The Imprinted Retrotransposon-Like Gene PEG11 (RTL1) Is Expressed as a Full-Length Protein in Skeletal Muscle from Callipyge Sheep

peer-reviewedMembers of the Ty3-Gypsy retrotransposon family are rare in mammalian genomes despite their abundance in invertebrates and some vertebrates. These elements contain a gag-pol-like structure characteristic of retroviruses but have lost their ability to retrotranspose into the mammalian genome and are thought to be inactive relics of ancient retrotransposition events. One of these retrotransposon-like elements, PEG11 (also called RTL1) is located at the distal end of ovine chromosome 18 within an imprinted gene cluster that is highly conserved in placental mammals. The region contains several conserved imprinted genes including BEGAIN, DLK1, DAT, GTL2 (MEG3), PEG11 (RTL1), PEG11as, MEG8, MIRG and DIO3. An intergenic point mutation between DLK1 and GTL2 causes muscle hypertrophy in callipyge sheep and is associated with large changes in expression of the genes linked in cis between DLK1 and MEG8. It has been suggested that over-expression of DLK1 is the effector of the callipyge phenotype; however, PEG11 gene expression is also strongly correlated with the emergence of the muscling phenotype as a function of genotype, muscle type and developmental stage. To date, there has been no direct evidence that PEG11 encodes a protein, especially as its anti-sense transcript (PEG11as) contains six miRNA that cause cleavage of the PEG11 transcript. Using immunological and mass spectrometry approaches we have directly identified the full-length PEG11 protein from postnatal nuclear preparations of callipyge skeletal muscle and conclude that its over-expression may be involved in inducing muscle hypertrophy. The developmental expression pattern of the PEG11 gene is consistent with the callipyge mutation causing recapitulation of the normal fetal-like gene expression program during postnatal development. Analysis of the PEG11 sequence indicates strong conservation of the regions encoding the antisense microRNA and in at least two cases these correspond with structural or functional domains of the protein suggesting co-evolution of the sense and antisense genes

The Imprinted Retrotransposon-Like Gene PEG11 (RTL1) Is Expressed as a Full-Length Protein in Skeletal Muscle from Callipyge Sheep

Members of the Ty3-Gypsy retrotransposon family are rare in mammalian genomes despite their abundance in invertebrates and some vertebrates. These elements contain a gag-pol-like structure characteristic of retroviruses but have lost their ability to retrotranspose into the mammalian genome and are thought to be inactive relics of ancient retrotransposition events. One of these retrotransposon-like elements, PEG11 (also called RTL1) is located at the distal end of ovine chromosome 18 within an imprinted gene cluster that is highly conserved in placental mammals. The region contains several conserved imprinted genes including BEGAIN, DLK1, DAT, GTL2 (MEG3), PEG11 (RTL1), PEG11as, MEG8, MIRG and DIO3. An intergenic point mutation between DLK1 and GTL2 causes muscle hypertrophy in callipyge sheep and is associated with large changes in expression of the genes linked in cis between DLK1 and MEG8. It has been suggested that over-expression of DLK1 is the effector of the callipyge phenotype; however, PEG11 gene expression is also strongly correlated with the emergence of the muscling phenotype as a function of genotype, muscle type and developmental stage. To date, there has been no direct evidence that PEG11 encodes a protein, especially as its anti-sense transcript (PEG11as) contains six miRNA that cause cleavage of the PEG11 transcript. Using immunological and mass spectrometry approaches we have directly identified the full-length PEG11 protein from postnatal nuclear preparations of callipyge skeletal muscle and conclude that its over-expression may be involved in inducing muscle hypertrophy. The developmental expression pattern of the PEG11 gene is consistent with the callipyge mutation causing recapitulation of the normal fetal-like gene expression program during postnatal development. Analysis of the PEG11 sequence indicates strong conservation of the regions encoding the antisense microRNA and in at least two cases these correspond with structural or functional domains of the protein suggesting co-evolution of the sense and antisense genes

Effect of DLK1 and RTL1 but Not MEG3 or MEG8 on Muscle Gene Expression in Callipyge Lambs

