Abnormal patterns of gene expression are a hallmark of cloned embryos and studies in cloned cattle and mice demonstrating genomic hypermethylation have begun to provide some explanation to these transcriptional abnormalities (Bourc'his et al., 2001
; Dean et al., 2001
; Kang et al., 2001
; Xue et al., 2002
). The key modulators of DNA methylation are the DNMTs and over-expression of this enzyme family has been hypothesized to lead to the observed hypermethylation and the aberrant patterns of X-chromosome inactivation and imprinted gene expression frequently seen in animals produced using nuclear transfer (Bestor, 1998
; Golding and Westhusin, 2003
). To distinguish between DNMT over-expression versus differing capacities of somatic and embryonic nuclei to biochemically regulate this enzyme family, we conducted real time quantitation of bovine Dnmt transcripts during IVF, NT and parthenote development. Results from this study demonstrate that within cloned embryos, members of the Dnmt family are not over-expressed above and beyond levels seen in IVF or parthenote embryos but rather demonstrate drastically reduced transcript levels encoding the most abundant methyltransferase, Dnmt1. The promoter of human dnmt1 contains several methylation responsive elements that have been hypothesized to modulate transcription in accordance with local levels of genomic methylation (Bigey et al., 2000
; Slack et al., 1999
; Slack et al., 2001
). Given the hypermethylated status of the genome in cloned embryos it is possible that transcription of Dnmt1 in clones becomes attenuated as a result of the high levels of CpG methylation within the donor genome.
During early development of cloned mice removal of the female pronucleus from the oocyte precludes any ability of the zygote to demethylate the donor genome and removes key elements controlling the trafficking of the oocyte specific isoform of Dnmt1 (Dnmt1o) (Howell et al., 2001
; Chung et al., 2003
; Kang et al., 2001
; Oswald et al., 2000
). Similarly, studies of nuclear breakdown and the dynamics of murine Dnmt1o in reconstructed embryos support the assertion that DNMT trafficking is disrupted in NT embryos. (Gonda et al., 2003
; Chung et al., 2003
). Given our observed down-regulation of bovine Dnmt1 transcription along with previous observations of early murine NT development, we postulate that the increasing levels of genomic methylation observed in cloned cattle may result from either abnormal trafficking or biochemical regulation of this gene family. Thus within the unique environment of an oocyte, somatic nuclei lack key elements controlling enzyme access or activity resulting in genomic hypermethylation.
In an effort to reduce genomic methylation levels we sought to suppress Dnmt1 activity in donor cell lines and in early embryos using RNAi. Utilizing siRNAs to block DNMT1 expression during preimplantation development we sought to develop protocols aimed at lowering levels of genomic methylation by inducing passive demethylation. However, similar to previously described studies using Dnmt1 targeting siRNAs in sheep, depletion of the most abundant methyltransferase within IVF and parthenote embryos resulted in a developmental arrest before the blastocyst stage (Taylor et al., 2009
). In contrast using a stably expressed shRNA to interfere with Dnmt1 expression in cloned embryos did not induce developmental arrest and produced development rates identical to the controls. We hypothesize that these observations can be explained by the differences in RNAi inducing molecule dosage and timing. SCNT embryos containing a shRNA expression cassette will not be transcribed until embryonic genome activation at the 8-cell stage where as siRNAs will immediately prime the RNAi machinery upon injection and suppress homologous transcripts. Moreover siRNAs were injected in picomolar amounts where as shRNAs will exhibit similar kinetics and abundance to other Polymerase II transcripts (Silva et al., 2005
; Stegmeier et al., 2005
). Thus in SCNT embryos generated using a Dnmt1-shRNA expressing cell line, maternal transcripts were likely sufficient to carry development beyond the blastocyst stage into post-implantation development at which point the depletion of DNMT1 was lethal. While ineffective at improving SCNT embryo development rates, our results clearly show that the techniques described above are effective as a tool to study functional genomics in a large animal model.
We were surprised that the reduction in global DNA methylation within the donor cell line was only on the order of 15% given the potent depletion of Dnmt1 transcripts to levels less than 5% of controls. Given the number of passages the transgenic fibroblasts were grown before analysis we fully expected to see a reduction in methylation levels by at least half. It is possible that DNMT3b, which does have some reported maintenance activity, could be compensating and maintaining methylation levels (Eilertsen et al., 2007
; Chen et al., 2003
). However our observations that levels of both Dnmt3a and Dnmt3b transcripts were reduced in Dnmt1-shRNAMir
cell lines do not wholly support this assertion. Moreover we are at a loss as to explain the down-regulation of both Dnmt3a and 3b in the absence of Dnmt1.
As the mammalian embryo develops, progressive changes in epigenetic modifications to DNA and histones gradually program cell specific patterns of gene expression and restrict both lineage potential and cell identity (Goldberg et al., 2007
). However, in contrast to cells within the inner cell mass of in vivo
preimplatation embryos, both ES and IPS cells in culture are relatively hypermethylated; yet clearly retain the capacity to generate all the somatic lineages. Moreover, while increased levels of genomic methylation are an impediment of SCNT reprogramming they are clearly not insurmountable. Therefore methods aimed at partially reducing these high levels should improve the “programability” of the donor nucleus and thus embryo survival. Conversely, a recent report suggests that bovine cloning efficiency is not correlated with altered levels of key epigenetic modifiers, including the Dnmts 1, 3a and 3b in the donor cell line (Zhou et al., 2009
). Our studies indicate that targeting maternal Dnmt1 transcripts within the preimplantation embryo using siRNAs does not likely represent a viable strategy for improving SCNT development rates as presumably the affect of RNAi persists past the 8-cell stage and block Dnmt1's essential role in developmental programming (Howell et al., 2001
). In contrast, transient siRNA mediated depletion of Dnmt1 within donor cell lines produces a ~30% increase in SCNT development rates to the blastocyst stage (Eilertsen et al., 2007
). It will be interesting to see whether this increase in development rates correlates with an increased ability to produce competent ES cells or live offspring.
The birth of Dolly and more recently the discovery of methods for the generation of induced pluripotent stem cells have raised fundamental questions concerning the irreversibility of the differentiated state (Campbell et al., 1996
; Takahashi and Yamanaka, 2006
). Since its discovery, induction of pluripotency within somatic cells in vitro
have shifted studies of the cellular reprogramming process from those restricted by the scale of embryo culture to large-scale studies in cell culture and greatly accelerated the pace of research within this field. It is now becoming clear that remodeling the epigenetic landscape of a cell represents the key to success for this reprogramming process and accordingly much effort has gone into identifying crucial factors involved in this process. Critical roles for genomic demethylating enzymes, polycomb proteins and chromatin remodeling factors in this process speak to the critical nature of epigenetics in IPS cell generation and highlight how essential a firm understanding of these processes are to IPS stability and therapeutic potential (Kim et al., 2010; Singhal et al., 2010
; Pereira et al., 2010
; Bhutani et al., 2009
). Thus, it is very likely that methods aimed at priming somatic cells for IPS programing will also serve to improve cell suitability for the SCNT process. The recent success in demethylating cellular genomes using proteins involved in elements of base excision repair is one example of potential methods that my be used to improve SCNT efficiency (Kim et al., 2010).