Post-fertilization transcription initiation in an ancestral LTR retrotransposon drives lineage-specific genomic imprinting of ZDBF2

  1. Hisato Kobayashi
  2. Tatsushi Igaki
  3. Soichiro Kumamoto
  4. Keisuke Tanaka
  5. Tomoya Takashima
  6. Shunsuke Suzuki
  7. Masaaki Hayashi
  8. Marilyn B. Renfree
  9. Manabu Kawahara
  10. Shun Saito
  11. Toshihiro Kobayashi
  12. Hiroshi Nagashima
  13. Hitomi Matsunari
  14. Kazuaki Nakano
  15. Ayuko Uchikura
  16. Hiroshi Kiyonari
  17. Mari Kaneko
  18. Hiroo Imai
  19. Kazuhiko Nakabayashi
  20. Matthew C. Lorincz
  21. Kazuki Kurimoto

  • Curated by eLife

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    eLife assessment

    The findings in the manuscript are important and the strength of evidences from the genomic analyses is convincing. However, the evidence for the existence of functional MER21B/C remnants in mice, as well as for the imprinting status of Zdbf2 in rabbits and non-human primates was viewed as mainly correlative and incomplete. This manuscript will be of interest to developmental biologists and those working on possible novel mechanisms of gene regulation.

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Abstract

The imprinted ZDBF2 gene is controlled by oocyte-derived DNA methylation, but its regulatory system is quite different from that of other canonically imprinted genes that are dependent on DNA methylation deposited in the gametes. At the ZDBF2 locus, maternal DNA methylation in the imprinted differentially methylated region (DMR) does not persist after implantation. Instead, a transient transcript expressed in the early embryo exclusively from the unmethylated paternal allele of the DMR, known as GPR1-AS , contributes to establishing secondary DMRs that maintain paternal expression of ZDBF2 in the somatic lineage. While the imprinting of ZDBF2 and its unique regulatory system are evident in humans and mice, whether this process is conserved in other mammals has not been addressed. Here, we show that the first exon of human GPR1-AS overlaps with that of a long terminal repeat (LTR) belonging to the MER21C subfamily of retrotransposons. Although this LTR family appears and is amplified in eutherians, the MER21C insertion into the GPR1-AS orthologous region occurred specifically in the common ancestor of Euarchontoglires, a clade that includes primates, rodents, and rabbits. Directional RNA sequencing of placental tissues from various mammalian species revealed GPR1-AS orthologs in rabbits and nonhuman primates, with their first exon embedded within the same ancestral LTR. In contrast, allele-specific expression profiling showed that cow and tammar wallaby, mammals outside the Euarchontoglires group, expressed both alleles in all tissues analyzed. Our previous studies showed that LTRs reactivated in oocytes drive lineage-specific imprinting during mammalian evolution. The data presented here suggest that LTR-derived sequence activation after fertilization can also contribute to the lineage-specific establishment of imprinted genes.

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  1. Version published to 10.7554/elife.94502.1 on eLife
  2. Version published to 10.7554/elife.94502 on eLife
  3. eLife

    eLife assessment

    The findings in the manuscript are important and the strength of evidences from the genomic analyses is convincing. However, the evidence for the existence of functional MER21B/C remnants in mice, as well as for the imprinting status of Zdbf2 in rabbits and non-human primates was viewed as mainly correlative and incomplete. This manuscript will be of interest to developmental biologists and those working on possible novel mechanisms of gene regulation.

  4. eLife

    Reviewer #1 (Public Review):

    Summary:
    The study tests the conservation of imprinting of the ZBDF2 locus across mammals. ZDBF2 is known to be imprinted in mice, humans, and rats. The locus has a unique mechanism of imprinting: although imprinting is conferred by a germline DMR methylated in oocytes, the DMR is upstream to ZDBF2 (at GPR1) and monoallelic methylation of the gDMR does not persist beyond early developmental stages. Instead, a lncRNA (GPR1-AS, also known as Liz in mouse) initiating at the gDMR is expressed transiently in embryos and sets up a secondary DMR (by mechanisms not fully elucidated) that then confers monoallelic expression of ZDBF2 in somatic tissues.