Callipyge sheep exhibit extreme postnatal muscle hypertrophy in the loin and hindquarters as a result of a single nucleotide polymorphism (SNP) in the imprinted DLK1-DIO3 domain on ovine chromosome 18. The callipyge SNP up-regulates the expression of surrounding transcripts when inherited in cis without altering their allele-specific imprinting status. The callipyge phenotype exhibits polar overdominant inheritance since only paternal heterozygous animals have muscle hypertrophy. Two studies were conducted profiling gene expression in lamb muscles to determine the down-stream effects of over-expression of paternal allele-specificDLK1 and RTL1 as well as maternal allele-specific MEG3, RTL1AS and MEG8, using Affymetrix bovine expression arrays. A total of 375 transcripts were differentially expressed in callipyge muscle and 25 transcripts were subsequently validated by quantitative PCR. The muscle-specific expression patterns of most genes were similar to DLK1 and included genes that are transcriptional repressors or affect feedback mechanisms in β-adrenergic and growth factor signaling pathways. One gene, phosphodiesterase 7A had an expression pattern similar to RTL1 expression indicating a biological activity for RTL1 in muscle. Only transcripts that localize to the DLK1-DIO3 domain were affected by inheritance of a maternal callipyge allele. Callipyge sheep are a unique model to study over expression of both paternal allele-specific genes and maternal allele-specific non-coding RNA with an accessible and nonlethal phenotype. This study has identified a number of genes that are regulated by DLK1 and RTL1expression and exert control on postnatal skeletal muscle growth. The genes identified in this model are primary candidates for naturally regulating postnatal muscle growth in all meat animal species, and may serve as targets to ameliorate muscle atrophy conditions including myopathic diseases and age-related sarcopenia

Dlk1 Is Necessary for Proper Skeletal Muscle Development and Regeneration

Delta-like 1homolog (Dlk1) is an imprinted gene encoding a transmembrane protein whose increased expression has been associated with muscle hypertrophy in animal models. However, the mechanisms by which Dlk1 regulates skeletal muscle plasticity remain unknown. Here we combine conditional gene knockout and over-expression analyses to investigate the role of Dlk1 in mouse muscle development, regeneration and myogenic stem cells (satellite cells). Genetic ablation of Dlk1 in the myogenic lineage resulted in reduced body weight and skeletal muscle mass due to reductions in myofiber numbers and myosin heavy chain IIB gene expression. In addition, muscle-specific Dlk1 ablation led to postnatal growth retardation and impaired muscle regeneration, associated with augmented myogenic inhibitory signaling mediated by NF-κB and inflammatory cytokines. To examine the role of Dlk1 in satellite cells, we analyzed the proliferation, self-renewal and differentiation of satellite cells cultured on their native host myofibers. We showed that ablation of Dlk1 inhibits the expression of the myogenic regulatory transcription factor MyoD, and facilitated the self-renewal of activated satellite cells. Conversely, Dlk1 over-expression inhibited the proliferation and enhanced differentiation of cultured myoblasts. As Dlk1 is expressed at low levels in satellite cells but its expression rapidly increases upon myogenic differentiation in vitro and in regenerating muscles in vivo, our results suggest a model in which Dlk1 expressed by nascent or regenerating myofibers non-cell autonomously promotes the differentiation of their neighbor satellite cells and therefore leads to muscle hypertrophy

Gene expression profiling of hypertrophied muscles in callipyge sheep

Callipyge sheep exhibit extreme postnatal hypertrophy in the loin and hindquarters as a result of single nucleotide polymorphism (SNP) in the imprinted DLK1-MEG3 gene cluster on ovine chromosome 18. The callipyge SNP up-regulates the expression of nearby imprinted transcripts when inherited in cis. This thesis contains phenotypic measurements of lambs possessing a callipyge allele from 10 days of age to 200 days of age. These animals were used in three studies designed to profile gene expression in lamb muscles to determine down-stream effects of paternal allele specific and maternal allele specific transcripts. Affymetrix Bovine Expression Arrays and quantitative PCR were used to discover transcripts differentially expressed as a result of the callipyge mutation. To determine the transcripts affected during callipyge muscle hypertrophy, two hypertrophied muscles (longissimus dorsi and semimembranosus) from callipyge (+/CPat ; n=8) and wild-type lambs (+/+; n=8) were compared across four ages that encompass the postnatal muscle hypertrophy development. Analysis methods by MAS5 and RMA identified 149 to 378 transcripts, respectively, that were differentially expressed in callipyge muscles. These included metabolic enzymes, apoptotic factors, proteins involved in regulating common signaling pathways, and transcription factors. Thirty-three novel transcripts were validated by qPCR to be differentially expressed between callipyge and normal muscles across additional ages. The third experiment aimed at detecting differences in genes expression affected by the presence of a maternal callipyge allele (CMat/+ and C/C). These lambs were contrasted against lambs with a wild-type maternal allele (+/ CPat and +/+). The microarray and qPCR data only validated significant differences in expression of MEG3 and MEG8, both genes within the callipyge imprinted cluster. These data indicate that the up-regulation of MEG3 and MEG8 does not influence gene expression of any transcripts on the bovine expression array which are not located near the callipyge mutation. High levels of DLK1 and RTL1 in the paternal heterozygous lambs are enhancing factors involved in existing muscle growth mechanisms including the AKT/mTOR signaling pathway

