NNAT and DIRAS3 genes are paternally expressed in pigs

Although expression and epigenetic differences of imprinted genes have been extensively characterised in man and the mouse, little is known on livestock species. In this study, the polymorphism-based approach was used to detect the imprinting status of NNAT and DIRAS3 genes in five heterozygous pigs (based on SNP) of Large White and Meishan F1 hybrids. The results show that both genes were paternally expressed in all the tested tissues (heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus, ovary and pituitary). In addition, the NNAT gene had two transcripts in all tested tissues, which is consistent with its counterpart in man and cattle.


INTRODUCTION
Genomic imprinting is a parent-of-origin-dependent epigenetic mechanism, in which a subset of autosomal genes is expressed from only one allele [15]. Imprinted genes have important roles in the regulation of foetal growth, development, function of the placenta and postnatal behaviour, in mammals in particular [14]. At present, more than 120 imprinted genes have been identified in man and mice, but only ten imprinted genes have been identified in sheep, seven in cattle and three in pigs (http://igc.otago.ac.nz/home.html). Therefore, it is of interest to identify other imprinted genes in pigs in order to analyse the conservation of genomic imprinting among different species. NNAT located on chromosomes 20q11.2-q12 in man and 2H1 in mice is preferentially expressed from the paternal allele [6,17]. The protein encoded by NNAT is a proteolipid that may be involved in the regulation of ion channels during brain development [5]. Chu and Tsai [3] have found that NNAT plays an important role in insulin secretion. In man and cattle, NNAT has two alternatively spliced transcripts (α and β), either with or without exon 2 [8,21]. DIRAS3 is a maternally imprinted tumour suppressor gene and its expression decreases dramatically in ovarian cancers [9,19]. Yu et al. [20] have reported that transgenic expression of the human DIRAS3 gene in the mouse produces a small stature and that the gene inhibits growth.
In this study, we obtained the complete coding region of porcine NNAT and DIRAS3 genes. The imprinted status of porcine NNAT and DIRAS3 genes was detected using SNP present in the exons of the two genes in five heterozygous pigs (based on SNP). We demonstrate that NNAT also has two transcripts in all tested tissues.

Experimental animals and DNA isolation
All animals in this study were from the Experimental pig station of Huazhong Agricultural University. Ten two-month F 1 hybrid pigs from Large White boars × Meishan sows and ten from Meishan boars × Large White sows were used to search for individuals heterozygous for the two genes. Genomic DNA from the twenty F 1 hybrids and their mothers were isolated according to the standard phenol-chloroform method.

RNA isolation and cDNA synthesis
Total RNA from twelve tissues (heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus, ovary and pituitary) of five heterozygous pigs of the twenty F 1 hybrids were isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, US) according to the manufacturer's instructions. First strand cDNA was synthesised from 2 µg total RNA treated with DNAse I (TaKaRa, Tokyo, Japan) in a 20 µL reaction volume containing 5 µM oligo(dT) 18

PCR of DNA and cDNA
The human NNAT cDNA sequence (GenBank: NM_005386) and DI-RAS3 cDNA sequence (GenBank: NM_004675) were used to identify porcine expressed sequence tags (EST) through standard BLAST (http://www.ncbi.nlm.nih.gov/blast/) searches in the 'EST-others' database. Pig EST sharing more than 85% sequence identity with the human cDNA sequences were assembled into EST-contigs. The exon-intron structures of porcine NNAT and DIRAS3 genes were estimated according to the structure of the two genes in man. All primers were designed from the consensus sequences of EST-contigs (Tab. I). The PCR conditions were as follows: 94 • C for 4 min, 35 cycles of 94 • C for 45 s, annealing at optimal temperature (Tab. I), 72 • C for 1 min and a final extension at 72 • C for 7 min. Primer pair HouseF/HouseR, which amplified the fragment spanning intron 8 of the GAPDH gene was applied to exclude the possibility of DNA contamination during all RT-PCR reactions (Fig. 1A).

