Expression of PEG11 and PEG11AS transcripts in normal and callipyge sheep

BMC Biology Expression of PEG11 and PEG11AS transcripts in normal and callipyge sheep Christopher A Bidwell 1 Lauren N Kramer 1 Allison C Perkins 1 Tracy S Hadfield 0 Diane E Moody 1 Noelle E Cockett 0 0 Department of Animal, Dairy and Veterinary Sciences, College of Agriculture, Utah State University , 4800 Old Main Hill, Logan UT 84322-4800 USA 1 Department of Animal Sciences, Purdue University , 125 South Russell Street, West Lafayette, IN 47907-2042 USA 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. - Background The mutation responsible for the callipyge trait is located within an imprinted gene cluster on the distal end of ovine chromosome 18 [1-3]. The callipyge phenotype is associated with an altered carcass composition including a 3040% increase in muscle mass, a 67% decrease in carcass fat, decreased organ weights, and a more compact skeleton, all without a net affect on animal growth [4-7]. There is a pronounced hypertrophy of muscles in the loin and pelvic limbs and a lesser degree of hypertrophy in muscles of the thoracic limbs [6-8]. The callipyge phenotype is inherited in a non-Mendelian mode termed polar overdominance [9,10] in which only animals that inherit a normal allele (wild type; +) from the dam and the mutant callipyge allele from the sire (CLPGPat) exhibit the callipyge phenotype. Maternal heterozygotes (CLPGMat/ +Pat) and callipyge allele homozygotes (CLPGMat/CLPGPat) have muscling and carcass compositions that are similar to normal sheep (wild type homozygotes; +/+). A physical contig spanning the region containing the callipyge mutation was constructed using overlapping ovine bacterial artificial chromosomes [11] and 215 kb of sequence was obtained from the contig [3]. Comparisons of the ovine sequence to the human genome sequence and to expressed sequence databases indicated the presence of at least six transcribed genes with allele-specific expression [3] (Figure 1). The gene order along the contig was found to be Delta-like 1 (DLK1), DLK associated transcript (DAT), gene-trap locus 2 (GTL2), paternal expressed gene 11 (PEG11 / PEG11AS) and maternal expressed gene 8 (MEG8). The same conserved gene order was also found as an imprinted domain on human chromosome 14 and mouse chromosome 12 [12-15]. The DLK1 locus (also referred to as PREF-1, Zog-1 and pG2) encodes a transmembrane protein that contains epidermal-growth factor repeats [16-19]. Cleavage of the extracellular domain of DLK1 produces the circulating protein fetal antigen-1 [20]. DAT is a short non-coding RNA that has been proposed to be a cleavage product of extended DLK1 transcripts [21]. Both DLK1and DAT are expressed from the paternal allele [3,13-15]. GTL2 (also referred to as MEG3) and MEG8 genes express non-coding RNA from the maternal allele [3,13-15]. The PEG11 gene contains an intronless open reading frame of 1333 amino acids in sheep [3]. The human and mouse orthologues known as retrotransposon-like 1 (RTL1/rtl1) encode 1358 and 1745 amino acids, respectively. RNA transcripts were detected from the opposite strand of the same gene, referred to as PEG11AS (formerly known as antiPEG11) [3]. In the mouse, two maternally expressed microRNAs have been identified with perfect complementarity to mouse Rtl1 [22]. The causative mutation for callipyge is a single base transition of A (wild type; +) to G (CLPG) in the intergenic region located between DLK1 and GTL2 [23] (Figure 1). This mutation has been shown to be 100% concordant with all animals of the +Mat/CLPGPat genotype based on haplotype analysis [23]. Analysis of sheep from 19 different breeds as well as 13 mammalian species revealed a highly conserved 12 base sequence that includes the single nucleotide polymorphism [24]. The G polymorphism is unique to direct descendents of the first known callipyge animal, a ram named "Solid Gold". This animal was mosaic for the mutation [24], providing strong evidence that this single nucleotide polymorphism is the causative mutation. Initial results indicate that the mutation alters the expression of several of the genes within the imprinted cluster [25,26] when they are inherited in cis without altering their parent-of-origin-specific expression [25]. In this study, we analyzed the expression of five genes within the callipyge cluster in the muscles of lambs of all four genotypes. Quantitative analysis of gene expression using a series of orthogonal contrasts showed that DLK1, PEG11 and MEG8 exhibited a polar overdominant pattern of gene expression. The expression of PEG11 and PEG11AS transcripts in the muscles of paternal heterozygous callipyge lambs (+Mat/CLPGPat) was different from the other three genotypes. A sense/antisense interaction of PEG11 and PEG11AS, such as an RNA interference mechanism, would (...truncated)