Human–Ovine Comparative Sequencing of a 250-kb Imprinted Domain Encompassing the Callipyge (clpg) Locus and Identification of Six Imprinted Transcripts: DLK1, DAT, GTL2, PEG11, antiPEG11, and MEG8

Abstract

Two ovine BAC clones and a connecting long-range PCR product, jointly spanning ∼250 kb and representing most of the MULGE5-OY3 marker interval known to contain the clpg locus, were completely sequenced. The resulting genomic sequence was aligned with its human ortholog and extensively annotated. Six transcripts, four of which were novel, were predicted to originate from within the analyzed region and their existence confirmed experimentally: DLK1, DAT, GTL2, PEG11, antiPEG11, and MEG8. RT-PCR experiments performed on a range of tissues sampled from an 8-wk-old animal demonstrated the preferential expression of all six transcripts in skeletal muscle, which suggests that they are under control of common regulatory elements. The six transcripts were also shown to be subject to parental imprinting: DLK1, DAT, and PEG11 were shown to be paternally expressed and GTL2, antiPEG11, and MEG8 to be maternally expressed.

Detection of Imprinted Genes by Single-Cell Allele-Specific Gene Expression

“…The 14q32 region has been associated with imprinting disorders 35 and tumorigenesis. 36 In particular, this genomic area harbors three differentially methylated regions 37,38 described to coordinately regulate (1) the paternally expressed genes DLK1 and RTL1 (retrosposon-like1) and (2) the maternally expressed genes MEG3 (long intergenic non-protein coding RNA 23 or GTL2), RTL-antisense transcript, MEG8, MEG9, and several polyadenylated C/D box small nucleolar (sno)RNAs 39,40 and microRNAs 20,[40][41][42][43] ( Figure 2, Tables S6 and S7). From our analysis, we validated the known imprinting statuses of MEG3, MEG8, MEG9, DLK1, and RTL-antisense transcripts (Tables S7), but the DIO3 and RTL1 genes were not detectable in fibroblasts.…”

The callipyge mutation and other genes that affect muscle hypertrophy in sheep

“…This imprinted gene cluster contains several paternally expressed protein-coding genes, including BEGAIN [54], DLK1 [31,49,55,65], PEG11 [14], and DIO3 [22,59,66], as well as several maternally expressed non-coding RNA genes, including GTL2 [44,49,55,65], antiPEG11 [14], MEG8 [14], and MIRG [50]. The BEGAIN, DLK1, PEG11, GTL2, antiPEG11, and MEG8 genes have been shown to be expressed and subject to genomic imprinting in ovine skeletal muscle tissues [14,54].…”

“…The DLK1 gene encodes a member of the delta-notch family of signalling molecules [33], and the PEG11 gene encodes for a protein product with similarity to the gag and pol polyproteins of retrotransposons [14]. Two lines of evidence currently point towards the involvement of DLK1 in muscle development.…”

Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome

“…] In recent years there have been increasing reports of functional non-protein-coding RNAs (ncRNAs) that are involved or implicated in developmental, tissue-specific, and disease processes, including X-chromosome dosage compensation, germ cell development and embryogenesis, neural and immune cell development, kidney and testis development, B-cell neoplasia, lung cancer, prostate cancer, cartilage-hair hypoplasia, spinocerebellar ataxia type 8, DiGeorge syndrome, autism, and schizophrenia (see Pang et al 2005). Many putative ncRNAs are alternatively spliced and/or polyadenylated (Sutherland et al 1996;Tam et al 1997;Bussemakers et al 1999;Raho et al 2000;Charlier et al 2001;Wolf et al 2001). Smaller ncRNAs, termed microRNAs, have also been shown to be involved in developmental processes in both plants and animals, as well as implicated in disease (Carrington and Ambros 2003;Mattick and Makunin 2005).…”