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.     

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.     

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.     

Expression and genomic imprinting of the porcine Rasgrf1 gene

Imprinted genes play important roles in mammalian growth, development and behavior. The Rasgrf1 (Ras protein-specific guanine nucleotide exchange factor 1) gene has been identified as an imprinted gene in mouse and rat. In the present study, we detected its sequence, imprinting status and expression pattern in the domestic pigs. A 228 bp partial sequence located in exon 14 and a 193 bp partial sequence located in exon 1 of the Rasgrf1 gene in domestic pigs were obtained. A G/A transition, was identified in Rasgrf1 exon 14, and then, the reciprocal Berkshire × Wannan black F₁ hybrid model and the RT-PCR-RFLP method were used to detect the imprinting status of porcine Rasgrf1 gene at the developmental stage of 1-day-old. The expression profile results indicated that the porcine Rasgrf1 mRNA was highly expressed in brain, pituitary and pancreas, followed by kidney, stomach, lung, testis, small intestine, ovary, spleen and liver, and at low levels of expression in longissimus dorsi, heart, and backfat. The expression levels of Rasgrf1 gene in brain, pituitary and pancreas tissues were significantly different between the two reciprocal F₁ hybrids. Imprinting analysis showed that porcine Rasgrf1 gene was maternally expressed in the liver, small intestine, paternally expressed in the lung, but biallelically expressed in brain, heart, spleen, kidney, stomach, pancreas, backfat, testis, ovary, longissimus dorsi and pituitary tissues.     

Imprinting analyses of the porcine GATM and PEG10 genes in placentas on days 75 and 90 of gestation

Imprinted genes are expressed monoallelically depending on their parental origin, and escape the Mendel's laws of heredity. They play important roles in the mammalian development, growth, and behavior. Placenta is a key tissue for the normal development and growth of fetus. It is also used to illuminate the evolution of genomic imprinting. In this study, we cloned the porcine GATM and PEG10 genes. Somatic cell hybrid panel (SCHP) and porcine radiation hybrid (IMpRH) panel were employed to locate GATM and PEG10 genes to SSC1q12-21 and SSC9p13-21, respectively. By sequencing PCR products, we detected several cSNPs in the two genes. The BseLI (GATM) and TaqI (PEG10) polymorphisms were used to investigate the allele frequencies in different pig breeds and the imprinting status in porcine placentas on days 75 and 90 of gestation. The results showed that for the GATM BseLI polymorphism, the Yorkshire and Duroc pigs had higher allele frequencies at the G allele, whereas the local pigs had higher allele frequencies at the A allele. Expression and sequencing analyses showed that both alleles were expressed for the GATM gene, indicating the GATM was not imprinted in the porcine placentas on days 75 and 90 of gestation. The allele frequencies of TaqI polymorphism for PEG10 gene were significantly different in native Chinese Erhualian breed comparing to Yorkshire. PEG10 was monoallelically expressed, showing the PEG10 gene may be imprinted in porcine placentas on days 75 and 90 of gestation.     

Conservation of imprinting of MKRN3 and NAP1L5 in rabbits

Maternally imprinted genes of makorin ring finger protein 3 (MKRN3) and nucleosome assembly protein 1‐like 5 (NAP1L5) have been identified in many species but have not yet been investigated in rabbits. In this study, a polymorphism‐based approach and bisulfite‐sequencing PCR (BSP) were used to determine the imprinting status of MKRN3 and NAP1L5 in rabbits. The single nucleotide polymorphism (SNP)‐based sequencing results demonstrated that MKRN3 and NAP1L5 were expressed preferentially from the paternal allele. Furthermore, the BSP results showed the gamete‐specific methylation patterns and hemimethylation in brain and full methylation in liver were observed in MKRN3 and NAP1L5 respectively. Thus, we provide the first evidence that MKRN3 and NAP1L5 are paternally expressed genes and that the CpG islands located in the promoter region may be the putative differentially methylated region of these two genes in rabbits.     

Localizing transcriptional regulatory elements at the mouse Dlk1 locus

Much effort has focused recently on determining the mechanisms that control the allele-specific expression of genes subject to genomic imprinting, yet imprinting regulation is only one aspect of configuring appropriate expression of these genes. Imprinting control mechanisms must interact with those regulating the tissue-specific expression pattern of each imprinted gene in a cluster. Proper expression of the imprinted Delta-like 1 (Dlk1)-Maternally expressed gene 3 (Meg3) gene pair is required for normal fetal development in mammals, yet the mechanisms that control tissue-specific expression of these genes are unknown. We have used a combination of in vivo and in vitro expression assays to localize cis-regulatory elements that may regulate Dlk1 expression in the mouse embryo. A bacterial artificial chromosome transgene encompassing the Dlk1 gene and 77 kb of flanking sequence conferred expression in most endogenous Dlk1-expressing tissues. In combination with previous transgenic data, these experiments localize the majority of Dlk1 cis-regulatory elements to a 41 kb region upstream of the gene. Cross-species sequence conservation was used to further define potential regulatory elements, several of which functioned as enhancers in a luciferase expression assay. Two of these elements were able to drive expression of a lacZ reporter transgene in Dlk1-expressing tissues in the mouse embryo. The sequence proximal to Dlk1 therefore contains at least two discrete regions that may regulate tissue-specificity of Dlk1 expression.     

