Role Of Histone Acetylation In Reprogramming Of Mouse Embryonic Genome After Nuclear Transfer

As known, efficiency for reprogramming of differentiated somatic nuclei into thetotipotent state by transfer into oocyte cytoplasm is very low. Although the somaticreconstructed embryos might reach the blastocyst stage in some experiments, theirdevelopment to full-term is limited. It is helpful to improve the efficiency of nucleartransfer to understanding mechanisms underlying the reprogramming process. In thepresent study, the pattern of gene transcription was investigated at the early stage of nucleartransfer embryos derived from breast cancer cells of mouse donor nuclei by real-timepolymerase chain reaction (PCR). In addition, roles of histone acetylation inreprogramming of mouse somatic nuclei after nuclear transfer were also studied.1 Development of nuclear transferred mouse embryos reconstructed withnuclei of breast cancer cellsIn the present study, we first constructed cloned mouse embryos derived from nucleiof donor mammary tumor cell. The results showed that the fusion rate was 76.3% (116/152)by electrofusion. Of the fused embryos, 90.5% (105/116) displayed pseudopronuclei afterbeing activated with strontium chloride. After a continuous culture for 24 h, 77.1% (81/105)of the reconstituted embryos developed to 2-cell stage; The developmental rates of 4-celland morula were 45.7% (48/105) and 17.1% (18/105), respectively.2 Expression of developmental related genes in genome reprogrammingof NT mouse embryos In order to assess the level of nuclear reprogramming in NT embryos reconstructedwith nuclei of mammary tumor cells, it is necessary to establish the pattern of genetranscription for the genes of interest in breast cancer cells and in IVF preimplantationembryos first. We detected the transcripts of ErbB2, Mucl, eIF-4C, MuERV-L, and c-mosin mammary tumor cells in the mouse by RT-PCR. To make the comparison of geneexpression pattern between somatic cells and NT embryos, we selected 50 mammary tumorcells (the same as NT embryos) for analysis. The results showed that transcriptions ofErbB2 and Muc1 were high in the mammary tumor cells, but transcriptions of eIF-4C,MuERV-L, and c-mos were not detected. The same RT-PCR methods were used forembryos reconstructed with mammary tumor cells. Transcripts of eIF-4C and MuERV-Lwere detectable in 1-cell and 2-cell embryos, and similar expression patterns to IVFembryos. However, expressions of eIF-4C and MuERV-L in the NT embryos wassignificantly down-regulated (P＜0.05). It was noted that c-mos which was normally silentin 2-cell derived from IVF was detected in the cloned 2-cell embryos, presumably due tothe degradation of maternal transcripts was repressed in the NT embryos, or are-transcription of c-mos resulted from the nuclear reprogramming event. Furthermore,transcripts of ErbB2 and Mucl were also not detected in the cloned and IVF embryos. Thisimplys that the interaction of nuclear-cytoplasm after NT inhibits the expression of specificgenes in the differentiated cells, and develops a new expression profile of genes.3 Role of histone acetylation in genome reprogramming of NT mouseembryos derived from mammary tumor cellsThe NT embryos were constructed by transferring mammary tumor cells intoenuleation oocytes, and treated with TSA (100nM) or aphidicolin (1.5μg/mL) for 6himmediately after electrofusion. Then the embryos were washed off TSA, activated withstrontium and cultured to 2-cell stage. The results indicated that transcripts of eIF-4C,MuERV-L, and c-mos had a modest, non-significant increase in 1-cell and 2-cell embryostreated with TSA for 6h in the pre-activation period (P＞0.05), and also had no significantincrease in transcripts of eIF-4C, MuERV-L, and c-mos when using aphidicolin to inhibitDNA replication. In addition, the transcription of ErbB2 and Mucl in treated with TSAand/or aphidicolin in the cloned embryos was not observed. This suggests that inhibition ofoocyte cytoplasm deacetylation to donor nuclei does not affect gene expression profile in reprogramming, and reprogramming of the genes responsible for embryonic developmentin the transfer somatic nuclei is independent from the acetylation events occurring beforeoocyte activation.The period of TSA treatment extended from 6h before activation to 6h afteractivation (the pronucleus formation in NT embryos), transcripts of eIF-4C, MuERV-L, andc-mos treated with TSA and TSA + aphidicolin were significant increased (P＜0.05).However, there was no any significant differences among treatments of TSA, TSA +aphidicolin, and aphidicolin only (P＞0.05). This implies that the increase of genetranscripts is because of the positive effect of TSA on acetylation in reprogramming. Inaddition, the transcripts of ErbB2 and Mucl in treated with TSA before and after 6h ofactivation were not observed. These data suggest that histone acetylation during nuclearremodeling (from the start of activation to the formation of pronuclei) is independent fromthe event that oocyte cytoplasm inhibits the transcription of specific genes in thedifferentiated cells, but induces reprogramming of gene expression.We further examined the effect of change of histone acetylation level in developmentof IVF and NT embryos. The results indicated that the level of gene expression wassiginifecantly improved in the IVF embryos, but the developmental rates of 2-cells wereobviously inhibitted 4-cell embryos treated with TSA continuously before and afteractivation. However, the developmental rates of 2-cells and 4-cells treated with TSAcontinuously before and after activation in the NT embryos, were 90.2% (110/122) and68.9% (84/122), respectively, which were significantly high (P＜0.01), especially the 4-cellrate was increased 52.4% comparing with the control group without TSA treatment. In theNT embryos, histone hyperacetylation induced by TSA conduced a significant increase ofgene expression level at the 2-cell stage, which was close to the level of genes in the IVFembryos. It may be a major reason for the improvement of developmental rates of the NTembryos. However, the increase of gene transcription and transcripts in IVF 2-cell embryosresulted from histone hyperacetylation induced by TSA decreased the embryonicdevelopment, it possibly repressed the development of 2-cell to 4-cell embryos.