Despite recent successes in cloning various animal species, the use of somatic cells as the source of donor nuclei has raised many practically relevant questions such as increased abortion rates, high birth weight and perinatal death. These anomalies may be caused by incomplete epigenetic reprogramming of donor DNA. Genome-wide demethylation occurs during early development, 'erasing' gamete-specific methylation patterns inherited from the parents. This process may be a prerequisite for the formation of pluripotent stem cells that are important for the later development. Here, we provide evidence that cloned bovine embryos may have impaired epigenetic reprogramming capabilities. We found highly aberrant methylation patterns in various genomic regions of cloned embryos. Cloned blastocysts closely resembled donor cells in their overall genomic methylation status, which was very different from that of normal blastocysts produced in vitro or in vivo. We found demethylation of the Bov-B long interspersed nuclear element sequence in normal embryos, but not in cloned embryos, in which the donor-type methylation was simply maintained during preimplantation development. There were also significant variations in the degree of methylation among individual cloned blastocysts. Our findings indicate that the developmental anomalies of cloned embryos could be due to incomplete epigenetic reprogramming of donor genomic DNA.

Methylation of histone H3 at lysine 9 (H3-K9) mediates heterochromatin formation by forming a binding site for HP1 and also participates in silencing gene expression at euchromatic sites. ESET, G9a, SUV39-h1, SUV39-h2, and Eu-HMTase are histone methyltransferases that catalyze H3-K9 methylation in mammalian cells. Previous studies demonstrate that the SUV39-h proteins are preferentially targeted to the pericentric heterochromatin, and mice lacking both Suv39-h genes show cytogenetic abnormalities and an increased incidence of lymphoma. G9a methylates H3-K9 in euchromatin, and G9a null embryos die at 8.5 days postcoitum (dpc). G9a null embryo stem (ES) cells show altered DNA methylation in the Prader-Willi imprinted region and ectopic expression of the Mage genes. So far, an Eu-HMTase mouse knockout has not been reported. ESET catalyzes methylation of H3-K9 and localizes mainly in euchromatin. To investigate the in vivo function of Eset, we have generated an allele that lacks the entire pre-and post-SET domains and that expresses lacZ under the endogenous regulation of the Eset gene. We found that zygotic Eset expression begins at the blastocyst stage and is ubiquitous during postimplantation mouse development, while the maternal Eset transcripts are present in oocytes and persist throughout preimplantation development. The homozygous mutations of Eset resulted in peri-implantation lethality between 3.5 and 5.5 dpc. Blastocysts null for Eset were recovered but in less than Mendelian ratios. Upon culturing, 18 of 24 Eset ؊/؊ blastocysts showed defective growth of the inner cell mass and, in contrast to the ϳ65% recovery of wild-type and Eset ؉/؊ ES cells, no Eset ؊/؊ ES cell lines were obtained. Global H3-K9 trimethylation and DNA methylation at IAP repeats in Eset ؊/؊ blastocyst outgrowths were not dramatically altered. Together, these results suggest that Eset is required for peri-implantation development and the survival of ES cells.

Cloning by nuclear transfer (NT) has been riddled with dif®culties: most clones die before birth and survivors frequently display growth abnormalities. The cross-species similarity in abnormalities observed in cloned fetuses/animals leads us to suspect the ®delity of epigenetic reprogramming of the donor genome. Here, we found that single-copy sequences, unlike satellite sequences, are demethylated in pre-implantation NT embryos. The differential demethylation pattern between genomic sequences was con®rmed by analyzing single blastocysts. It suggests selective demethylation of other developmentally important genes in NT embryos. We also observed a reverse relationship between methylation levels and inner cell mass versus trophectoderm (ICM/TE) ratios, which was found to be a result of another type of differential demethylation occurring in NT blastocysts where unequal methylation was maintained between ICM and TE regions. TE-localized methylation aberrancy suggests a widespread gene dysregulation in an extra-embryonic region, thereby resulting in placental dysfunction familiar to cloned fetuses/animals. These differential demethylations among genomic sequences and between differently allocated cells produce varied overall, but speci®ed, methylation patterns, demonstrating that epigenetic reprogramming occurs in a limited fashion in NT embryos.