Identification and characterization of ERV transcripts in goat embryos

in Reproduction
Correspondence should be addressed to Y Zhang or J Liu; Email: zhy1956@263.net or liujun2013@nwsuaf.edu.cn

*(R Deng and C Han contributed equally to this work)

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Endogenous retroviruses (ERVs), which are abundant in mammalian genomes, can modulate the expression of nearby genes, and their expression is dynamic and stage-specific during early embryonic development in mice and humans. However, the functions and mechanisms of ERV elements in regulating embryonic development remain unclear. Here, we utilized several methods to determine the contribution of ERVs to the makeup and regulation of transcripts during embryonic genome activation (EGA). We constructed an ERV library and embryo RNA-seq library (IVF_2c and IVF_8c) of goat to serve as our research basis. The GO and KEGG analysis of nearby ERV genes revealed that some ERV elements may be associated with embryonic development. RNA-seq results were consistent with the features of EGA. To obtain the transcripts derived from the ERV sequences, we blasted the ERV sequences with embryonic transcripts and identified three lncRNAs and one mRNA that were highly expressed in IVF-8c rather than in IVF-2c (q-value <0.05). Then, we validated the expression patterns of nine ERV-related transcripts during early developmental stages and knocked down three high-expression transcripts in EGA. The knockdown of lncRNA TCONS_00460156 or mRNA HSD17B11 significantly decreased the developmental rate of IVF embryos. Our findings suggested that some transcripts from ERVs are essential for the early embryonic development of goat, and analyzing the ERV expression profile during goat EGA may help elucidate the molecular mechanisms of ERV in regulating embryonic development.

Downloadable materials

  • Supplementary Table 1 Information: 645 intact ERV sequences using LTRharvest and MGEScan-LTR methods. Tables containing all intact ERV sequences which were obtained by LTRharvest and MGEScan-LTR methods ( see Methods for details).
  • Supplementary Table 2 Information: LTR length and position of 645 potential complete ERV. Table containing information of LTR position in ERV. Each ERV has 5’LTR and 3’LTR. LTR_id: ERV number, chr: chromosome which ERV located, 5’LTR_start: the starting position of 5’LTR in chromosome, 5’LTR_end: the ending position of 5’LTR in chromosome, 3’LTR_start: the starting position of 3’LTR in chromosome, 3’LTR_end: the ending position of 3’LTR in chromosome, 5’LTR_length = 5’LTR_end - 5’LTR_start, 3’LTR_length = 3’LTR_end - 3’LTR_start, length: ERV overall length.
  • Supplementary Table 3 Information: The postion of RT domain of ERV sequence. Table illustrates the information of the RT domain of ERV sequence. Annotated methods: using different methods to annotated different ERVs. Structure name: RT domains which different ERVs contain. Start positions: RT domain starting position in chromosome. End positions: RT domain ending position in chromosome.
  • Supplementary Table 4 Information: Functional enrichment analysis of the upstream–downstream genes of ERVs within 10 kb. Table containing GO and KEGG information of different ERVs nearby genes, which can provide some indications for readers about different ERVs.
  • Supplementary Table 5 Information: Summary of RNA-seq data and reads mapped to Capra hircus reference genome. A table summarizing the raw data and clean data quality of our samples(see Methods for details of how gene reads were determined from RNA-seq data).
  • Supplementary Table 6 Information: FPKM scores for the lncRNAs and the mRNAs in IVF-2c and IVF-8c. The table containing all genes analysed using Cuffdiff ( see Methods for details).
  • Supplementary Table 7 Information: List of differentially expressed lncRNAs and mRNAs from IVF-2c and IVF-8c. Tables containing differentially expressed genes between IVF-2cell and IVF-8cell. The significance is determined by qvalue < 0.05.
  • Supplementary Table 8 Information: ERV-derived transcripts based on Blastall which was used to bidirectionally blast ERV library and RNA-seq library. Tables containing information of ERV-derived lncRNA and mRNA, including their ERVs and transcripts position in chromosome.
  • Supplementary Table 9 Information: Differentially expressed ERV-derived transcripts in goat IVF-2c and IVF-8c. Tables containing differentially expressed genes between IVF-2cell and IVF-8cell. The significance is determined by qvalue < 0.05.

