Expression of PEG11 and PEG11AS transcripts in normal and callipyge sheep

BACKGROUND: The callipyge mutation is located within an imprinted gene cluster on ovine chromosome 18. The callipyge trait exhibits polar overdominant inheritance due to the fact that only heterozygotes inheriting a mutant paternal allele (paternal heterozygotes) have a phenotype of muscle hypertrophy, reduced fat and a more compact skeleton. The mutation is a single A to G transition in an intergenic region that results in the increased expression of several genes within the imprinted cluster without changing their parent-of-origin allele-specific expression. RESULTS: There was a significant effect of genotype (p < 0.0001) on the transcript abundance of DLK1, PEG11, and MEG8 in the muscles of lambs with the callipyge allele. DLK1 and PEG11 transcript levels were elevated in the hypertrophied muscles of paternal heterozygous animals relative to animals of the other three genotypes. The PEG11 locus produces a single 6.5 kb transcript and two smaller antisense strand transcripts, referred to as PEG11AS, in skeletal muscle. PEG11AS transcripts were detectable over a 5.5 kb region beginning 1.2 kb upstream of the PEG11 start codon and spanning the entire open reading frame. Analysis of PEG11 expression by quantitative PCR shows a 200-fold induction in the hypertrophied muscles of paternal heterozygous animals and a 13-fold induction in homozygous callipyge animals. PEG11 transcripts were 14-fold more abundant than PEG11AS transcripts in the gluteus medius of paternal heterozygous animals. PEG11AS transcripts were expressed at higher levels than PEG11 transcripts in the gluteus medius of animals of the other three genotypes. CONCLUSIONS: The effect of the callipyge mutation has been to alter the expression of DLK1, GTL2, PEG11 and MEG8 in the hypertrophied skeletal muscles. Transcript abundance of DLK1 and PEG11 was highest in paternal heterozygous animals and exhibited polar overdominant gene expression patterns; therefore, both genes are candidates for causing skeletal muscle hypertrophy. There was unique relationship of PEG11 and PEG11AS transcript abundance in the paternal heterozygous animals that suggests a RNA interference mechanism may have a role in PEG11 gene regulation and polar overdominance in callipyge sheep

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

Identification of Genes Directly Responding to DLK1 Signaling in Callipyge Sheep

Background In food animal agriculture, there is a need to identify the mechanisms that can improve the efficiency of muscle growth and protein accretion. Callipyge sheep provide excellent machinery since the up-regulation of DLK1 and RTL1 results in extreme postnatal muscle hypertrophy in distinct muscles. The aim of this study is to distinguish the genes that directly respond to DLK1 and RTL1 signaling from the genes that change as the result of muscle specific effects. Results The quantitative PCR results indicated that DLK1 expression was significantly increased in hypertrophied muscles but not in non-hypertrophied muscles. However, RTL1 was up-regulated in both hypertrophied and non-hypertrophied muscles. Five genes, including PARK7, DNTTIP1, SLC22A3, METTL21E and PDE4D, were consistently co-expressed with DLK1, and therefore were possible transcriptional target genes responding to DLK1 signaling. Treatment of myoblast and myotubes with DLK1 protein induced an average of 1.6-fold and 1.4-fold increase in Dnttip1 and Pde4d expression respectively. Myh4 expression was significantly elevated in DLK1-treated myotubes, whereas the expression of Mettl21e was significantly increased in the DLK1-treated myoblasts but reduced in DLK1-treated myotubes. DLK1 treatment had no impact on Park7 expression. In addition, Park7 and Dnttip1 increased Myh4 and decreased Myh7 promoter activity, resemble to the effects of Dlk1. In contrast, expression of Mettl21e increased Myh7 and decreased Myh4 luciferase activity. Conclusion The study provided additional supports that RTL1 alone was insufficient to induce muscle hypertrophy and concluded that DLK1 was likely the primary effector of the hypertrophy phenotype. The results also suggested that DNTTIP1 and PDE4D were secondary effector genes responding to DLK1 signaling resulting in muscle fiber switch and muscular hypertrophy in callipyge lamb

Extending the Ehresmann-Schein-Nambooripad Theorem

We extend the `join-premorphisms' part of the Ehresmann-Schein-Nambooripad Theorem to the case of two-sided restriction semigroups and inductive categories, following on from a result of Lawson (1991) for the `morphisms' part. However, it is so-called `meet-premorphisms' which have proved useful in recent years in the study of partial actions. We therefore obtain an Ehresmann-Schein-Nambooripad-type theorem for meet-premorphisms in the case of two-sided restriction semigroups and inductive categories. As a corollary, we obtain such a theorem in the inverse case.Comment: 23 pages; final section on Szendrei expansions removed; further reordering of materia

