Identification of an Imprinted Gene Cluster in the X-Inactivation Center

<div><p>Mammalian development is strongly influenced by the epigenetic phenomenon called genomic imprinting, in which either the paternal or the maternal allele of imprinted genes is expressed. Paternally expressed <i>Xist</i>, an imprinted gene, has been considered as a single <i>cis</i>-acting factor to inactivate the paternally inherited X chromosome (Xp) in preimplantation mouse embryos. This means that X-chromosome inactivation also entails gene imprinting at a very early developmental stage. However, the precise mechanism of imprinted X-chromosome inactivation remains unknown and there is little information about imprinted genes on X chromosomes. In this study, we examined whether there are other imprinted genes than <i>Xist</i> expressed from the inactive paternal X chromosome and expressed in female embryos at the preimplantation stage. We focused on small RNAs and compared their expression patterns between sexes by tagging the female X chromosome with green fluorescent protein. As a result, we identified two micro (mi)RNAs–miR-374-5p and miR-421-3p–mapped adjacent to <i>Xist</i> that were predominantly expressed in female blastocysts. Allelic expression analysis revealed that these miRNAs were indeed imprinted and expressed from the Xp. Further analysis of the imprinting status of adjacent locus led to the discovery of a large cluster of imprinted genes expressed from the Xp: <i>Jpx</i>, <i>Ftx</i> and <i>Zcchc13</i>. To our knowledge, this is the first identified cluster of imprinted genes in the <i>cis</i>-acting regulatory region termed the X-inactivation center. This finding may help in understanding the molecular mechanisms regulating imprinted X-chromosome inactivation during early mammalian development.</p></div

Abnormal behavior in the <i>Sirh11/Zcchc16</i> KO mice.

<p><b>(</b>A) Light/Dark transition test. The left panel shows the latency time before entering into the light chamber. The right panel shows the number of transitions. The white and black bars represent WT and KO, respectively. Each data point represents the mean ± S. E. M. (N = 7 each). The asterisks indicate significant differences between the male WT and KO mice (*: p < 0.05). <b>(</b>B) Home-cage activity test. Upper: The plots show the activity counts every hour over 5 days. The white and grey areas indicate the light and dark phases, respectively. Middle: The white and black bars represent the activity counts in the WT and KO, respectively (mean ± S. D. (N = 7 each)). Zeitgeiber time (ZT) is shown on the x-axis. The asterisks indicate significant differences between the male WT and KO mice (**: p < 0.01, *: p < 0.05). Lower: the table shows the p-values of the two-way ANOVA at each ZT. The yellow columns indicate a significant difference in genotype (p < 0.05). (C) Y-maze test. Left: each plot shows the percentage of alternation behavior. Right: each plot shows the number of total arm entries. The white and black plots represent the WT and KO, respectively. The asterisk indicates a significant difference between the male WT (N = 6) and KO (N = 8) mice (*: p < 0.05).</p

Cognitive Function Related to the <i>Sirh11/Zcchc16</i> Gene Acquired from an LTR Retrotransposon in Eutherians

<div><p>Gene targeting of mouse <u><i>S</i></u><i>ushi-</i><u><i>i</i></u><i>chi-related</i><u><i>r</i></u><i>etrotransposon</i><u><i>h</i></u><i>omologue</i><u><i>11</i></u><i>/</i><u><i>Z</i></u><i>inc finger</i><u><i>CCHC</i></u><i>domain-containing</i><u><i>16</i></u> (<i>Sirh11/Zcchc16</i>) causes abnormal behaviors related to cognition, including attention, impulsivity and working memory. <i>Sirh11/Zcchc16</i> encodes a CCHC type of zinc-finger protein that exhibits high homology to an LTR retrotransposon Gag protein. Upon microdialysis analysis of the prefrontal cortex region, the recovery rate of noradrenaline (NA) was reduced compared with dopamine (DA) after perfusion of high potassium-containing artificial cerebrospinal fluid in knockout (KO) mice. These data indicate that <i>Sirh11/Zcchc16</i> is involved in cognitive function in the brain, possibly via the noradrenergic system, in the contemporary mouse developmental systems. Interestingly, it is highly conserved in three out of the four major groups of the eutherians, euarchontoglires, laurasiatheria and afrotheria, but is heavily mutated in xenarthran species such as the sloth and armadillo, suggesting that it has contributed to brain evolution in the three major eutherian lineages, including humans and mice. <i>Sirh11/Zcchc16</i> is the first <i>SIRH</i> gene to be involved in brain function, instead of just the placenta, as seen in the case of <i>Peg10</i>, <i>Peg11/Rtl1</i> and <i>Sirh7/Ldoc1</i>.</p></div

Abnormality in brain of <i>Sirh11/Zcchc16</i> KO mice.

