More with less Xist

Since the birth of Dolly, the first mammal produced from somatic cell nuclear transfer (SCNT), little progress has been made in the efficiency of cloning. Overall, about 0.5–2% of the embryos reconstructed by SCNT develop to term after transfer of reconstructed embryos to recipient females. In PNAS, Matoba et al. (1) report significant improvement in cloning efficiency by manipulation of the level of the Xist transcript.

SCNT involves replacement of the metaphase II chromosomes of a mature oocyte with the nucleus of a differentiated cell. Without any additional intervention, the nuclear envelop breaks down and the chromatin condenses into a structure that is similar to the recently removed metaphase II chromosomes. A variety of methods can be used to mimic the early events of fertilization such that the oocyte is activated to begin the embryonic developmental program in the absence of fertilization. However, the transferred nucleus was actively participating in a somatic developmental program at the time that it was harvested or transferred. To begin, the newly initiated embryonic developmental program requires an event similar to a computer reboot—the somatic program must be restarted as an embryonic program. In contrast to the computer analogy, factors that affect cell identity or result from the cell's history can still associate with the genome such that genes that are not normally expressed in early development can be inappropriately expressed in the reconstructed embryo (2). Similarly, because the somatic nucleus does not pass through a germ cell developmental program in which epigenetic modifications are normally reset, the epigenetic modifications that have occurred over the history of the somatic nucleus are not erased and reestablished (Fig. 1). As a result of retained transcription factors and epigenetic marks, the transferred nucleus must be actively reprogrammed amid mixed developmental signals.

Fig. 1.

In normal development, the two-cell embryo is the first instance in which a maternal and paternal genome reside in the same nucleus. During preimplantation development, the two-cell embryo divides and begins the first differentiation events to give rise to a blastocyst, a hollow structure with an undifferentiated inner cell mass and a polarized outer cell layer called the trophectoderm. The outer cells of a blastocyst give rise to the placenta, and the inner cell mass gives rise to all of the cell types of the fetus, including the primordial germ cells. During germ cell development, the epigenetic status of the genome is reset in a manner that is specific to the sex of the fetus. During this process, the entire genome is erased of the parent of origin imprints, and a sex-specific imprint is established in only this lineage. Germ cells of a female will give rise to oocytes or eggs, and germ cells of a male will give rise to sperm. On fertilization, a new embryo is created with specific parent of origin imprints. In the process of nuclear transfer, a somatic nucleus is thrust into an oocyte environment and then activated. The resulting two-cell embryo has an ill-defined parent of origin imprint and retains somatic cell-specific epigenetic modifications.

Because the oocyte volume is large compared with most somatic cells or isolated nuclei, most somatic transcription factors are likely severely diluted at nuclear transfer or during subsequent cell divisions. However, because maintenance of epigenetic marks is not well-described, it is unclear how modified histones and DNA methylation are reset or reprogrammed during early development of reconstructed embryos. In the systems that are most studied (3, 4), it seems that placental inefficiencies are a major cause of embryonic loss in cloned mammals. However, those embryos that develop to term can still display abnormalities. Most of the abnormalities observed in clones are reminiscent of conditions observed in humans that are associated with altered parent of origin genomic imprinting. Although there may be many factors that contribute to a lack of viability in SCNT embryos, it seems that genomic imprinting is involved.

Xist is an X-linked gene that produces a noncoding RNA, and it is one of the first imprinted genes to be expressed in the early embryo with expression beginning at zygotic genome activation (5). Although Xist is known to be involved in X-chromosome inactivation in females, the function of Xist RNA is not fully understood (6). Many genes are aberrantly expressed in cloned embryos (2, 7), and Xist is among these genes (8). Using nuclear donor cells that harbor a defective Xist gene, it has been shown that, in the absence of Xist expression, several aberrantly expressed genes approach normal expression patterns in cloned mouse embryos (9). In PNAS, the work by Matoba et al. (1) extends this line of investigation by a demonstration that an siRNA approach can temporarily reduce Xist RNA in the early preimplantation embryo. Remarkably, although the Xist RNA knockdown effect is temporary, early knockdown of Xist RNA results in an order of magnitude improvement in developmental competence of the reconstructed embryos. Importantly, this improvement is observed in live offspring as opposed to blastocyst development or early pregnancies.

Because male somatic cells are used in the work by Matoba et al. (1), overexpression of Xist is caused by increased expression at the single Xist gene as opposed to induction of Xist expression from the inactive Xist gene that would only be found in female cells. Because transcription of Xist physically overlaps with transcription of another gene, Tsix, the work by Matoba et al. (1) also shows that siRNA knockdown is specific to Xist noncoding RNA.

If the approach by Matoba et al. (1) proves applicable to species other than the mouse, this observation could have a positive impact in multiple fields. Potentially, cloning of agriculturally important species could allow for more efficient amplification of superior genetics or more consistent production schemes. Similarly, because SCNT has become an integral part of genetic engineering in livestock, this work may provide more efficient methods of genetic engineering for the production of biomedical models or improved agricultural animals. If proven robust and safe, manipulation of early expression of Xist may even prove useful in human-assisted reproductive technologies. In any case, the work by Matoba et al. (1) is likely to spur additional experimental approaches for improvement of SCNT and the study of epigenetics.

In addition to a potential strategy for improvement of SCNT, these results produce additional questions. Matabo et al. (1) use male cells for their work. Because male cells have a single X chromosome, this work strongly suggests additional roles of Xist beyond X inactivation. What are these additional functions of Xist, and will the approach prove useful with female reconstructed embryos? Because the aberrant Xist expression was only temporarily suppressed, these results imply that some of the functions of Xist are temporally sensitive and that continuous appropriate expression is not necessarily required for some of this gene's functions. What are these additional functions? Can other imprinting phenomena be corrected by temporary suppression of gene expression. Do these observations provide insight to alternative therapeutic strategies for imprinting disorders such as Beckwith–Wiedemann syndrome, which presents with many of the symptoms observed in nonviable clones?

As normally observed in scientific endeavors, the work by Matoba et al. (1) provides some hope of a practical application of their work (SCNT efficiency) as well as a long list of additional questions and new investigational directions. The impact of this work remains to be seen. At minimum, however, replication and extension of this research is all but guaranteed.