DOT1 And Histone H3 Lysine 79 Methylation Are Required For Meiosis Progression In Mouse Oocytes

The N-terminal tails of core histones are subjected to multiple covalent modificationsthat include acetylation, methylation, phosphorylation and ubiquitination. One suchmodification, histone methylation, plays important functions in regulation of chromatindynamics and gene activity. Recently, a distinct class of histone lysine methyltransferaseDOT1 was found to mediate histone H3 lysine79 (H3-K79) methylation. The DOT1proteins do not contain a SET domain, a conserved sequence motif found in all previouslycharacterized histone lysine methyltransferases. Histone H3 lysine 79 residue is beyond theN-terminal tails of core histones and located on the accessible face of the nucleosome core.Previous studies have reported that DOT1-mediated H3-K79 methylation playimportant roles in regulation of chromatin dynamics and gene activity from fungi tomammal. Furthermore, recent reports have shown that histone methylation such as H3-K4,H3-K9 play important roles in mammalian oocytes meiosis progression or earlyembryogenesis. Nevertheless, the roles of H3-K79 methylation in meiosis in mammal areunelucidated.In the present study, we examed the H3-K79 methylation pattern and it's roles inmeiosis progression in mouse oocytes. The results indicated that:(1) Immunocytochemistry with specific antibody against methylated lysine79 onhistone H3 (meH3-K79) indicate that there are intense fluorescence signals in the nuclei ofthe GV stage, GVBD stage and Mâ…¡stage oocytes. Histone H3 lysine79 of chromosomeshighly methylate in GVBD stage and Mâ…¡stage oocytes. Interestingly, the fluorescencesignals preferentially focus on several dots disperse in the germinal vesicles of the GVstage oocytes. Simultaneous staining with anti-centromeric proteins antibody CRESTindicate that the methylation of H3-K79 only occur on centromeres in the germinal vesiclesof the GV stage oocytes.(2) By using RNAi, we successfully inhibited mdotla expression and histone H3lysine79 methylation in GV stage oocytes. Quantitative analysis indicate that injection ofmdotla siRNA triggered an drastic reduction in amount of mdotla mRNA in the GV stage oocytes after 24h culture in CZB medium containing 0.2 mM IBMX. The relative amountof mdotla mRNA of siRNA injection group significantly reduce to 15.49% compared withthe not injection group. In contrast, there is no significant difference between the controlsiRNA injection group and the not injection group. The immunocytochemistry results ofRNAi group demonstrate that there are faint fluorescence signals from most of the siRNAinjection oocytes. There is a significant difference between the siRNA injection group andthe control siRNA injection group, the H3-K79 methylation level of RNAi group reducedto 19.54% compared with the control group. However, there is no significant differencebetween control siRNA injection group and the not injection group.(3) Our results reveal that the oocytes of siRNA injection group exhibit seriousabnormal phenotype in chromosome congression and chromosome segregation. The resultsindicate that 61.15% (85/139) oocytes in siRNA injection group exhibit abnormalchromosome congression and chromosome segregation. In contrast, only 16.89% (25/148)and 15.92% (25/157) oocytes in control siRNA injection group and not injection groupexhibit abnormal chromosome congression and chromosome segregation, respectively.There is no significant difference between control siRNA injection group and not injectiongroup.(4) Our results reveal that the oocytes of siRNA injection group exhibit seriousabnormal phenotype in spindle organization in the first meiosis. The results indicate that49.64% (69/139) oocytes in the siRNA injection group exhibit abnormal spindleorganization. In contrast, only 16.22% (24/148) and 14.01% (22/157) oocytes in the controlsiRNA injection group and the not injection group exhibit abnormal spindle organization,respectively. There is no significant difference between the control siRNA injection groupand the not injection group.(5) Inhibition of mdotla expression and histone H3 lysine79 methylation preventedthe oocytes meiotic maturation. The statistic results indicate that the rate of oocytesmaturation in the siRNA injection group is much lower than it in the control siRNAinjection group and not injection group. In the siRNA injection group, only 22.32% (25/112)oocytes accomplish meiotic maturation, however, in the control siRNA injection group andthe not injection group, there is 66.13% (82/124) and 69.47% (91/131) ooeytes accomplishmeiotic maturation, respectively. There is no significant difference between the controlsiRNA injection group and the not injection group.In conclusion, inhibition of mdotla expression and histone H3 lysine79 methylation prevented the oocytes meiotic maturation, cause abnormal spindle organization andchromosome congression, and then resulted in abnormal chromosome segregation inmeiosisâ… . These revealed that mDOT1a and H3 lysine79 methylation are required formeiosis progression in mouse oocytes.