Molecular cloning, mRNA expression and imprinting status of PEG3, NAP1L5 and PPP1R9A genes in pig

Imprinted genes are expressed monoallelically depending on their parental origin, and play important roles in the regulation of fetal growth, development, and postnatal behavior. Most genes known to be imprinted have been identified and studied in the human and the mouse. However, there are only a small number of reported imprinted genes in pigs. Therefore, identification and characterization of more imprinted genes in pigs is useful for comparative analysis of genomic imprinting across species. In this study, we cloned the porcine PEG3, NAP1L5 and PPP1R9A genes. The imprinting status of these genes was determined using sequencing directly and single nucleotide polymorphisms (SNPs) identified in individuals from reciprocal cross of Meishan and Large White pigs. Imprinting analysis was carried out in 13 different tissues (skeletal muscle, fat, pituitary gland, heart, lung, liver, kidney, spleen, stomach, small intestine, uterus, ovary and testis) from twelve 2-month-old piglets. Imprinting analysis showed that PEG3 and NAP1L5 were exclusively expressed from the paternal allele whereas PPP1R9A was biallelically expressed in all tissues tested where the genes were expressed. The study is of interest to understand the conservation of genomic imprinting among mammals at the 3 loci.     

Molecular characterization of porcine NECD, SNRPN and UBE3A genes and imprinting status in the skeletal muscle of neonate pigs

Imprinted genes are expressed monoallelically depending on their parental origin, and play important roles in embryo survival and postnatal growth regulation. In this study, we characterized the porcine NECD (necdin), SNRPN (small nuclear ribonucleoprotein polypeptide N) and UBE3A (UBE3A ubiquitin protein ligase E3A) genes, analyzed their expression in nine tissues including liver, lung, small intestine, skeletal muscle, heart, kidney, spleen, inguinal lymph nodes and fat, and also examined their imprinting status in the skeletal muscle of neonate pigs. Results indicated that these three genes were highly homologous between pigs and cattle, being 95.02?% in nucleotide and 99.17?% in amino acid with the cattle SNRPN gene, and 96.46?% in nucleotide and 98.63?% in amino acid with the cattle UBE3A gene, respectively. The three genes were expressed in all the tissues investigated. Three single nucleotide polymorphisms (SNPs) in the coding region of these genes, i.e. g.263G>C, g.402T>C and g.340A>G for porcine NECD, SNRPN and UBE3A genes, respectively, were revealed; and imprinting analysis with which indicated that, in the skeletal muscle of neonate pigs, both NECD and SNRPN were maternally imprinted, while UBE3A was not imprinted.     

Assessment of genomic imprinting of PPP1R9A, NAP1L5 and PEG3 in pigs

Imprinted genes play significant roles in the regulation of fetal growth and development, function of the placenta, and maternal nurturing behaviour in mammals. At present, few imprinted genes have been reported in pigs compared to human and mouse. In order to increase understanding of imprinted genes in swine, a polymorphism-based approach was used to assess the imprinting status of three porcine genes in 12 tissue types, obtained from F1 pigs of reciprocal crosses between Rongchang and Landrace pure breeds. In contrast to human and mouse homologues, porcine PPP1R9A was not imprinted, and was found to be expressed in all tissues examined. The expression of porcine NAP1L5 was detected in pituitary, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, ovary, and uterus, but undetectable in heart. Furthermore, porcine NAP1L5 was paternally expressed in the tissues where it's expression was observed. For PEG3, pigs expressed the paternal allele in skeletal muscle, liver, spleen, kidney, and uterus, but biallele in heart, lung, fat, stomach, small intestine, and ovary. Our data indicate that tissue distribution of the three gene differs among mammals, and the imprinting of NAP1L5 and PEG3 is well conserved.     