Sequencing and SNP detection
PCR products amplified by primer pairs NN1F/NN1R, NN2F/NN2R and DIF/DIR were purified with the Wizard prep PCR purification system (Promega) and sequenced commercially. The site was considered as a heterozygous mutation if double peaks appeared at similar heights in the sequence bands.  Table I.

Imprinting analysis and transcript identification of the NNAT gene
RT-PCR of primer pair NN1F/NN1R indicated that the NNAT gene has two transcripts in all tissues examined (heart, liver, spleen, lung, kidney, stomach, small intestine skeletal muscle, fat, uterus, ovary and pituitary) (Fig. 1B). Sequencing of the two RT-PCR products showed that the two transcripts differ in exon 2. Transcript α has 81 amino acids and transcript β has 54 amino acids. Primer pair NN2F/NN2R amplified DNA from the heterozygous pigs and their mothers and cDNA from various tissues of the heterozygous pigs. Sequencing of the PCR and RT-PCR products revealed that the NNAT gene is paternally expressed in all the tissues tested. The three heterozygous pig hybrids of Large White boars × Meishan sows expressed the T allele but their mothers showed the C allele in genomic DNA, and the two heterozygous pig hybrids of Meishan boars × Large White sows expressed the C allele but their mothers showed the T allele in genomic DNA (Fig. 2). The five heterozygous pigs had the same imprinted status.

Imprinting analysis of the DIRAS3 gene
RT-PCR of the same tissues as above from the five pigs with primer pair DIIF/DIIR indicates that the DIRAS3 gene was expressed in all tissues examined (Fig. 1C). The primer pair also amplified DNA from the heterozygous pigs and their mothers. Sequencing of the PCR and RT-PCR products showed that the DIRAS3 gene is also paternally expressed in all tissues tested. The three heterozygous pigs of Large White boars × Meishan sow hybrids expressed the T allele but their mothers showed the G allele in genomic DNA, and the two heterozygous pigs of Meishan boars × Large White sows expressed the G allele but their mothers showed the T allele in genomic DNA (Fig. 2). There was no difference in the imprinted status of the gene in all tissues from the five heterozygous pigs.

DISCUSSION
At present, most imprinted genes have been identified and studied in man and mice and few reports exist on imprinting in livestock species. Therefore, identifying more imprinted genes in livestock species should be useful for the study of the conservation and function of imprinted genes. It has been reported that in the adult mouse, NNAT is expressed in the brain, pituitary gland, lungs, adrenal glands, uterus, skeletal muscles, ovaries, and pancreas [2,7,12,16]. Our study, as compared with these studies, shows that in the pig, NNAT is expressed in the pituitary gland, lungs, uterus, skeletal muscles and ovaries, but also in the heart, liver, spleen, kidney, stomach, small intestine and fat. These observations indicate that NNAT is expressed in a wide range of tissues. Xu et al. [18] showed that the DIRAS3 gene acts as a negative regulator in mouse growth and development. Luo et al. [10] reported that an appropriate methylation status of the CpG islands in the promoter region may play a role in the down-regulation of DIRAS3 gene expression. Therefore, our study should be useful to study whether the appropriate methylation status of the CpG islands affects porcine growth and development.
NNAT and DIRAS3 genes are both preferentially expressed paternal alleles in the human and mouse foetus [6,17]. In the present study, the imprinting analysis of the two genes in five heterozygous pigs shows that the two genes are both paternally expressed in all tested tissues. The results confirm the conservation of genomic imprinting among different species, although there are some studies reporting species-specific and tissue-specific imprinting [4,11].
Aikawa et al. [1] and Zaitoun et al. [21] have reported that the NNAT gene has two transcripts in the pig and bovine foetus, and that the two transcripts are paternally expressed in cattle. Our results were consistent with these reports since they indicate that in all tissues tested from the five two-month old pigs, the NNAT gene also has two transcripts and both express paternal alleles. Alternative splicing has recently emerged as a major mechanism of generating protein diversity in higher eukaryotes [13]. The alternative splicing of the NNAT gene may be useful to study the function of the NNAT protein.