Characterisation of marsupial PHLDA2 reveals eutherian specific acquisition of imprinting

<p>Abstract</p> <p>Background</p> <p>Genomic imprinting causes parent-of-origin specific gene expression by differential epigenetic modifications between two parental genomes. We previously reported that there is no evidence of genomic imprinting of <it>CDKN1C </it>in the <it>KCNQ1 </it>domain in the placenta of an Australian marsupial, the tammar wallaby (<it>Macropus eugenii</it>) whereas tammar <it>IGF2 </it>and <it>H19</it>, located adjacent to the <it>KCNQ1 </it>domain in eutherian mammals, are imprinted. We have now identified and characterised the marsupial orthologue of <it>PHLDA2</it>, another gene in the <it>KCNQ1 </it>domain (also known as <it>IPL </it>or <it>TSSC3</it>) that is imprinted in eutherians. In mice, <it>Phlda2 </it>is a dose-sensitive negative regulator of placental growth, as <it>Cdkn1c </it>is for embryonic growth.</p> <p>Results</p> <p>Tammar <it>PHLDA2 </it>is highly expressed in the yolk sac placenta compared to other fetal tissues, confirming a similar expression pattern to that of mouse <it>Phlda2</it>. However, tammar <it>PHLDA2 </it>is biallelically expressed in both the fetus and yolk sac placenta, so it is not imprinted. The lack of imprinting in tammar <it>PHLDA2 </it>suggests that the acquisition of genomic imprinting of the <it>KCNQ1 </it>domain in eutherian mammals, accompanied with gene dosage reduction, occurred after the split of the therian mammals into the marsupials and eutherians.</p> <p>Conclusions</p> <p>Our results confirm the idea that acquisition of genomic imprinting in the <it>KCNQ1 </it>domain occurred specifically in the eutherian lineage after the divergence of marsupials, even though imprinting of the adjacent <it>IGF2-H19 </it>domain arose before the marsupial-eutherian split. These data are consistent with the hypothesis that genomic imprinting of the <it>KCNQ1 </it>domain may have contributed to the evolution of more complex placentation in the eutherian lineage by reduction of the gene dosage of negative regulators for both embryonic and placental growth.</p

Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation

Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes

Ono, R., Ishii, M., Fujihara, Y. et al. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes. Sci Rep 5, 12281 (2015). https://doi.org/10.1038/srep1228

A trans-homologue interaction between reciprocally imprinted miR-127 and Rtl1 regulates placenta development.

The paternally expressed imprinted retrotransposon-like 1 (Rtl1) is a retrotransposon-derived gene that has evolved a function in eutherian placentation. Seven miRNAs, including miR-127, are processed from a maternally expressed antisense Rtl1 transcript (Rtl1as) and regulate Rtl1 levels through RNAi-mediated post-transcriptional degradation. To determine the relative functional role of Rtl1as miRNAs in Rtl1 dosage, we generated a mouse specifically deleted for miR-127. The miR-127 knockout mice exhibit placentomegaly with specific defects within the labyrinthine zone involved in maternal-fetal nutrient transfer. Although fetal weight is unaltered, specific Rtl1 transcripts and protein levels are increased in both the fetus and placenta. Phenotypic analysis of single (ΔmiR-127/Rtl1 or miR-127/ΔRtl1) and double (ΔmiR-127/ΔRtl1) heterozygous miR-127- and Rtl1-deficient mice indicate that Rtl1 is the main target gene of miR-127 in placental development. Our results demonstrate that miR-127 is an essential regulator of Rtl1, mediated by a trans-homologue interaction between reciprocally imprinted genes on the maternally and paternally inherited chromosomes.This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) and Medical Research Council (MRC) and EU FP7 Marie Curie Action 290123 (INGENIUM). This work was partly funded by a National Health and Medical Research Council (NHMRC) CJ Martin Biomedical Fellowship to A.N.S.P.This is the accepted manuscript. The final version is available at http://dev.biologists.org/content/early/2015/07/01/dev.121996.abstract

The origin and evolution of genomic imprinting and viviparity in mammals

Genomic imprinting is widespread in eutherian mammals. Marsupial mammals also have genomic imprinting, but in fewer loci. It has long been thought that genomic imprinting is somehow related to placentation and/or viviparity in mammals, although neither is restricted to mammals. Most imprinted genes are expressed in the placenta. There is no evidence for genomic imprinting in the egg-laying monotreme mammals, despite their short-lived placenta that transfers nutrients from mother to embryo. Post natal genomic imprinting also occurs, especially in the brain. However, little attention has been paid to the primary source of nutrition in the neonate in all mammals, the mammary gland. Differentially methylated regions (DMRs) play an important role as imprinting control centres in each imprinted region which usually comprises both paternally and maternally expressed genes (PEGs and MEGs). The DMR is established in the male or female germline (the gDMR). Comprehensive comparative genome studies demonstrated that two imprinted regions, PEG10 and IGF2-H19, are conserved in both marsupials and eutherians and that PEG10 and H19 DMRs emerged in the therian ancestor at least 160 Ma, indicating the ancestral origin of genomic imprinting during therian mammal evolution. Importantly, these regions are known to be deeply involved in placental and embryonic growth. It appears that most maternal gDMRs are always associated with imprinting in eutherian mammals, but emerged at differing times during mammalian evolution. Thus, genomic imprinting could evolve from a defence mechanism against transposable elements that depended on DNA methylation established in germ cells

