In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. Imprinting is linked to therian reproduction, that is, the placenta and imprinting emerged at roughly the same time and potentially co-evolved. We assessed the transcriptome-wide and ontology effect of maternally versus paternally methylated ICRs at the developmental stage of setting of the chorioallantoic placenta in the mouse (8.5dpc), using two models of imprinting deficiency including completely imprint-free embryos. Paternal and maternal imprints have a similar quantitative impact on the embryonic transcriptome. However, transcriptional effects of maternal ICRs are qualitatively focused on the fetal-maternal interface, while paternal ICRs weakly affect non-convergent biological processes, with little consequence for viability at 8.5dpc. Moreover, genes regulated by maternal ICRs indirectly influence genes regulated by paternal ICRs, while the reverse is not observed. The functional dominance of maternal imprints over early embryonic development is potentially linked to selection pressures favoring methylation-dependent control of maternal over paternal ICRs. We previously hypothesized that the different methylation histories of ICRs in the maternal versus the paternal germlines may have put paternal ICRs under higher mutational pressure to lose CpGs by deamination. Using comparative genomics of 17 extant mammalian species, we show here that, while ICRs in general have been constrained to maintain more CpGs than non-imprinted sequences, the rate of CpG loss at paternal ICRs has indeed been higher than at maternal ICRs during evolution. In fact, maternal ICRs, which have the characteristics of CpG-rich promoters, have gained CpGs compared to non-imprinted CpG-rich promoters. Thus, the numerical and, during early embryonic development, functional dominance of maternal ICRs can be explained as the consequence of two orthogonal evolutionary forces: pressure to tightly regulate genes affecting the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline.

X chromosome inactivation is controlled by the cis-acting X-inactivation centre (Xic). In addition to initiating inactivation, Xic, which includes the XIST: gene, is involved in both a counting process that senses the number of X chromosomes and the choice of X chromosome to inactivate. Controlling elements lying 3' to XIST: include the DXPas34 locus. Deletion of DXPas34 in undifferentiated embryonic stem (ES) cells eliminates expression of both XIST: and the antisense transcript TSIX:, thought to initiate from a CpG island lying close to, but telomeric to, the DXPas34 locus itself. Deletion of DXPas34 leads to non-random inactivation on ES cell differentiation and disrupts imprinted X-inactivation in vivo. In order to investigate the role of methylation at DXPas34 in the initial steps of X-inactivation, we studied its methylation status during pre- and post-implantation embryonic development and ES cell differentiation, using the bisulphite sequencing technique. Analysis of the methylation status of both the DXPas34 locus and the associated downstream CpG island shows that extensive hypermethylation of the DXPas34 locus is a relatively late event in differentiation and embryogenesis. We conclude that methylation of DXPas34 cannot be the X chromosome imprint, nor can it be involved in the parent-of-origin effects associated with deletion of the DXPas34 locus and the neighbouring CpG island.

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We have sequenced to high levels of accuracy 714-kb and 233-kb regions of the mouse and bovine X-inactivation centers (Xic), respectively, centered on the Xist gene. This has provided the basis for a fully annotated comparative analysis of the mouse Xic with the 2.3-Mb orthologous region in human and has allowed a three-way species comparison of the core central region, including theXist gene. These comparisons have revealed conserved genes, both coding and noncoding, conserved CpG islands and, more surprisingly, conserved pseudogenes. The distribution of repeated elements, especially LINE repeats, in the mouse Xic region when compared to the rest of the genome does not support the hypothesis of a role for these repeat elements in the spreading of X inactivation. Interestingly, an asymmetric distribution of LINE elements on the two DNA strands was observed in the three species, not only within introns but also in intergenic regions. This feature is suggestive of important transcriptional activity within these intergenic regions. In silico prediction followed by experimental analysis has allowed four new genes, Cnbp2, Ftx, Jpx, and Ppnx, to be identified and novel, widespread, complex, and apparently noncoding transcriptional activity to be characterized in a region 5′ of Xist that was recently shown to attract histone modification early after the onset of X inactivation.[The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AJ421478, AJ421479, AJ421480, andAJ421481. Online supplemental data are available athttp://pbil.univ-lyon1.fr/datasets/Xic2002/data.html andwww.genome.org.]