Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat-/- mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation. © 2013 McGraw et al

A role of Pumilio 1 in mammalian oocyte maturation and maternal phase of embryogenesis

Abstract Background RNA binding proteins play a pivotal role during the oocyte-to-embryo transition and maternal phase of embryogenesis in invertebrates, but their function in these processes in mammalian systems remain largely understudied. Results Here we report that a member of the Pumilio/FBF family of RNA binding proteins in mice, Pumilio 1 (Pum1), is a maternal effect gene. The absence of maternal PUM1 in the oocyte does not affect meiotic maturation but leads to abnormal preimplantation development. Furthermore, genome-wide transcriptome analysis of oocytes and embryos revealed that there is a concomitant perturbation of the mRNA milieu. Of note, putative PUM1 mRNA targets were equally perturbed as non-direct targets, which indicates that PUM1 regulates the stability of maternal mRNAs both directly and indirectly. We show Cdk1 mRNA, a known PUM1 target essential for meiosis and preimplantation development, is not degraded appropriately during meiosis, leading to an increase in CDK1 protein in mature oocytes, which indicates that PUM1 post-transcriptionally regulates Cdk1 mRNA; this could partially explain the observed abnormal preimplantation development. Furthermore, our results show that maternal and zygotic PUM1 are required for postnatal survival. Conclusions These findings indicate that PUM1 is essential in the process of cytoplasmic maturation and developmental competence of the oocyte. These results reveal an important function of maternal PUM1 as a post-transcriptional regulator during mammalian embryogenesis

Model of the dynamic regulation of methylation maintenance by DNMT1o and establishment of imprinted XCI during preimplantation development.

<p>Wild-type XX: Maternally produced DNMT1o translocates into the nucleus of 8-cell embryos. Nuclear DNMT1o produces a ‘boost’ to maintain methylation marks on DMDs, and sequences on the X chromosome, repeats and other specific sequences. In the blastocyst, a reprogramming and <i>de novo</i> methylation phase takes place in the inner cell mass (ICM) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003873#pgen.1003873-Sado4" target="_blank">[65]</a> and high levels of global genome methylation are observed in this cell lineage. Imprinted XCI is maintained on the Xp in the extraembryonic derivatives, while in the ICM the reprogramming activity reactivates the Xp and random XCI causes inactivation of either the Xm or Xp. <i>Dnmt1o^mat−/−</i> XY and <i>Dnmt1o^mat−/−</i> XX: Lack of DNMT1o at the 8-cell stage prevents the methylation ‘boost’ and causes a failure in the maintenance of methylation marks on DMDs and other sequences including repeat sequences and X-linked genes. This failure of maintenance methylation at the 8-cell stage results in expression from both Xp and Xm in the extraembryonic lineage (relaxation of imprinted XCI). Activation of both X chromosomes in extraembryonic tissues is associated with methylation loss at repeat elements as well as other sequences across the genome. In contrast, the reprogramming event in the ICM restores proper epigenetic patterns and normal random XCI is established in XX embryos. Following the reprogramming and <i>de novo</i> methylation phase, the global DNA methylation levels in the XY and XX cells derived from ICM are similar to wild-type.</p

Early <i>Dnmt1o^mat−/−</i> blastocysts exhibit abnormal methylation of <i>Xite</i> but normal <i>Xist</i> expression.

<p>(A) Perturbed imprinted XCI model following <i>Dnmt1o</i>-deficiency. Lack of DNMT1o activity initiates hypomethylation events on <i>Xite</i> sequences, which activates and sustains the <i>Tsix</i> expression on the paternal X chromosome. This chain of events leads to repression of <i>Xist</i> expression on the paternal X chromosome. (B) Bisulfite cloning and sequencing results in sexed control and <i>Dnmt1o^mat−/−</i> blastocysts for two regions that exhibited hypomethylation in female <i>Dnmt1o^mat−/−</i> extraembryonic tissues (9.5dpc). Each line represents one sequenced allele, and the number at the left indicates the number of clones sequenced for that allele. Filled circles refer to methylated CpG dinucleotides. Percentages of methylated CpGs are shown. (C) Graphs represent the means of methylation percentages obtained for single sexed blastocysts. Mean ± SEM. **p<0.001. (D) Combined confocal microscopy images from RNA-DNA FISH experiments. Localization of <i>Xist</i> RNA (green) marks the inactive X chromosome. Specific X chromosome staining with Dxwas70 (red). Nuclei were counterstained with DAPI (blue).</p