Conservation of genomic imprinting at the NDN, MAGEL2 and MEST loci in pigs

Imprinted genes have important effects on the regulation of fetal growth, development, and postnatal behavior. However, the study of imprinted genes has been limited in mammalian species other than human and mouse. Therefore, the study of porcine imprinted genes is useful for defining the extent of conservation of genomic imprinting among different species. In this study, the imprinting status of porcine NDN, MAGEL2 and MEST genes was determined by direct sequencing of the cDNAs and detection of single nucleotide polymorphisms (SNPs) identified in individuals from reciprocal crosses between Meishan and Large White pigs for allele discrimination. The analysis was carried out in 13 different tissues (skeletal muscle, fat, pituitary gland, heart, lung, liver, kidney, spleen, stomach, small intestine, uterus, ovary and testis) from 12 two-month-old piglets. Imprinting analysis showed that NDN and MAGEL2 were paternally expressed in all tissues where the genes were expressed as in human and mouse. Interestingly, MEST showed tissue-specific imprinting, being paternally expressed in skeletal muscle, fat, pituitary gland, heart, kidney, lung, stomach and uterus, and maternally expressed in spleen and liver.     

Maternal inheritance of the Gnas cluster mutation Ex1A-T affects size, implicating NESP55 in growth

Genes subjected to genomic imprinting are often associated with prenatal and postnatal growth. Furthermore, it has been observed that maternally silenced/paternally expressed genes tend to favour offspring growth, whilst paternally silenced/maternally expressed genes will restrict growth. One imprinted cluster in which this has been shown to hold true is the Gnas cluster; of the three proteins expressed from this cluster, two, Gsα and XLαs, have been found to affect postnatal growth in a number of different mouse models. The remaining protein in this cluster, NESP55, has not yet been shown to be involved in growth. We previously described a new mutation, Ex1A-T, which upon paternal transmission resulted in postnatal growth retardation due to loss of imprinting of Gsα and loss of expression of the paternally expressed XLαs. Here we describe maternal inheritance of Ex1A-T which gives rise to a small but highly significant overgrowth phenotype which we attribute to reduction of maternally expressed NESP55.     

Identification of a methylation imprint mark within the mouse Gnas locus

The imprinted mouse gene Gnas produces the G protein alpha-subunit G(S)alpha and several other gene products by using alternative promoters and first exons. G(S)alpha is maternally expressed in some tissues and biallelically expressed in most other tissues, while the gene products NESP55 and XLalphas are maternally and paternally expressed, respectively. We investigated the mechanisms of Gnas imprinting. The G(S)alpha promoter and first exon are not methylated on either allele. A further upstream region (approximately from positions -3400 to -939 relative to the G(S)alpha translational start site) is methylated only on the maternal allele in all adult somatic tissues and in early postimplantation development. Within this region lies a fourth promoter and first exon (exon 1A) that generates paternal-specific mRNAs of unknown function. Exon 1A and G(S)alpha mRNAs have similar expression patterns, making competition between their promoters unlikely. Differential methylation in this region is established during gametogenesis, being present in oocytes and absent in spermatozoa, and is maintained in preimplantation E3. 5d blastocysts. Therefore, this region is a methylation imprint mark. In contrast, differential methylation of the NESP55 and XLalphas promoter regions (Nesp and Gnasxl) is not established during gametogenesis. The methylation imprint mark that we identified may be important for the tissue-specific imprinting of G(S)alpha.     

PHLDA2 is an imprinted gene in cattle

Genomic imprinting is an epigenetic non-Mendelian phenomenon found predominantly in placental mammals. Imprinted genes display differential expression in the offspring depending on whether the gene is maternally or paternally inherited. Currently, some 100 imprinted genes have been reported in mammals, and while some of these genes are imprinted across most mammalian species, others have been shown to be imprinted in only a few species. The PHLDA2 gene that codes for a pleckstrin homology-like domain, family A (member 2), protein has to date been shown to be a maternally expressed imprinted gene in humans, mice and pigs. Genes subject to imprinting can have major effects on mammalian growth, development and disease. For instance, disruption of imprinted genes can lead to aberrant growth syndromes in cloned domestic mammals, and it has been demonstrated that PHLDA2 mRNA expression levels are aberrant in the placenta of somatic clones of cattle. In this study, we demonstrate that PHLDA2 is expressed across a range of cattle foetal tissues and stages and provide the first evidence that PHLDA2 is a monoallelically expressed imprinted gene in cattle foetal tissues, and also in the bovine placenta.     

