Epigenetic reprogramming is a prerequisite process during mammalian development that is aberrant in cloned embryos. However, mechanisms that evolve abnormal epigenetic reprogramming during preimplantation development are unclear. To trace the molecular event of an epigenetic mark such as DNA methylation, bovine fibroblasts were epigenetically altered by treatment with trichostatin A (TSA) and then individually transferred into enucleated bovine oocytes. In the TSA-treated cells, expression levels of histone deacetylases and DNA methyltransferases were reduced, but the expression level of histone acetyltransferases such as Tip60 and histone acetyltransferase 1 (HAT1) did not change compared with normal cells. DNA methylation levels of non-treated (normal) and TSA-treated cells were 64.0 and 48.9% in the satellite I sequence (P!0.05) respectively, and 71.6 and 61.9% in the a-satellite sequence respectively. DNA methylation levels of nuclear transfer (NT) and TSA-NT blastocysts in the satellite I sequence were 67.2 and 42.2% (P!0.05) respectively, which was approximately similar to those of normal and TSA-treated cells. In the a-satellite sequence, NT and TSA-NT embryos were substantially demethylated at the blastocyst stage as IVF-derived embryos were demethylated. The in vitro developmental rate (46.6%) of TSA-NT embryos that were individually transferred with TSA-treated cells was higher than that (31.7%) of NT embryos with non-treated cells (P!0.05). Our findings suggest that the chromatin of a donor cell is unyielding to the reprogramming of DNA methylation during preimplantation development, and that alteration of the epigenetic state of donor cells may improve in vitro developmental competence of cloned embryos.

Global reduction of DNA methylation, a part of genome reprogramming processes, occurs in a gradual manner until before implantation and is recognized as a conserved process in mammals.Here, we reported that in bovine, satellite regions exhibited varied patterns of methylation changes when one-cell egg advanced to the blastocyst; a maintenance methylation was observed in satellite I sequences, a decrease in alpha satellites, and an increase in satellite II regions. Cloned embryos exhibited similar changes for DNA methylation in the satellite I and alpha. We also observed that the satellite I and a sequences were methylated more in inner cell mass region of the blastocyst whereas the satellite II showed selective demethylation in this region. Together, these findings point that individual satellite sequences carry their own methylation patterns under the pressure of global demethylation, suggesting that local methylation control system acts on the satellite regions in early bovine embryos. Mol. Reprod. Dev. 71: 29-35, 2005. ß 2005 Wiley-Liss, Inc.

Certain bone and soft tissue (BST) tumours harbour a chromosomal translocation [t(6;22)(p21;q12)], which fuses the Ewing's sarcoma (EWS) gene at 22q12 with the octamer‐binding transcription factor 4 (Oct‐4) gene at 6p21, resulting in the chimeric EWS‐Oct‐4 protein that possesses high transactivation ability. Although abnormal activation of signalling pathways can lead to human cancer development, the pathways underlying these processes in human BST tumours remain poorly explored. Here, we investigated the functional significance of fibroblast growth factor (FGF) signalling in human BST tumours. To identify the gene(s) involved in the FGF signalling pathway and potentially regulated by EWS‐Oct‐4 (also called EWS‐POU5F1), we performed RNA‐Seq analysis, electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and xenograft assays. Treating GBS6 or ZHBTc4 cells‐expressing EWS‐Oct‐4 with the small molecule FGF receptor (FGFR) inhibitors PD173074, NVPBGJ398, ponatinib, and dovitinib suppressed cellular proliferation. Gene expression analysis revealed that, among 22 Fgf and four Fgfr family members, Fgf‐4 showed the highest upregulation (by 145‐fold) in ZHBTc4 cells‐expressing EWS‐Oct‐4. Computer‐assisted analysis identified a putative EWS‐Oct‐4‐binding site at +3017/+3024, suggesting that EWS‐Oct‐4 regulates Fgf‐4 expression in human BST tumours. Fgf‐4 enhancer constructs showed that EWS‐Oct‐4 transactivated the Fgf‐4 gene reporter in vitro, and that overexpression of EWS‐Oct‐4 stimulated endogenous Fgf‐4 gene expression in vivo. Finally, PD173074 significantly decreased tumour volume in mice. Taken together, these data suggest that FGF‐4 signalling is involved in EWS‐Oct‐4‐mediated tumorigenesis, and that its inhibition impairs tumour growth in vivo significantly.

Epigenetic reprogramming such as DNA methylation is incomplete in cloned embryos during early development as compared with normal embryos. The increased methylation levels of cloned bovine blastocysts are showed in centromeric heterochromatin. The aim of the present study was to investigate the change of methylation state by treatment of trichostatin A (TSA), a specific inhibitor of histone deacetylase in somatic donor nuclei and cloned blastocyst reconstructed with TSA-treated cells or nontreated cells. Bovine ear skin fibroblast cells (bESF) were used as donor cell and treated with TSA for 60 h at a final concentration of 1 �M. To methylation analysis of satellite I as specific DNA sequence, genomic DNA from 7 � 104 cells and a blastocyst were isolated, and then the genomic DNA was analyzed by bisulfite sequencing. The reduction of HDAC1, 2 and Dnmt family such as Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3L after TSA treatment were shown by Western blot in bESF, but histone acetyltransferases such as Tip60 and HAT1 were not changed. Satellite I DNA in nontreated cells was highly methylated in CpG sequences, whereas methylation level of TSA-treated cells was significantly decreased (64 vs. 48%, P < 0.05). After nuclear transfer using normal or altered donor cells, methylation levels of satellite were measured at the blastocyst stage of NT and TSA-NT embryos as compared with IVF embryos. In nontreated NT blastocysts, methylation levels were significantly higher than IVF blastocysts (66 vs. 29%, P < 0.05) and were similar to that of nontreated bESF cells. The reduction of methylation levels in TSA-NT blastocysts were showed and were significantly lower than NT blastocyst derived with nontreated cells (37 vs. 66%, P < 0.05), but no significant differences were found between TSA-NT and IVF blastocysts. Also, the levels of methylation were similar to that of TSA-treated donor cells. In blatocyst formation, TSA-NT embryos were improved significantly compared with NT or IVF embryos (45.9 vs. 31.7 or 28%, P < 0.05). These results demonstrated that somatic methylation status after epigenetic alteration affect in early cloned embryo development, suggesting epigenetic control may help to solve of inherent problems in cloning.