Retrotransposon Silencing by DNA Methylation Can Drive Mammalian Genomic Imprinting

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation

Study on the usefulness of Meg1Crb10 transgenic mouse as aType2 diabetes mellitus model animal. -Analysis of blood plasma component and expression of genes related to onset of diabetes -

Meg1/Grb10遺伝子導入マウス（Meg1マウス）はインスリンのシグナル伝達阻害による高インスリン血症を呈することから2型糖尿病モデルと考えられている。Meg1マウスは肥満を伴なわずに高血糖を発症するが､脂肪･カロリーの過剰摂取によっても糖尿病の発症が著しく増加する｡本研究はMeg1マウスの2型糖尿病モデルとしての有用性を検討するために､Meg1マウスと対照マウスを高脂肪･高カロリー飼料（HFD）及び対照飼料（NFD）で飼育した時の血漿アディポネクチン量とBMI値を比較するとともに糖尿病関連遺伝子の発現量について他の糖尿病モデルマウスと比較検討した。血漿アディポネクチン量はMeg1マウスのHFDが最も高く､対照マウスのNFDが最も低い値を示した。一方､BMI値は対照マウスのHFDが最も高い値を示し､血漿アディポネクチン量とBMI値は逆相関が認められ､ヒト2型糖尿病と類似することが認められた。また､Grb10､Glut4遺伝子の発現量はMeg1マウスと他の糖尿病モデルマウスでは異なる値を示し､Meg1マウスでのGrb10遺伝子の発現量は高く､Glut4遺伝子の発現量は低かった。以上のことから､Meg1マウスには他の糖尿病モデルと異なる発症機構の存在が示唆され､Meg1マウスは2型糖尿病モデルとしての有用性が考えられた

Analysis of plasma components related to onset of diabetes in Meg1/Grb10 transgenic mice

2型糖尿病は肥満から糖尿病を発症すると考えられており､Meg1マウスは通常飼料では肥満を伴わないが､高カロリー飼料を摂取することによりグルコース代謝の異常によって糖尿病を発症する｡Meg1/Grb10遺伝子導入マウス(Meg1マウス)はインスリンのシグナル伝達阻害によるインスリン抵抗性を呈することから2型糖尿病モデルと考えられている｡本研究は､糖尿病発症機序の解明を目的として､Meg1/Grb10遺伝子導入マウス(Meg1マウス､TG+)とコントロールのマウス(Meg1マウスの親系統であるC57BL/6N;TG-)の雄を用いて高カロリー･高脂肪飼料を給餌した場合のグルコース代謝におけるシグナル伝達阻害の影響について血漿成分の面から検討した｡その結果､血漿中のIGF､BUN値はいずれもTG+がTG-より有意に低く､一方､中性脂肪量はTG+ がTG-より有意に高かった｡Meglマウスで認められたインスリン値の増加とIGF-1値の減少はインスリン伝達経路においてGrb10がインスリン受容体への結合を阻害していることの証明といえる｡Meg1マウスにおける高インスリン血症はインスリン抵抗性の結果と考えられる

Effect of High-Fat and High-Calorie Diets on the Onset of Diabetes in Meg1/Grb10 Transgenic Mice

Meg1/Grb10遺伝子導入マウスはインスリンのシグナル伝達阻害による高インスリン血症を呈することから､II型糖尿病モデルと考えられている｡そこで､本モデルマウスを用いてII型糖尿病発症に及ぼす飼料の影響について検討した結果､ヒトII型糖尿病モデルとして有用性を確認することが出来た

The Evolution of Mammalian Genomic Imprinting Was Accompanied by the Acquisition of Novel CpG Islands

Parent-of-origin–dependent expression of imprinted genes is mostly associated with allele-specific DNA methylation of the CpG islands (CGIs) called germ line differentially methylated regions (gDMRs). Although the essential role of gDMRs for genomic imprinting has been well established, little is known about how they evolved. In several imprinted loci, the CGIs forming gDMRs may have emerged with the insertion of a retrotransposon or retrogene. To examine the generality of the hypothesis that the CGIs forming gDMRs were novel CGIs recently acquired during mammalian evolution, we reviewed the time of novel CGI emergence for all the maternal gDMR loci using the novel data analyzed in this study combined with the data from previous reports. The comparative sequence analyses using mouse, human, dog, cow, elephant, tammar, opossum, platypus, and chicken genomic sequences were carried out for Peg13, Meg1/Grb10, Plagl1/Zac1, Gnas, and Slc38a4 imprinted loci to obtain comprehensive results. The combined data showed that emergence of novel CGIs occurred universally in the maternal gDMR loci at various time points during mammalian evolution. Furthermore, the analysis of Meg1/Grb10 locus provided evidence that gradual base pair–wise sequence change was involved in the accumulation of CpG sequence, suggesting the mechanism of novel CGI emergence is more complex than the suggestion that CpG sequences originated solely by insertion of CpG-rich transposable elements. We propose that acquisition of novel CGIs was a key genomic change for the evolution of imprinting and that it usually occurred in the maternal gDMR loci