Identified loci that display altered DNA methylation in <i>Dnmt1o^mat−/−</i> embryos and placentae.

a<p>Number of profiles with methylation change for specific loci (total placenta samples : 4XX + 4XY).</p>b<p>Methylation changed in all XX profiles and unchanged in all XY profiles.</p>c<p>Loci methylation changed in all profiles.</p

Morphological phenotypes in 9.5dpc extraembryonic tissues associated with DNMT1o deficiency of <i>Dnmt1o^mat−/−</i> offspring.

<p>(A) Representative examples of normal and hyperplastic extraembryonic tissues (magnification 16×). Scale bar equals 1 mm. (B) Gross morphological assessment of extraembryonic tissues from <i>Dnmt1o^mat+/+</i> (wild-type), <i>Dnmt1o^mat+/−</i> and <i>Dnmt1o^mat−/−</i> females. Extraembryonic hyperplasia: severe (ectoplacental cone encompassing 2/3 of the embryo), mild (3×8 mm to 6×8 mm), normal (3×4 mm). XX <i>Dnmt1o^mat−/−</i> vs XX <i>Dnmt1o^mat+/−</i>, *p<0.001. (C) Sex-specific analysis of imprinted DMDs in 9.5dpc control and <i>Dnmt1o^mat−/−</i>conceptuses. Analysis of imprinted gene DMD methylation using MassARRAY. Mean ± SEM. p<0.05.</p

Map of selected elements within or surrounding the X-inactivation center region.

<p>The <i>Xite</i> (yellow) regulatory element is a positive enhancer of <i>Tsix</i> (green) expression, which is an antisense transcript that represses <i>Xist</i> (red). Numbers below arrows indicate regions amplified for DNA methylation studies. 1- <i>Xist</i>; 2-<i>Tsix</i>-CTCFc; 3- <i>Tsix</i> CGI, (major promoter); 4- <i>Tsix</i> (upstream major promoter); 5- <i>Xite</i>-DHS2; 6- <i>Xite-</i>DHS4, 7- <i>Xite</i>-DHS6 (<i>Tsix</i> minor promoter) and 8- <i>Chic1</i> promoter.</p

DNA methylation of X-CGI and <i>Xic</i> loci in <i>Dnmt1o^mat−/−</i> female placentae.

<p>(A) Analysis of X-CGI and <i>Xic</i> loci in <i>Dnmt1o^mat−/−</i> female placentae using MassARRAY. The positions of 17 CpG islands and 7 regions of the <i>Xic</i> that were analysed are depicted on the left of the figure. Each tick mark equals 10 Mb. Methylation values are displayed relative to the average of the control samples for each gene (blue, hypomethylation; yellow, hypermethylation). Samples are clustered according to their methylation values revealing two distinct groups (green, control; pink, <i>Dnmt1o^mat−/−</i> normal morphology; red, <i>Dnmt1o^mat−/−</i> abnormal morphology). (*p<0.05 control versus <i>Dnmt1o^mat−/−</i>; ^†p<0.05 control versus <i>Dnmt1o^mat−/−</i> abnormal morphology). Loci marked with “<b>#</b>” were identified through RLGS experiments. (B) Histograms displaying the absolute methylation values for <i>Xite</i>-DHS6, <i>Tsix</i>-CTCFc and <i>Xist</i> genes separated into placenta morphology groups (*p<0.05 control versus normal or abnormal <i>Dnmt1o^mat−/−</i>).</p