Genomic imprinting at the mammalian Dlk1-Dio3 domain

Genomic imprinting causes genes to be expressed or repressed depending on their parental origin. The majority of imprinted genes identified to date map in clusters and much of our knowledge of the mechanisms, function and evolution of imprinting have emerged from their analysis. The cluster of imprinted genes delineated by the delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (Dlk1-Dio3) is located on distal mouse chromosome 12 and human chromosome 14. Its developmental importance is exemplified by severe phenotypes associated with altered dosage of these genes in mice and humans. The domain contains three imprinted protein-coding genes, Dlk1, Rtl1 and Dio3, expressed from the paternally inherited chromosome and several imprinted large and small noncoding RNA genes expressed from the maternally inherited homolog. Here, we discuss the function and regulation of imprinting at this domain.     

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.     

Post-natal imprinting: evidence from marsupials

Genomic imprinting has been identified in therian (eutherian and marsupial) mammals but not in prototherian (monotreme) mammals. Imprinting has an important role in optimising pre-natal nutrition and growth, and most imprinted genes are expressed and imprinted in the placenta and developing fetus. In marsupials, however, the placental attachment is short-lived, and most growth and development occurs post-natally, supported by a changing milk composition tailor-made for each stage of development. Therefore there is a much greater demand on marsupial females during post-natal lactation than during pre-natal placentation, so there may be greater selection for genomic imprinting in the mammary gland than in the short-lived placenta. Recent studies in the tammar wallaby confirm the presence of genomic imprinting in nutrient-regulatory genes in the adult mammary gland. This suggests that imprinting may influence infant post-natal growth via the mammary gland as it does pre-natally via the placenta. Similarly, an increasing number of imprinted genes have been implicated in regulating feeding and nurturing behaviour in both the adult and the developing neonate/offspring in mice. Together these studies provide evidence that genomic imprinting is critical for regulating growth and subsequently the survival of offspring not only pre-natally but also post-natally.     

Variable allelic expression of imprinted genes in human pluripotent stem cells during differentiation into specialized cell types in vitro

Genomic imprinting is an epigenetic phenomenon by which a subset of genes is asymmetrically expressed in a parent-of-origin manner. However, little is known regarding the epigenetic behaviors of imprinted genes during human development. Here, we show dynamic epigenetic changes in imprinted genes in hESCs during in vitro differentiation into specialized cell types. Out of 9 imprinted genes with single nucleotide polymorphisms, mono-allelic expression for three imprinted genes (H19, KCNQ1OT1, and IPW), and bi- or partial-allelic expression for three imprinted genes (OSBPL5, PPP1R9A, and RTL1) were stably retained in H9-hESCs throughout differentiation, representing imprinting stability. Three imprinted genes (KCNK9, ATP10A, and SLC22A3) showed a loss and a gain of imprinting in a lineage-specific manner during differentiation. Changes in allelic expression of imprinted genes were observed in another hESC line during in vitro differentiation. These findings indicate that the allelic expression of imprinted genes may be vulnerable in a lineage-specific manner in human pluripotent stem cells during differentiation.     

Parental genomic imprinting in plants: significance for reproduction

Parental genomic imprinting is an epigenetic phenomenon causing the expression of a gene from one of the two parental alleles. Imprinting has been identified in plants and mammals. Recent evidence shows that DNA methylation and histone modifications are responsible for this parent-of-origin dependent expression of imprinted genes. We review the mechanisms and functions of imprinting in plants. We further describe the significance of imprinting for reproduction and discuss potential models for its evolution.     

Genome-wide analysis of genomic imprinting in the endosperm and allelic variation in flax

Genomic imprinting is an epigenetic phenomenon that causes biased expression of maternally and paternally inherited alleles. In flowering plants, genomic imprinting predominantly occurs in the triploid endosperm and plays a vital role in seed development. In this study, we identified 248 candidate imprinted genes including 114 maternally expressed imprinted genes (MEGs) and 134 paternally expressed imprinted genes (PEGs) in flax (Linum usitatissimum L.) endosperm using deep RNA sequencing. These imprinted genes were neither clustered in specific chromosomal regions nor well conserved among flax and other plant species. MEGs tended to be expressed specifically in the endosperm, whereas the expression of PEGs was not tissue-specific. Imprinted single nucleotide polymorphisms differentiated 200 flax cultivars into the oil flax, oil-fiber dual purpose flax and fiber flax subgroups, suggesting that genomic imprinting contributed to intraspecific variation in flax. The nucleotide diversity of imprinted genes in the oil flax subgroup was significantly higher than that in the fiber flax subgroup, indicating that some imprinted genes underwent positive selection during flax domestication from oil flax to fiber flax. Moreover, imprinted genes that underwent positive selection were related to flax functions. Thirteen imprinted genes related to flax seed size and weight were identified using a candidate gene-based association study. Therefore, our study provides information for further exploration of the function and genomic variation of imprinted genes in the flax population.