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

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

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

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

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

Mating experiment.

<p>Both the null <i>Sirh11/Zcchc16</i> KO (–/Y) and <i>Sirh11/Zcchc16</i> homo KO (–/–) mice exhibited normal reproductive ability. The number of pups in each genotype is shown.</p><p>Mating experiment.</p

Characteristics of Sirh11/Zcchc16.

<p>(A) Structure of the Sirh11/Zcchc16 protein. The scheme shows the alignment of the sushi-ichi gag domain and Sirh11/Zcchc16 proteins, which overall exhibit 21% identity and 37.5% similarity, respectively. The zinc finger CCHC domain (purple) in the C-terminus is conserved, but the coiled-coil motif (green) and gag domain (blue) are absent in the Sirh11/Zcchc16 protein. (B) Chromosomal Location. <i>SIRH11/ZCCH16</i> is conserved in an orthologous chromosomal region between <i>TRPC5</i> and <i>LHFPL1</i> in eutherian mammals. The upper panel shows that <i>SIRH11/ZCCHC16</i> is absent in the chicken (birds), platypus (monotremes) and opossum (marsupials). The identically colored boxes represent orthologous genes and the light blue boxes represent <i>psuedoSIRH11/ZCCHC16</i> genes. The lower panel provides a comparison of <i>SIRH11/ZCCHC16</i> and its flanking genome sequences with 50~100% homology to the mouse genome in several eutherian mammals. The purple (<i>SIRH11/ZCCHC16</i>) and red (others) areas indicate evolutionarily conserved sequences (ECSs). The shaded area indicates the regions that correspond to the mouse <i>Sirh11/Zcchc16</i> open reading frame. The yellow line in the Armadillo column represents the gap region in the genome sequence. (C) Conservation of <i>Sirh11/Zcchc16</i> in eutherian mammals. The <i>SIRH11/ZCCHC16</i> sequence was confirmed in four major eutherian groups (red), euarchontoglires, laurasiatheria, xenarthra and afrotheria, but became a pseudogene in xenarthra (the dashed line), indicating that the insertion of <i>SIRH11/ZCCHC16</i> occurred in a common eutherian ancestor. The number of species possessing <i>SIRH11/ZCCHC16</i> (front) and those which in total were analyzed (back) are noted in parentheses. (D) PAML analysis. Two models, the one-ratio model based on the assumption that ω₁ (dN/dS) is the same for all of the branches (model 1) and the two-ratio model based on the assumption that ω₂ in the xenathran lineage is different from ω₁ in all the others (model 2) were compared. The model 2 was statistically significant and is shown. The <i>p</i>-value was calculated by the likelihood ratio test. Armadillo 1 and 2 represent <i>Dasypus novemcinctus</i> and <i>Tolypeutes matacus</i>, respectively. Sloth 1 and 2 represent <i>Choloepus hoffmanni</i> and <i>Choloepus didactylus</i>, respectively.</p

Expression profile of mouse <i>Sirh11/Zcchc16</i> mRNA.

<p>(A) Exon-intron structure of the <i>Sirh11/Zcchc16</i> gene. The full-length cDNA sequence was identified by 5'- and 3'-RACE (Rapid Amplification of cDNA Ends) experiments using brain RNA at 8 w. The <i>Sirh11/Zcchc16</i> gene consists of 7 exons and the protein coding sequence is in exon 7. The identified cDNA sequence corresponds to Genbank Accession No. NM_001033795.4. The white and black boxes represent the exons and ORF, respectively. (B) <i>Sirh11</i> expression in fetuses and adults. qRT-PCR analyses were carried out using C57BL/6J cDNA from various tissues and organs at d14.5 and 8 w. The black and white bars indicate males and females, respectively. Data represents the mean ± S. D. (N = 3 each). <i>Sirh11/Zcchc16</i> expression was observed in the brain, testis, ovary and kidney, but not in the placenta. qRT-PCR primers (F9R9) were designed in the 3’ UTR region of <i>Sirh11/Zcchc16</i> mRNA (see arrows in Fig 2A). (C) Overexpression of the <i>Sirh11/Zcchc16</i> transcript without any ORF in KO mice. The graph shows the <i>Sirh11/Zcchc16</i> expression levels at 8 weeks of age using F9R9 (3’ UTR) primer sets relative to <i>Actb</i> mRNA in the male brain, kidney and testis. The white and black bars represent WT and KO, respectively. Each data point represents the mean ± S. D. (N = 4 each). The asterisks indicate significant differences between the WT and KO mice (**: p < 0.01).</p