A screen for retrotransposed imprinted genes reveals an association between X chromosome homology and maternal germ-line methylation

Abstract

Imprinted genes undergo epigenetic modifications during gametogenesis, which lead to transcriptional silencing of either the maternally or the paternally derived allele in the subsequent generation. Previous work has suggested an association between imprinting and the products of retrotransposition, but the nature of this link is not well defined. In the mouse, three imprinted genes have been described that originated by retrotransposition and overlap CpG islands which undergo methylation during oogenesis. Nap1l5, U2af1-rs1, and Inpp5f_v2 are likely to encode proteins and share two additional genetic properties: they are located within introns of host transcripts and are derived from parental genes on the X chromosome. Using these sequence features alone, we identified Mcts2, a novel candidate imprinted retrogene on mouse Chromosome 2. Mcts2 has been validated as imprinted by demonstrating that it is paternally expressed and undergoes promoter methylation during oogenesis. The orthologous human retrogenes NAP1L5, INPP5F_V2, and MCTS2 are also shown to be paternally expressed, thus delineating novel imprinted loci on human Chromosomes 4, 10, and 20. The striking correlation between imprinting and X chromosome provenance suggests that retrotransposed elements with homology to the X chromosome can be selectively targeted for methylation during mammalian oogenesis.

Figure 1. The Genomic Environment and Germ-Line Methylation Status of Three Imprinted Retrogenes

The chromosome maps on the left-hand side show the position of each imprinted retrogene relative to other imprinted domains on the same chromosome. For each of the three loci, the top right-hand section shows the exonic structure and splicing pattern, the middle section shows the intron within which the retrogene is situated, and the bottom section shows the methylation status in oocytes and sperm, as determined by bisulphite sequencing. Circles on horizontal lines depict CpG dinucleotides on individual strands of genomic DNA. Filled circles represent methylated CpGs and open circles are unmethylated CpGs. The horizontal bar underneath each section marks the extent of the region below to depict scale. For U2af1-rs1, the methylation data is a summary of previously published results [15].

Figure 2. Multi-Species Comparative Sequence Analysis of Introns Containing Retrogenes and gDMRs in the Mouse Genome

The genomic DNA sequence of the entire host gene was used to generate VISTA plots using mouse as the base genome. (A) Inpp5f/Inpp5f_v2 and (B) Murr1/U2af1-rs1. Conserved sequences corresponding to coding exonic regions in the mouse are shaded purple, noncoding exons are light-blue, and conserved nontranscribed regions are pink. The position of exonic mouse sequences is indicated at the bottom of each plot. Presence or absence of the retroposed sequence in each species can be used to determine the approximate point in the mammalian radiation at which each retrogene originated. (C) Maximum likelihood (ML) tree showing members of the Nap1l family in mouse, human, and chicken. The alignment from which this tree was generated can be found in Figure S1. The imprinted mouse gene is within a shaded box, the two X-linked monoexonic paralogues are within open boxes, and the two autosomal and multi-exonic members are underlined. 100 trial bootstrap resampling scores are given for nodes relevant to the chromosomal origin of Nap1l5. (D) Multi-species comparative sequence analysis of Herc3, containing the imprinted Nap1l5 retrogene. The VISTA plot is annotated in the same manner as (A and B).

Figure 3. Identification of a Novel Imprinted Retrogene and gDMR

(A) Allele-specific RT-PCR sequencing assays in inter-specific mouse hybrids. SNPs were identified between C57BL/6J (B6) and Mus mus castaneus (cast), such that the parental origin of the expressing allele(s) could be determined in F1 hybrids. The maternal allele is indicated first in the hybrid crosses. (B) Allele-specific RT-PCR sequencing assay for the U2af1-rs1 and Inpp5f_v2 genes in mouse testes. cDNA was prepared from whole testes. (C) Comparative analysis of the H13 genomic sequence in multiple species, using mouse as the base genome. For clarity only the intron containing the imprinted murine retrogene is shown. Purple shading indicates coding exonic sequence, light-blue shading indicates noncoding exonic sequence, and pink shading indicates conserved nontranscribed sequence. Positions of mouse exons are shown as horizontal lines underneath the plot. (D) Methylation status of the Mcts2 promoter region in germ cells and E13.5 embryo, determined by the sequencing of bisulphite-modified genomic DNA. Closed circles indicate methylated CpGs, open circles are unmethylated. E13.5 embryos were derived from B6 mothers and cast fathers, so the parental origin of each strand could be determined using a SNP within the PCR amplicon.

Figure 4. Evolutionary Tree for Placental Mammals, Using the Topology Determined in [54]

(A) Based on the multi-species comparative sequence analysis in Figures 2 and 3, the approximate points in the mammalian radiation at which each of the four imprinted retrogenes originated are superimposed as grey boxes. The genome sequence of Dasypus novemcictus (armadillo) and Echinops telfari (tenrec) are currently only available in draft format and were therefore not used for the comparative analyses. (B) Allele-specific RT-PCR sequencing assays for H13 in B6 × cast reciprocal F1 hybrids. The maternal allele is given first in the hybrid crosses. (C) Transcriptional overview of the H13 locus. Protein-coding regions are shown as thick blocks, UTR regions as thin blocks, and introns as thin lines. Splice patterns are indicated. The paternally expressed Mcts2 is shown in blue and the maternally expressed H13 is in red. Arrows indicate the orientation of transcription.

Figure 5. Allele-Specific RT-PCR Sequencing Assays for the Human INPP5F_V2 (A), NAP1L5 (B), and MCTS2 (C) Transcripts

The maternal and fetal genotype was determined for each family. Where mother and fetus were both heterozygous, the parental origin of the single expressing fetal allele could not be determined (“not informative”). For all three genes, the first family shows paternal-allele-specific expression in fetal spinal cord. In each case, the remaining two families exhibited monoallelic expression.