 

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    Overview of ERV-derived transcripts pipeline. Left: identification pipeline for potentially complete ERVs. Using LTRharvest, we identified 64,159 unique LTR candidates within LTR and TSD and annotated the candidates. In addition, we used the other pipeline MGEScan-LTR to identify known complete ERVs. By combining these two pipelines, we obtained 645 potential complete ERVs. All ERVs were classified into 18 families on the basis of the polymorphisms of the RT genes and the similarity of the exogenous retrovirus. Right: lncRNA and mRNA discovery pipeline for RNA-Seq. Bottom, ERV-derived transcripts. We used Blastall to blast goat lncRNA and mRNA with potentially complete ERVs.

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    Comprehensive analysis of ERV involvement and chromosome distribution. (A) Phylogenetic tree of goat ERVs constructed by analyzing the polymorphisms of reverse transcriptase. Class III was flanked by classes I and II, and different families with different colors and bootstrap values are shown above the branches with neighbor-joining bootstrap values of >70%. MMTV, mouse mammary tumor virus; GH-G18, Golden Hamster intracisternal A-partide H18; BLV, bovine leukemia virus; HFV, human endogenous retrovirus; ZFERV, zebrafish endogenous retrovirus; FELV, feline leukemia virus; MuLV, murine leukemia virus; MDEV, Mus dunni endogenous virus; GaLV, gibbon ape leukemia virus; BaEV, baboon endogenous virus; KoRV, koala retrovirus; SnRV, snakehead fish retrovirus; SRV-1, Simian type-D retrovirus-1; JSRV, Jaagsiekte sheep retrovirus; HML, human MMTV-like; WDSV, walleye dermal sarcoma virus; HIV-1, human immunodeficiency virus; ALV, avian leukemia virus. (B) Distribution of different classes of ERVs in the chromosome. Each chromosome compromises a similar percentage of different classes except for the Y chromosome, which can only be found in class I. (C) ERV sequence structure feature display. Gag, pol, and pro represent the ERV repeat region.

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    Enrichment analysis of ERV elements in nearby genes. The input gene number is small, and the significance is determined by P < 0.05. (A) GO analysis of the target of ERV upstream–downstream elements within 10 kb. (B) KEGG pathway analysis of the target of ERVs upstream–downstream elements within 10 kb.

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    RNA-seq results of IVF-two-cell and IVF-eight-cell embryos. (A) FPKM distributions of lncRNA and mRNA. (B) Volcano plot. Blue dots present upregulated transcripts, red dots denote downregulated transcripts, and green dots indicate no significant difference. (C) Hierarchical clustering analysis of 743 differentially expressed lncRNAs and mRNAs between IVF-2c and IVF-8C. Data are expressed as FPKM. Red: relatively high expression. Blue: relatively low expression.

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    Expression profile of transcripts. β-Actin functioned as the control. The error bars represent s.e.m. Thirty embryos from each stage were used, and three experimental replications were performed. GFF, goat fetal fibroblast; MII, metaphase II; 2C, two-cell; 8C, eight-cell; MO, morula; BL, blastocyst.

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    Function of ERV-derived transcripts in the goat embryonic development. (A) SiRNA and Smart Silencer efficiently mediated the mRNA and lncRNA knockdown, respectively, and decreased the mRNA expression of CHD1L, which is the target gene of TCONS_00460156. Embryos were collected at the eight-cell stage for reverse-transcription qPCR analysis. Error bars represent s.e.m. Thirty embryos from each stage were used in each group. (B) Embryos injected with the control SiRNA were successfully developed to hatched blastocyst stage. SiRNA-depleted embryos arrested at the eight-cell stage. Scale bar, 100 μm. A minimum of three experimental replications were performed. (C) Effects of TCONS_00460156 and HSD17B11 knockdown on the development of embryos to the blastocyst stage. (D) Knockdown of HSD17B11 and TCONS_00460156 considerably decreased the protein levels of HSD17B11 and CHD1L, respectively. Embryos were collected at the eight-cell stage for Western blot analysis. Approximately 100 eight-cell embryos were used in each group for GAPDH.

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