<p>(A) Microdialysis analysis in the prefrontal cortex in the cerebrum. The levels of various monoamines, including DA, NA, 3-MT and DOPAC were measured after perfusion of high potassium-containing artificial cerebrospinal fluid in the prefrontal cortex. Each plot shows the ratio of the DA metabolites, NA, 3-MT and DOPAC, to DA. The asterisk indicates a significant difference between the WT and KO mice (*: p < 0.05). The three lines on the plots indicate the mean ± S. E. M. (B) <i>Dbh</i> mRNA expression in each part of the brain. The white and black bars represent the relative expression levels of <i>Dbh</i> to <i>Actb</i> mRNA in WT and KO, respectively (mean ± S. D., N = 4 each). The asterisk indicates a significant difference between the WT and KO (*: p < 0.05). The expression levels in the mesencephalon and brainstem are shown in a separate figure with a different scale. <b>(</b>C) <i>Sirh11</i> mRNA expression in each part of the brain. The white and black bars represent the relative expression levels of <i>Sirh11</i> (3’ UTR) to <i>Actb</i> mRNA in the WT and KO mice, respectively (mean ± S. D., N = 4 each). The asterisk indicates a significant difference between the WT and KO mice (*: p < 0.05). (D) Negative correlation between <i>Sirh11/Zcchc16</i> and <i>Dbh</i> mRNA expression levels in the brainstem. The plots show the relative expression levels of <i>Sirh11</i> (x-axis) and <i>Dbh</i> (y-axis) to <i>Actb</i> mRNA in the brainstem. The white, black and grey circles indicate the WT, KO and B6, respectively. The Pearson correlation coefficient (r) is shown in the plots. The p-value was calculated by the test for non-correlation.</p

Identification of a cluster of imprinted genes in the X-inactivation center (Xic).

<p>A newly identified cluster of imprinted genes on the X chromosome is shown in the mouse genome (UCSC mm9; <a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>). This cluster is located within the Xic. The arrows show the direction of each transcription unit. <i>Ftx</i> and Z<i>cchc13</i> are transcribed in opposite directions from the same putative bidirectional promoter. Imprinted genes are shown by the thick black arrows; <i>Xist, Jpx, Ftx, Zcchc13</i>, miR-374-5p and miR-421-3p were paternally expressed, while <i>Tsix</i> was reported previously to be maternally expressed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222-Lee1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222-Sado1" target="_blank">[29]</a>. The thick black bar indicates the position of the Tg53 (460 kb) transgene reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222-Okamoto2" target="_blank">[35]</a>.</p

Verification of imprinting of two miRNAs predominantly expressed in female embryos.

<p>(A) Schematic representation of two mature miRNAs, miR-374-5p and miR-421-3p and their preceding transcript pri-miRNA. Both miRNAs were clustered and located in the intron of the <i>Ftx</i> (B230206F22Rik) gene on the X chromosome. Both are thought to be transcribed as pri-miRNAs and then processed to form mature miRNAs. The location of the primers used to assess the pri-miRNA and <i>Ftx</i> expression levels are indicated: qRT–PCR primers 207 and 208 were used for <i>Ftx</i>; primers used for allelic discrimination were 057 and 058 for pri-miRNA, 116 and 117 for <i>Ftx</i>. The exons for <i>Ftx</i> are numbered according to a previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222-Chureau1" target="_blank">[22]</a>. DNA polymorphisms used for allelic discrimination are indicated with red characters. PCR primers correspond to the list in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222.s004" target="_blank">Table S3</a> (B) Production of mature miRNAs from pre-miRNAs in female blastocysts. Although two mature forms of miRNA, miR-374-5p/miR-374-3p and miR-421-5p/miR-421-3p are registered in miRBase, only miR-374-5p and miR-421-3p were detected at the blastocyst stage. Orange coloring shows mismatch sequences compared with the reference sequences, suggesting miRNA editing or misreading by the 454 sequencer. (C) Expression of pri-miRNA in wild-type male and female blastocysts obtained from the uterus. Female-predominant expression was observed as well as previously reported X-linked imprinted genes such as <i>Xist and Rhox5</i>. (D) Verification of imprinting of the pri-miRNA for miR-374-5p and miR-421-3p in preimplantation mouse embryos. Pri-miRNA was expressed from the paternal B6 allele in JF1× C57BL/6 crosses and from the paternal JF1 allele in the reciprocal B6× (JF1× B6) cross. The arrows indicate the polymorphism sites that were used in this assay.</p

The dN/dS ratio between the mouse and the seven other eutherian species expect xenarthral.

<p>Pairwise dN/dS analysis was performed using PAML [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005521#pgen.1005521.ref031" target="_blank">31</a>].</p><p>The dN/dS ratio between the mouse and the seven other eutherian species expect xenarthral.</p

List of differentially expressed small RNAs between male and female blastocysts (2).

†<p>Small RNAs showing more than a 10-fold (1000%) change in sequence reads are listed. Small RNAs showing fewer than 20 reads have been excluded from this list.</p

Classification of annotated small RNAs in male and female blastocysts.

*<p>The number of genes mapped is not always equal to the “Total ncRNA” plus “Only genomic” counts because of multiple annotations for single read sequences.</p><p>Abbreviations: miRNA_mature, micro RNA mature form; Mt_tRNA, mitochondrial transfer RNA; Mt_rRNA, mitochondrial ribosomal RNA; snRNA, small nuclear RNA; rRNA, ribosomal RNA; snoRNA, small nucleolar RNA; miscRNA, miscellaneous RNA; lincRNA, large intergenic noncoding RNA; miRNA_hairpin: micro RNA stem-loop sequence obtained from miRBase.</p>**<p>The sequences classified as “Only genomic” were further annotated against RepeatMasker and the tRNA database. The results are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071222#pone.0071222.s002" target="_blank">Table S1</a>.</p

Postnatal growth.

<p><i>Sirh11/Zcchc16</i> KO mice exhibited normal growth. The mean body weight (grams) in the male mice is shown.</p><p>Postnatal growth.</p