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Interestingly we found that IG DMR was
Interestingly, we found that IG-DMR was hypermethylated in the MEFs and mouse tail cells in comparison to ES cells (Figs. 2A and 3SA). This may be a result of IG-DMR imprinting in a tissue-specific manner. Indeed, it had been reported before that IG-DMR is hypermethylated in postnatal neural stem cells (NSCs) and niche-astrocytes but differentially methylated on the paternal chromosome in embryonic NSCs (Ferron et al., 2011). Likewise, IG-DMR may not be imprinted in MEFs or in mouse tail, meaning that both maternal and paternal IG-DMR regions are partially or completely methylated. But it is imprinted in ES cells and only the paternal IG-DMR is methylated. That is why we found IG-DMR exhibited hypermethylation in the MEF and mouse tail samples, compared with about 50% of methylation in the ES cell sample (Fig. 3SA). It has been documented that pluripotent stem cells obtained via iPS reprogramming harbor more residual DNA methylation signatures of the parental somatic cells than nuclear-transfer-derived pluripotent stem cells (Kim et al., 2010). Thus, it is possible that some iPS clones isolated with certain approaches may have retained more parental imprinting memory at this Dlk1-Dio3 imprinted region than other iPS clones derived with other approaches.
We generated multiple iPS clones from the hybrid MEF cells derived from the cross between a DBA/2 female mouse and a 129 male mouse. The SNPs between these two mouse strains allowed us to distinguish the parental origins of several ICRs. Indeed, we found that maternally inherited DNA methylation imprint at both the Snrpn and Zac1 DMRs was partially or completely lost in all iPS clones examined. Even for several iPS clones with relatively high methylation levels at these two DMR regions, partial loss of maternally inherited DNA methylation imprint was still evident. These results indicate that DNA methylation imprint is susceptible to the genome-wide demethylation process during iPS derivation. Thus, an approach using iPS cell derivation may be employed to dissect the underlying mechanism in the maintenance and erasure of genomic DNA methylation imprint in somatic cells.
Curiously, one or two DNA molecules carrying the SNPs of an apparent 129 origin were methylated at the Snrpn or Zac1 DMR based on bisulfite sequencing data (Figs. 4, 5 and 5S). This could mean that DNA methylation imprint was established de novo on the paternal chromosome for these two imprinted regions during iPS cell derivation. Alternatively, the DNA molecules containing the SNPs of a 129 origin could come from the trace amount of contaminated genomic DNA of the SNL feeder cells carried over with the iPS cells when they were diluted onto the gelatin-coated plates for genomic DNA isolation. However, only a small number of feeder cells were typically transferred to the gelatin-coated plates and the irradiated feeder cells did not proliferate because of radiation-induced AMI5 inhibitor arrest. By contrast, iPS cells proliferate rapidly. We usually did not harvest the iPS cells on gelatin-coated plates for DNA preparation until they reached confluency. Thus, it is unlikely that the carryover feeder cells would comprise a significant portion of the iPS cell culture on gelatin-coated plates before genomic DNA preparation.
The results for allele-specific RT-PCR analysis of both Snrpn and Zim1 imprinted genes are consistent with the DNA methylation analysis of these two ICRs in most iPS clones with a few notable exceptions. The iPS clones with more intact DNA methylation imprint had the tendency to maintain the preferential mono-allelic expression patterns of both imprinted genes. This was true for Snrpn in the iPS cells at early-passage (