Assessment of genomic imprinting of <Emphasis Type="Italic">PPP1R9A</Emphasis>, <Emphasis Type="Italic">NAP1L5</Emphasis> and <Emphasis Type="Italic">PEG3</Emphasis> in pigs

Imprinted genes play significant roles in the regulation of fetal growth and development, function of the placenta, and maternal nurturing behaviour in mammals. At present, few imprinted genes have been reported in pigs compared to human and mouse. In order to increase understanding of imprinted genes in swine, a polymorphism-based approach was used to assess the imprinting status of three porcine genes in 12 tissue types, obtained from F1 pigs of reciprocal crosses between Rongchang and Landrace pure breeds. In contrast to human and mouse homologues, porcine PPP1R9A was not imprinted, and was found to be expressed in all tissues examined. The expression of porcine NAP1L5 was detected in pituitary, liver, spleen, lung, kiduey, stomach, small intestine, skeletal muscle, fat, ovary, and uterus, but undetectable in heart. Furthermore, porcine NAP1L5 was paternally expressed in the tissues where it’s expression was observed. For PEG3, pigs expressed the paternal allele in skeletal muscle, liver, spleen, kidney, and uterus, but biallele in heart, lung, fat, stomach, small intestine, and ovary. Our data indicate that tissue distribution of the three gene differs among mammals, and the imprinting of NAP1L5 and PEG3 is well conserved.     

An unexpected function of the Prader-Willi syndrome imprinting center in maternal imprinting in mice

Genomic imprinting is a phenomenon that some genes are expressed differentially according to the parent of origin. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders caused by deficiency of imprinted gene expression from paternal and maternal chromosome 15q11-q13, respectively. Imprinted genes at the PWS/AS domain are regulated through a bipartite imprinting center, the PWS-IC and AS-IC. The PWS-IC activates paternal-specific gene expression and is responsible for the paternal imprint, whereas the AS-IC functions in the maternal imprint by allele-specific repression of the PWS-IC to prevent the paternal imprinting program. Although mouse chromosome 7C has a conserved PWS/AS imprinted domain, the mouse equivalent of the human AS-IC element has not yet been identified. Here, we suggest another dimension that the PWS-IC also functions in maternal imprinting by negatively regulating the paternally expressed imprinted genes in mice, in contrast to its known function as a positive regulator for paternal-specific gene expression. Using a mouse model carrying a 4.8-kb deletion at the PWS-IC, we demonstrated that maternal transmission of the PWS-IC deletion resulted in a maternal imprinting defect with activation of the paternally expressed imprinted genes and decreased expression of the maternally expressed imprinted gene on the maternal chromosome, accompanied by alteration of the maternal epigenotype toward a paternal state spread over the PWS/AS domain. The functional significance of this acquired paternal pattern of gene expression was demonstrated by the ability to complement PWS phenotypes by maternal inheritance of the PWS-IC deletion, which is in stark contrast to paternal inheritance of the PWS-IC deletion that resulted in the PWS phenotypes. Importantly, low levels of expression of the paternally expressed imprinted genes are sufficient to rescue postnatal lethality and growth retardation in two PWS mouse models. These findings open the opportunity for a novel approach to the treatment of PWS.     

Epigenetic states and expression of imprinted genes in human embryonic stem cells

AIM: To investigate the epigenetic states and expression of imprinted genes in five human embryonic stem cell (hESC) lines derived in Taiwan.METHODS: The heterozygous alleles of single nucleotide polymorphisms (SNPs) at imprinted genes were analyzed by sequencing genomic DNAs of hESC lines and the monoallelic expression of the imprinted genes were confirmed by sequencing the cDNAs. The expression profiles of 32 known imprinted genes of five hESC lines were determined using Affymetrix human genome U133 plus 2.0 DNA microarray.RESULTS: The heterozygous alleles of SNPs at seven imprinted genes, IPW, PEG10, NESP55, KCNQ1, ATP10A, TCEB3C and IGF2, were identified and the monoallelic expression of these imprinted genes except IGF2 were confirmed. The IGF2 gene was found to be imprinted in hESC line T2 but partially imprinted in line T3 and not imprinted in line T4 embryoid bodies. Ten imprinted genes, namely GRB10, PEG10, SGCE, MEST, SDHD, SNRPN, SNURF, NDN, IPW and NESP55, were found to be highly expressed in the undifferentiated hESC lines and down-regulated in differentiated derivatives. The UBE3A gene abundantly expressed in undifferentiated hESC lines and further up-regulated in differentiated tissues. The expression levels of other 21 imprinted genes were relatively low in undifferentiated hESC lines and five of these genes (TP73, COPG2, OSBPL5, IGF2 and ATP10A) were found to be up-regulated in differentiated tissues.CONCLUSION: The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.     

