|Freking, Bradley - Brad|
|Smith, Timothy - Tim|
Submitted to: Genome Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/21/2005
Publication Date: 1/13/2006
Citation: Murphy, S.K., Nolan, C.M., Huang, Z., Kucera, K.S., Freking, B.A., Smith, T.P.L., Leymaster, K.A., Weidman, J.R., Jirtle, R.L. 2006. Callipyge mutation affects gene expression in cis: A potential role for chromatin structure. Genome Research. 16:340-346. Interpretive Summary: The mechanism linking a single base mutation in sheep, named callipyge, with large effects on lean and fat development as well as eating quality of meat was investigated at the molecular level. A better understanding of a model for coordinated regulation of gene expression in this imprinted region has further implications to the human medical field. Muscle hypertrophy in callipyge sheep is expressed in a unique parent-of-origin manner referred to as polar overdominance. Specifically, animals exhibiting muscle hypertrophy must inherit the mutated allele from the sire, and not from the dam, making callipyge a unique phenomenon. We previously provided evidence that the callipyge phenotype correlated with abnormal maintenance of high expression levels of the nearby DLK1 gene, specifically in fast-twitch muscle. We also previously reported detection of a novel RNA transcript, named CLPG1, spanning the region containing the mutation. In this manuscript, we investigated the expression of CLPG1 in both fetal and adult sheep tissues as well as the influence of the mutation on cytosine methylation in the immediately vicinity. We were able to document that CLPG1 was expressed in the affected longissimus dorsi muscle of all four possible genotypes during the prenatal stage, but postnatal expression was exclusive to sheep that carried the mutation. The CLPG1 transcript was expressed in adult muscle tissue from either parental allele as long as it also contained the mutation. Normal adult sheep do not express this RNA transcript. Regulation of genes in an allele-specific manner can be accomplished by differential cytosine methylation. As a consequence of the mutation, we observed a repression of the normal postnatal increase in methylation that is typically two-fold greater in adults compared to fetal sheep for this location in the genome. Heterozygous individuals had intermediate levels of methylation, while homozygous mutants had the lowest. This attenuation of methylation is specific to the affected tissues and results from the presence of the mutation. A conceptual model has been refined incorporating these observations and their potential influences on chromatin structure and regional deregulation of gene expression. Establishing how this mutation works at the molecular level will lead to a better understanding of mechanisms associated with genes regulated by imprinting, in addition to the specific role of this particular genetic region with increased lean tissue growth and altered meat quality.
Technical Abstract: Muscular hypertrophy in callipyge sheep results from a single nucleotide substitution located in the genomic interval between the imprinted Delta, Drosophila, Homolog-Like 1 (DLK1) and Maternally Expressed Gene 3 (MEG3). The mechanism linking the mutation to muscle hypertrophy is unclear but involves DLK1 overexpression. The mutation is contained within CLPG1 transcripts produced from this region. Herein we show that CLPG1 is expressed prenatally in the hypertrophy-responsive longissimus dorsi muscle by all four possible genotypes, but postnatal expression is restricted to sheep carrying the mutation. Surprisingly, the mutation results in non-imprinted monoallelic transcription of CLPG1 from only the mutated allele in adult sheep, whereas it is expressed biallelically during prenatal development. Normal adult sheep do not express CLPG1. We further demonstrate that local CpG methylation is altered by the presence of the mutation in longissimus dorsi of postnatal sheep. For ten CpG sites flanking the mutation, methylation is similar prenatally across genotypes, but doubles postnatally in normal sheep. This normal postnatal increase in methylation is significantly repressed in sheep carrying one copy of the mutation, and repressed even further in sheep with two mutant alleles. The attenuation in methylation status in the callipyge sheep correlates with the onset of the phenotype, continued CLPG1 transcription and high-level expression of DLK1. In contrast, normal sheep exhibit hypermethylation of this locus after birth and CLPG1 silencing, which coincides with DLK1 transcriptional repression. These data are consistent with the notion that the callipyge mutation inhibits perinatal nucleation of regional chromatin condensation resulting in continued elevated transcription of prenatal DLK1 levels in adult callipyge sheep. We propose a model incorporating these results that can also account for the enigmatic normal phenotype of homozygous mutant sheep.