    In this study, the authors first interrogate existing placental RNA-seq datasets from multiple mammalian species, and detect GPR1-AS1 candidate transcripts in humans, baboons, macaques and mice, but not in about a dozen other mammals. Because of the varying depth, quality, and nature of these RNA-seq libraries, the ability to definitely detect the GPR1-AS1 lncRNA is not guaranteed; therefore, they generate their own deep, directional RNA-seq data from tissues/embryos from five species, finding evidence of GPR1-AS in rabbits and chimpanzees, but not bovine animals, pigs or opossums. From these surveys, the authors conclude that the lncRNA is present only in Euarchontoglires mammals. To test the association between GPR1-AS and ZDBF2 imprinting, they perform RT-PCR and sequencing in tissue from wallabies and cattle, finding biallelic expression of ZDBF2 in these species that also lack a detected GPR1-AS transcript. From inspection of the genomic location of the GPR1-AS first exon, the authors identify an overlap with a solo LTR of the MER21C retrotransposon family in those species in which the lncRNA is observed, except for some rodents, including mice. However, they do detect a degree of homology (46%) to the MER21C consensus at the first exon on Liz in mouse. Finally, the authors explore public RNA-seq datasets to show that GPR1-AS is expression transiently during human preimplantation development, an expression dynamic that would be consistent with the induction of monoallelic methylation of a somatic DMR at ZDBF2 and consequent monoallelic expression.

    Strengths:
    -The analysis uncovers a novel mechanism by which a retrotransposon-derived LTR may be involved in genomic imprinting.
    -The genomic analysis is very well executed.
    -New directional and deeply-sequenced RNA-seq datasets from the placenta or the trophectoderm of five mammalian species and marsupial embryos, that will be of value to the community.

    Weaknesses:
    Although the genomic analysis is very strong, the study remains entirely correlative. All of the data are descriptive, and much of the analysis is performed on RNA-seq and other datasets from the public domain; a small amount of primary data is generated by the authors.
    Evidence that the residual LTR in mouse is functionally relevant for Liz lncRNA expression is lacking.

  5. eLife

    Reviewer #2 (Public Review):

    Summary:
    This work concerns the evolution of ZDBF2 imprinting in mammalian species via initiation of GPR1 antisense (AS) transcription from a lineage-specific long-terminal repeat (LTR) retrotransposon. It extends previous work describing the mechanism of ZDBF2 imprinting in mice and humans by demonstrating conservation of GPR1-AS transcripts in rabbits and non-human primates. By identifying the origin of GPR1-AS transcription as the LTR MER21C, the authors claim to account for how imprinting evolved in these species but not in those lacking the MER21C insertion. This illustrates the principle of LTR co-option as a means of evolving new gene regulatory mechanisms, specifically to achieve parent-of-origin allele specific expression (i.e., imprinting). Examples of this phenomenon have been described previously, but usually involve initiation of transcription during gametogenesis rather than post-fertilization, as in this work. The findings of this paper are therefore relevant to biologists studying imprinted genes or interested more generally in the evolution of gene regulatory mechanisms.

    Strengths:
    (1) The authors convincingly demonstrate the existence of GPR1-AS orthologs in specific mammalian lineages using deeply sequenced, stranded, and paired-end RNA-seq libraries collected from diverse mammalian species.

    Weaknesses:
    (1) The authors do not directly demonstrate imprinting of the ZDBF2 locus in rabbits and non-human primates, which would greatly strengthen their model linking ZDBF2 imprinting to transcription from MER21C.

    (2) Experimental evidence linking GPR1-AS transcription to ZDBF2 imprinting in rabbits and non-human primates is currently lacking. Consideration should be given to the challenges associated with studying non-model species and manipulating repeat sequences, which may explain the absence of experimental evidence in this case. Further, this mechanism is established in humans and mice, so the authors' model is arguably sufficiently supported merely by the existence of GPR1-AS orthologs in other mammalian lineages.

  6. eLife

    Reviewer #3 (Public Review):

    Summary:
    Kobayashi et al identify MER21C as a common promoter of GPR1-AS/Liz in Euarchontoglires, which establishes a somatic DMR that controls ZFDB2 imprinting. In mice, MER21C appears to have diverged significantly from its primate counterparts and is no longer annotated as such.

    Strengths:
    The authors used high-quality cross-species RNA-seq data to characterise GPR1-AS-like transcripts, which included generating new data in five different species. The association between MER21C/B elements and the promoter of GPR1-AS in most species is clear and convincing. The expression pattern of MER21C/B elements overall further supports their role in enabling correct temporal expression of GPR1-AS during embryonic development.

    Weaknesses:
    A deeper comparison of syntenic regions to the GPR1-AS promoter could be performed to provide a clearer picture of how the MER21C/B element evolved. The use of alternative TE annotation software may also be helpful. These analyses would be particularly useful to drive home the conclusion that the mouse (Liz) promoter is derived from the same insertion.

  7. Version published to 10.1101/2023.10.30.564869v2 on bioRxiv
  8. Version published to 10.1101/2023.10.30.564869v1 on bioRxiv