Secretion and molecular forms of NESP55, a novel genomically imprinted neuroendocrine-specific protein from AtT-20 cells

NESP55 (neuroendocrine secretory protein of M(r) 55,000) is a paternally imprinted proteoglycan, expressed specifically in endocrine cells and the nervous system. We investigated the subcellular localization and secretion of NESP55 in AtT-20 cells. NESP55 accumulated in the medium linearly over 24 h exceeding its intracellular content 3.7-fold by that time. Incubation of cells at 16 degrees C, to block protein export, inhibited basal secretion by 79%. Stimulation of AtT-20 cells with 8-Br-cAMP increased secretion of NESP55 by only 45%. The NESP55 secretory vesicles sedimented at a density of 1.2-1.4 M, which is slightly lighter than that of the large dense core vesicles. Immunofluorescence studies revealed immunoreactivity in the Golgi apparatus and a punctuate staining of processes or neurites. Our data demonstrate that NESP55 is mainly sorted to and released from a population of constitutive secretory vesicles, which are transported out of the perikarya into processes or axons. In addition, some NESP55 is also routed to the regulated pathway. The signal peptide of NESP55, as determined with peptide antisera, is 46 amino acids long and represents the best conserved region of this molecule suggesting that the signal peptide may have a function of its own. The subcellular localization and export of NESP55 from cells are reminiscent of neuronal proteoglycans forming the extracellular matrix, which are implicated in the development and maintenance of neuronal circuits and mechanisms of axonal guidance.     

Snx14 Regulates Neuronal Excitability,Promotes Synaptic Transmission,and Is Imprinted in the Brain of Mice

Genomic imprinting describes an epigenetic process through which genes can be expressed in a parent-of-origin-specific manner. The monoallelic expression of imprinted genes renders them particularly susceptible to disease causing mutations. A large proportion of imprinted genes are expressed in the brain, but little is known about their functions. Indeed, it has proven difficult to identify cell type-specific imprinted genes due to the heterogeneity of cell types within the brain. Here we used laser capture microdissection of visual cortical neurons and found evidence that sorting nexin 14 (Snx14) is a neuronally imprinted gene in mice. SNX14 protein levels are high in the brain and progressively increase during neuronal development and maturation. Snx14 knockdown reduces intrinsic excitability and severely impairs both excitatory and inhibitory synaptic transmission. These data reveal a role for monoallelic Snx14 expression in maintaining normal neuronal excitability and synaptic transmission.     

Isolation and imprinting analysis of the porcine DLX5 gene and its association with carcass traits

Imprinted genes play important roles in mammalian growth and development. However, reports on imprinted genes are limited in livestock. In this study, the complete ORF containing 289 amino acids of the porcine DLX5 gene was obtained. A C-to-T SNP mutation in exon 1 of the DLX5 gene was used to detect imprinting status with an RT-PCR/RFLP test (using HhaI) in eight heterozygous pigs from a population of Large White × Meishan F₁ hybrids. Imprinting analysis showed that the porcine DLX5 gene was maternally expressed in skeletal muscle, fat, lung, spleen, stomach and small intestine, but not imprinted in heart, liver, kidney, uterus, ovary, testicle or pituitary. A PCR–RFLP test was also used to detect the polymorphism in 310 pigs of a Large White × Meishan F₂ resource population. The statistical results showed significant association ( P  < 0.01) of the genotypes and fat meat percentage, carcass length, bone percentage, 6–7 rib fat thickness, average backfat thickness, thorax-waist fat thickness and buttock fat thickness.