“Mouse ooplasm confers context-specific reprogramming capacity”

The mammalian oocyte mediates one of the largest periods of DNA methylation dynamics; global demethylation of the paternal genome, a process required for the establish­ment of totipotency and developmental competence (Reik et al 2011). Somatic cell nuclear transfer (SCNT) attempts to recapitulate this artificially by using enucleated oocytes to reprogram a somatic cell nucleus, but only achieves this with very low efficiency, likely compromised by the retention of uncharacterised somatic epigenetic modifications (Rideout et al andWakayama et al).   Now, researchers from the laboratory of Alexander Meissner have reported the results from their study of genome-scale DNA methylation patterns after SCNT and their comparison to observed dynamics during normal fertilization, and identify specific targets for DNA demethylation during SCNT and identify some unique limitations (Chan et al).

Using reduced representation bisulphite sequencing, ideal for analysis of small DNA samples (Meissner et al), single base pair–resolution maps of DNA methylation were generated from fibroblasts and SCNT mouse embryos. In what the authors consider to be the “first genome-scale measurement in SCNT”, 15% of the host oocyte genome was detected which affected ~35% and ~13% of loci in reconstructed embryos generated using the inbred and hybrid donors, respec­tively. Comparison of these findings to what is understood to occur during normal fertilization (Smith et al) demonstrated that following SCNT, the fibroblast DNA methylation landscape becomes demethylated, as occurs for the paternal genome upon fertilization. However, the SCNT-mediated demethylation was of a smaller magnitude resulting in SCNT embryos more closely resembling donor fibroblasts. However, the SCNT embryos were similar to each other suggesting that the majority of methylation changes which occur during SCNT are consistent across experiments.

Upon consideration of the genomic context of the differences in DNA methylation dynamics, it was discovered that methyla­tion levels at repetitive elements such as long interspersed elements (LINEs) and long terminal repeats (LTRs) only slightly decreased or did not change after SCNT. This suggests that these elements and their resistance to demethylation could be the cause of unsuccessful reprogramming in SCNT, as these elements account for a large proportion of demeth­ylation events during fertilization. DNA methylation at promoter sequences was next evaluated to identify the most consistently changing promoters during SCNT, leading to the identification of 15 promoters which undergo consistent changes in methylation during SCNT. These included promoters for genes which function during meiosis, which are unmethylated in gametes and the early embryo, suggesting the presence of defined tar­geting factors in the ooplasm that ensure the unmethylated status of these sites (Egli et al). However, approximately 2/3 of fertilization-specific targets of demethylation retained DNA methylation after SCNT, suggesting that only regions in certain genomic contexts are equivalently demethylated in both processes, which likely affects embryonic development through the persistence of somatic epigenetic DNA methylation. Further differences between SCNT-reconstructed embryos and true embryos were observed; 102 previously identified differentially methylated promoters that are hypermethyl­ated in the oocyte and remain transiently methylated on the maternal allele during pre-implantation (Smith et al) were unmethylated in SCNT-reconstructed embryos, similar to fibroblasts.

This study has provided an excellent resource by starting to identify some of the core problems behind the inefficiency of SCNT, the lack of widespread DNA demethylation, and also some of the main reprogramming abilities of the mammalian oocyte. The researchers also note that their future investigations will hopefully expand on this work by studying the prevalence and dynamics of hydroxymethylcytosine, an important intermediate in a proposed DNA demethylation pathway (Booth et al) and important histone modifications.   Altogether this should illuminate the role of the oocyte in promoting widespread epigenetic modifications and the failings of SCNT in this sense, perhaps even allowing the construction of strategies to rectify these failings.

References

Booth, M.J. et al.
Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution.
Science 336, 934–937 (2012).

Chan MM, et al.
Mouse ooplasm confers context-specific reprogramming capacity.
Nat Genet. 44:978-80 (2012).

Egli, D. et al.
Reprogramming within hours following nuclear transfer into mouse but not human zygotes.
Nat. Commun. 2, 488 (2011).

Meissner, A. et al.
Genome-scale DNA methylation maps of pluripotent and differentiated cells.
Nature 454, 766–770 (2008).

Reik, W. et al.
Epigenetic Reprogramming in Mammalian Development.
Science 293, 1089–1093 (2001).

Rideout, W.M. III. et al.
Nuclear Cloning and Epigenetic Reprogramming of the Genome.
Science 293, 1093–1098 (2001).

Smith, Z.D. et al.
A unique regulatory phase of DNA methylation in the early mammalian embryo.
Nature 484, 339–344 (2012).

Wakayama, T. et al.
Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.
Nature 394, 369–374 (1998).

Original article appeared in Nature Genetics.

STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.