Analysis of Gene Expression during the Onset of Muscle Hypertrophy in Callipyge Lambs

The callipyge mutation causes postnatal muscle hypertrophy in heterozygous lambs that inherit a paternal callipyge allele (+/CLPG). Our hypothesis was that the up-regulation of one or both of the affected paternally expressed genes (DLK1 or PEG11) initiates changes in biochemical and physiological pathways in skeletal muscle to induce hypertrophy. the goal of this study was to identify changes in gene expression during the onset of muscle hypertrophy to identify the pathways that are involved in the expression of the callipyge phenotype. Gene expression was analysed in longissimus dorsi total RNA from lambs at 10, 20, and 30 days of age using the Affymetrix Bovine Expression Array. an average of 40.6% of probe sets on the array was detected in sheep muscle. Data were normalized and analysed using a two-way anova for genotype and age effects with a false discovery rate of 0.10. From the anova, 13 genes were significant for the effect of genotype and 13 were significant for effect of age (P \u3c 0.10). No significant age-by-genotype interactions were detected (P \u3e 0.10). of the 13 genes indicating an effect of genotype, quantitative PCR assays were developed for all of them and tested on a larger group of animals from 10 to 200 days of age. Nine genes had significantly elevated transcript levels in callipyge lambs. These genes included phosphofructokinase, a putative methyltransferase protein, a cAMP phosphodiesterase, and the transcription factor DNTTIP1

Relative expression levels of <i>PEG11</i>/<i>PEG11as</i> mRNA in wild type (<i>NN</i>) and callipyge (<i>NC^pat</i>) genotypes during skeletal muscle developmental.

<p>Expression levels were measured by qRT-PCR and normalised to <i>RPLPO</i>. (A) <i>PEG11/PEG11as</i> mRNA expression in SM muscle taken at 12 weeks (230 days post-fertilisation) of age from four wild-type (<i>NN</i>) (grey) and four callipyge paternal heterozygote (<i>NC^pat</i>) individuals (black). The mean expression values of the four individuals representing each genotype are illustrated in the inset. The qRT-PCR assay measured expression of both <i>PEG11</i> and <i>PEG11as</i> as the former is wholly contained within the latter. However, the contribution of <i>PEG11as</i> is relatively minor in these genotypes and thus the assay primarily measures expression of <i>PEG11</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008638#s4" target="_blank">Materials and Methods</a>). (B) <i>PEG11/PEG11as</i> expression in LD muscle during muscle development as a function of genotype. The samples were taken at 80, 100, 120, 150 and 230 days of development. Birth is at 147 days. The error bars denote the standard error of mean (n = 4). The asterisks denote significant (P<0.001) differences in expression levels between the <i>NC^pat</i> (black) and <i>NN</i> (grey) genotypes at each developmental stage.</p

Conserved regions of the ovine <i>PEG11</i> gene correspond with antisense miRNA.

<p>The <i>PEG11</i> open reading frame is shown in blue while the locations of antisense miRNA (mir-431, mir-433, mir-127, mir-432 and mir-136) are shown in red. The black arrow denotes the direction of transcription of <i>PEG11</i>. The graph shows the extent of <i>PEG11</i> gene conservation using the UCSC PhastCons Conserved Elements 17-way Vertebrate Multiz Alignment and Conservation tool <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008638#pone.0008638-Kent1" target="_blank">[34]</a>.</p

Ovine PEG11 peptides detected by mass spectrometry¹.

1<p>Data from the seven different peptide fragments identified by LC-MALDI-MS/MS and LC-ESI-MS/MS are tabulated with their corresponding theoretical and experimental peptide masses (Da), as well as the difference in mass (Δm).</p