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Biallelic Expression of Tssc4, Nap1l4, Phlda2 and Osbpl5 in Adult Cattle

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Biallelic Expression of Tssc4, Nap1l4, Phlda2 and Osbpl5 in Adult Cattle c Indian Academy of Sciences RESEARCH ARTICLE Biallelic expression of Tssc4, Nap1l4, Phlda2 and Osbpl5 in adult cattle MENGNAN WANG1, DONGJIE LI2, MINGYUE ZHANG1, WENZHI YANG1, GUOJIANG WU1, YALI CUI1 and SHIJIE LI1∗ 1College of Life Science, Agricultural University of Hebei, Baoding 071001, People’s Republic of China 2College of Life Science and Life Engineering, Science and Technology University of Hebei, Shijiazhuang 050018, People’s Republic of China Abstract Genomic imprinting of the Cdkn1c/Kcnq1ot1 region shows lack of conservation between human and mouse. This region has been reported to be associated with Beckwith–Wiedemann syndrome (BWS) and cancer. To increase our understanding of imprinted genes in bovine Cdkn1c/Kcnq1ot1 imprinting cluster, we assessed the imprinting status of four cattle genes (Tssc4, Nap1l4, Phlda2 and Osbpl5) in seven types of tissues: heart, liver, spleen, lung, kidney, skeletal muscle and subcutaneous fat using polymorphism-based sequencing approach. It was found that all the four genes showed biallelic expression in tissues in which transcripts were detected. Nap1l4 and Tssc4 were detected in all examined tissues, while the expression of Phlda2 and Osbpl5 was tissue-specific. Phlda2 was not detected in heart and subcutaneous fat, and Osbpl5 was not detected in spleen and skeletal muscle. In addition, identification of species-specific imprinted genes is necessary to understand the evolution of genomic imprinting and to elucidate mechanisms leading to allele-specific expression. [Wang M., Li D., Zhang M., Yang W., Wu G., Cui Y. and Li S. 2015 Biallelic expression of Tssc4, Nap1l4, Phlda2 and Osbpl5 in adult cattle. J. Genet. 94, 391–395] Introduction Characterization of the allele-specific expression profile of imprinted genes at different developmental stages is neces- Genomic imprinting is an epigenetic process where imprin- sary to understand the function of imprinted genes and to ted genes are expressed monoallelically depending on their elucidate the mechanisms behind imprinting. parental origin (see Morison et al. 2005; Hasan et al. 2007). The Cdkn1c/Kcnq1ot1 region is one of the largest known At present, around 200 genes are known to be imprin- imprinted clusters. In humans, it is located on chromosome ted in mice and humans, but only a few have been iden- 11p15.5 and in mouse, on distal chromosome 7. It also har- tified in cattle (http://otago.ac.nz/IGC/). The imprinting bours multiple imprinted genes (Reik et al. 2003). Altered status of many genes is conserved among mammalian imprinting of this region is associated with human diseases species, but some genes show species-specific imprinting such as Beckwith–Wiedemann syndrome (BWS) and many (Paulsen et al. 2000). Examining the extent of genetic types of tumours (Mitsuya et al. 1999). Cattle are a better imprinting among mammalian species is useful in under- model organism than mouse, since shorter evolutionary dis- standing the evolution of genomic imprinting. Many tance to human and similar timing of preimplantation devel- imprinted genes play significant roles in embryonic growth, opment. In the present study, the imprinting status of four development, adult behaviour, meat yield, lean meat percent- genes (Tssc4, Nap1l4, Phlda2 and Osbpl5) was investigated age and feed efficiency in mammals. In addition, imprinted in cattle. Tumour suppressing subtransferable candidate 4 genes are closely related to disease susceptibility and immu- (Tssc4), an important tumour-suppressor gene, plays a role in nity (Reik and Walter 2001;Reiket al. 2003; Kawahara malignancies and diseases that involve the Cdkn1c/Kcnq1ot1 and Kono 2012). Currently, imprinting status is gener- region (Paulsen et al. 2000). Nucleosome assembly protein ally studied in mammalian embryos and placental tissues. 1-like 4 (Nap1l4) also known as NAP-2, encodes a nucleo- Some imprinted genes exhibit development stage-specific some assembly protein that belongs to the NAPs family of imprinting patterns (Ohlsson et al. 1994;Leeet al. 1997). proteins (Rodriguez et al. 1997; Okuwaki et al. 2010). Pleck- strin homology-like domain, family A, member 2 (Phlda2), ∗For correspondence. E-mail: [email protected]. also known as Ipl or Tssc3, encodes a cytoplasmic protein Keywords. bovine; Tssc4; Nap1l4; Phlda2; Osbpl5; allele-specific expression; imprinting. Journal of Genetics, Vol. 94, No. 3, September 2015 391 Mengnan Wang et al. and negatively controls placental development (Frank et al. identified in heterozygous individuals were used to assess 2002; Salas et al. 2004; Tunster et al. 2010). Oxysterol- genomic imprinting. binding protein-like 5 (Osbpl5) play a key role in maintain- ing the cholesterol balance in the body. These four genes are known to be maternally imprinted in mouse placenta at the Total RNA extraction and reverse transcription Cdkn1c/Kcnq1ot1 region. Total RNA was isolated from tissue samples of heterozygous calves using the Trizol RNA Extracted Kit (Invitrogen, Shanghai, China) and treated with RNase-free DNase to Materials and methods remove possible contaminating genomic DNA. The quality and quantity of the RNA was tested using a ND-1000 spectro- Animals and tissues photometer (NanoDrop, Wilmington, USA). Approximately Tissue samples of heart, liver, spleen, lung, kidney, skele- 2 µg RNA was reverse transcribed using an RT kit (Promega, tal muscle and subcutaneous fat of 32 dairy cattle (Holstein) Beijing, China). The RT reaction was conducted according to were collected from a local abattoir. After collecting all tissue the manufacturer’s guidelines using oligo (dT) primers and 5 samples, they were immediately frozen in liquid nitrogen and units of Superscript RT enzyme in 20 µL total volume. stored at –70◦C until further analysis. All protocols involving use of animals were approved by the Agriculture Research Animal Care Committee of Hebei Agriculture University. RT-PCR RT-PCR was used to determine the expression patterns of Tssc4, Nap1l4, Phlda2 and Osbpl5 in tissues: heart, liver, DNA extraction and PCR amplification spleen, lung, kidney, skeletal muscle and subcutaneous fat. Genomic DNA was extracted from liver tissues of 32 All primers were designed according to the retrieved adult cattle using DNA Extraction Kit (Sangon Biotech, sequences of Tssc4 (GenBank accession no. NM_0010754 Shanghai, China) following the manufacturer’s instruction. 