Fixed-point elimination in the intuitionistic propositional calculus

It is a consequence of existing literature that least and greatest fixed-points of monotone polynomials on Heyting algebras-that is, the algebraic models of the Intuitionistic Propositional Calculus-always exist, even when these algebras are not complete as lattices. The reason is that these extremal fixed-points are definable by formulas of the IPC. Consequently, the $\mu$-calculus based on intuitionistic logic is trivial, every $\mu$-formula being equivalent to a fixed-point free formula. We give in this paper an axiomatization of least and greatest fixed-points of formulas, and an algorithm to compute a fixed-point free formula equivalent to a given $\mu$-formula. The axiomatization of the greatest fixed-point is simple. The axiomatization of the least fixed-point is more complex, in particular every monotone formula converges to its least fixed-point by Kleene's iteration in a finite number of steps, but there is no uniform upper bound on the number of iterations. We extract, out of the algorithm, upper bounds for such n, depending on the size of the formula. For some formulas, we show that these upper bounds are polynomial and optimal

A gene network switch enhances the oxidative capacity of ovine skeletal muscle during late fetal development

Background: The developmental transition between the late fetus and a newborn animal is associated with profound changes in skeletal muscle function as it adapts to the new physiological demands of locomotion and postural support against gravity. The mechanisms underpinning this adaption process are unclear but are likely to be initiated by changes in hormone levels. We tested the hypothesis that this developmental transition is associated with large coordinated changes in the transcription of skeletal muscle genes.Results: Using an ovine model, transcriptional profiling was performed on Longissimus dorsi skeletal muscle taken at three fetal developmental time points (80, 100 and 120 d of fetal development) and two postnatal time points, one approximately 3 days postpartum and a second at 3 months of age. The developmental time course was dominated by large changes in expression of 2,471 genes during the interval between late fetal development (120 d fetal development) and 1-3 days postpartum. Analysis of the functions of genes that were uniquely up-regulated in this interval showed strong enrichment for oxidative metabolism and the tricarboxylic acid cycle indicating enhanced mitochondrial activity. Histological examination of tissues from these developmental time points directly confirmed a marked increase in mitochondrial activity between the late fetal and early postnatal samples. The promoters of genes that were up-regulated during this fetal to neonatal transition were enriched for estrogen receptor 1 and estrogen related receptor alpha cis-regulatory motifs. The genes down-regulated during this interval highlighted de-emphasis of an array of functions including Wnt signaling, cell adhesion and differentiation. There were also changes in gene expression prior to this late fetal - postnatal transition and between the two postnatal time points. The former genes were enriched for functions involving the extracellular matrix and immune response while the latter principally involved functions associated with transcriptional regulation of metabolic processes.Conclusions: It is concluded that during late skeletal muscle development there are substantial and coordinated changes in the transcription of a large number of genes many of which are probably triggered by increased estrogen levels. These changes probably underpin the adaption of muscle to new physiological demands in the postnatal environment

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

An advanced sheep (Ovis aries, 2n = 54) cytogenetic map and assignment of 88 new autosomal loci by fluorescence in situ hybridization and R-banding

Presented herein is an updated sheep cytogenetic map that contains 452 loci (291 type I and 161 type II) assigned to specific chromosome bands or regions on standard R-banded ideograms. This map, which significantly extends our knowledge of the physical organization of the ovine genome, includes new assignments for 88 autosomal loci, including 74 type I loci (known genes) and 14 type II loci (SSRs/microsatellite marker/STSs), by FISH-mapping and R-banding. Comparison of the ovine map to the cattle and goat cytogenetic maps showed that common loci were located within homologous chromosomes and chromosome bands, confirming the high level of conservation of autosomes among ruminant species. Eleven loci that were FISH-mapped in sheep (B3GAT2, ASCC3, RARSL, BRD2, POLR1C, PPP2R5D, TNRC5, BAT2, BAT4, CDC5L and OLA-DRA) are unassigned in cattle and goat. Eleven other loci (D3S32, D1S86, BMS2621, SFXN5, D5S3, D5S68, CSKB1, D7S49, D9S15, D9S55 and D29S35) were assigned to specific ovine chromosome (OAR) bands but have only been assigned to chromosomes in cattle and goat

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