA region of HeLa cells triggers the nucleolar disruptionI performed ChIP assay on ActD-treated HeLa cells using anti-RNA: DNA hybrid/R-loop antibody S9.6,and found that the level of R-loops at the rDNA site increased evidently after treated with transcription inhibitor for different times.Ribonuclease H1(RNase H1)is capable of specifically degrading R-loops in the genome.The role of R-loop in nucleolar disruption was investigated by using small interfering RNA to transiently knockdown RNase H1 in HeLa cells.After RNase H1 knockdown,ChIP data showed that the level of R-loops at the rDNA locus increased significantly,and IF staining showed that there was also a significant number of interphase nuclei with more than three nucleoli in HeLa cells.Construction of a vector overexpressing RNase H1 and transient transfection of HeLa cells significantly attenuated the effects of inducing nucleolar disruption by ActD.These data indicated that the level of R-loops at the rDNA region in HeLa cells can regulate the structure of the nucleolus and excessive R-loops can lead to an increase in the number of nucleoli.4.The level of R-loops at the rDNA locus of HeLa cells is regulated by H3K9me2ChIP assay indicated that knockdown of RNase H1 led to an increase of R-loops at the rDNA site,but the level of H3K9me2 at rDNA region remained essentially unchanged.The treatment of BIX reduced the overall level of H3K9me2 at the rDNA region and simultaneously increased the number of R-loops.Our data indicated that H3K9me2 at the rDNA locus regulates the level of R-loops and both of them are involved in the maintenance of nucleolus structural integrity and organization.