10), Nap1l4 (GenBank accession no. NM_001038094), Single-nucleotide polymorphisms (SNPs) were identified in Phlda2 (GenBank accession no. NM_001076521) and heterozygous individuals and confirmed by sequencing the Osbpl5 (GenBank accession no. XM_002699428) in cat- PCR products. All primers were designed using Primer5 and tle. In RT-PCR reactions, all primers were designed to Oligo6 (table 1). PCR sample mixtures were as follows: 1 µL cross introns to exclude genomic DNA. A 367 bp fragment primers (10 µM), 1 µL genomic DNA, 9.5 µL ddH2Oand of the Gapdh gene was amplified as the internal control 12.5 µL ES Tap MasterMix (CWBio, Beijing, China). PCR using primers (Gap-F: GCACAGTCAAGGCAGAA AC and amplification conditions were as follows: initial denaturation Gap-R: GCGTGGACAGTGGTCATAAG) designed based at 94◦C for 5 min, followed by 35 cycles of denaturation at on the GenBank sequence (accession no. BTU85042). The 94◦C for 30 s, annealing at 56◦CforTssc4,50◦CforNap1l4 cDNA of Tssc4, Nap1l4, Phlda2, Osbpl5 and Gapdh were 48◦CforPhlda2 and 53◦CforOsbpl5 for 30 s, elongation amplified using the following programme: initial denatura- at 72◦C for 70 s, and a final extension at 72◦C for 10 min. tion at 94◦C for 5 min, followed by 35 cycles of denatu- Table 1 shows the primer sequences used to amplify the ration at 94◦C for 30 s, annealing at 56◦CforTssc4,47◦C genomic DNA and the expected PCR product sizes. SNPs for Nap1l4,48◦CforPhlda2,55◦CforOsbpl5 and 50◦C Table 1. Primer sequences and PCR information. Gene Primers (5′–3′) Annealing temp. (◦C) PCR size (bp) Product type Tssc4 Tss-Fg: CCTTCGTCAGCATTCA 56 1453 DNA Tss-Rg: TGGTCCACACAGATACAC Tss-F: CCTTCGTCAGCATTCA 56 752 cDNA Tss-R: TCCTGGATACCCTCTTC Nap1l4 Nap-Fg : GCTGGAGAAGCGAAGTGACA 50 1119 DNA Nap-Rg: ACTCTGGAGGCTCAGTGAAT Nap-F: AGGGGATGGAGAATCACTGG 47 751 cDNA Nap-R: TTGGAGGACAAGCGAGAGAG Phlda2 Phl-Fg: AGCTGGATAAGCGAAGTGAC 48 937 DNA Phl-Rg: AGAAAAGAGAATGAACCGAG Phl-F: GCTGGAGAAGCGAAGTGACA 48 539 cDNA Phl-R: ACTCTGGAGGCTCAGTGAAT Osbpl5 Osb-Fg: CTCCGTTTTCACCAGA 53 946 DNA Osb-Rg: GAAGGGAAGATCGTGC Osb-F: GTCACCGAGAGCAGCG 55 825 cDNA Osb-R: AGAGGGCAGAAGGAAGC 392 Journal of Genetics, Vol. 94, No. 3, September 2015 Expression of bovine Tssc4, Nap1l4, Phlda2 and Osbpl5 for Gapdh for 30 s, elongation at 72◦C for 50 s, and a final extension at 72◦C for 10 min. The PCR reaction mixture (25 µL) contained 1 µL primers (10 µM), 1 µL cDNA, 9.5 µL ddH2O and 12.5 µL ES Tap MasterMix (CWBio). Table 1 shows the primer sequences used to amplify the cDNA and the expected PCR product sizes. The PCR prod- ucts were purified using UNIQ-10 column DNA gel extrac- tion kit (Sangon, China) and sequenced directly using the dideoxy chain-termination method. Allele-specific imprinting patterns analysis For imprinting assays, 12 heterozygous individuals (three heterozygous individuals per line per time point) of Tssc4, Figure 2. Expression analysis of cattle Tssc4, Nap1l4, Phlda2 and Nap1l4, Phlda2 and Osbpl5 were slaughtered to extract Osbpl5 genes. M, DNA marker DL2000 (1000, 750, 500 and 250 RNA samples. All cDNA samples were reverse transcribed bp). Lanes 1–7 are RT-PCR products obtained from heart, liver, spleen, lung, kidney, muscle and subcutaneous fat. The size of from total RNA prepared from the organs of three heterozy- RT-PCR production were 367bp for GADPH. gous animals. PCR products were purified and sequenced directly. Imprinted genes are expected to exhibit hemizy- Nap1l4, Phlda2 and Osbpl5 genes were expressed virtually gosity, whereas a biallelically-expressed gene would exhibit in all analysed tissues, with the exception of Phlda2,which heterozygosity at the SNP. was not detected in heart and subcutaneous fat tissues, as well as Osbpl5, which was not detected in spleen and skeletal Results muscle tissues (figure 2).
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