猪MKRN3基因的印记表达和DNA甲基化状态分析

doi:10.11843/j.issn.0366-6964.2024.09.011

Abstract

Abstract:

The aim of this study was to investigate the imprinting status of the MKRN3 gene and its DNA methylation level in wild type and cloned pigs. The imprinting status of the MKRN3 gene in the 3 wild-type crossbred pigs, which were born at full term, were examined using interspecies single nucleotide polymorphism(SNP) present in Western (Duroc) and local (Bama or Rongchang) pig breeds. CpG islands near the MKRN3 promoter region were predicted using the MethPrimer website, and CpGs were verified as differentially methylated region (DMR) in oocyte and sperm genomes. The genomes of the 3 wild-type hybrid and 2 cloned pigs were sequenced after bisulfite transformation to detect the methylation status of MKRN3-DMR in wild-type and cloned pigs, and to analyze the relationship between methylation status and expression. The results showed that a single nucleotide polymorphism (SNP) existed in the coding region of the MKRN3 gene between Western (Duroc) and local (Bama and Rongchang) pig breeds: A/G; RT-PCR sequencing showed that MKRN3 was bi-allelic expressed in the heart tissue of one of the wild-type individual (offspring of a cross between Duroc pig and Bama pig: DB2), and monoallelic expressed in organs such as liver, spleen, lungs, and brain tissues, and all of them expressed the allele from paternal origin. MKRN3-DMR showed hypomethylation (0%) in spermatozoa and showed hypermethylation (70.9%) in oocytes. The average methylation levels of MKRN3-DMR in the six tissues of hybrid pigs were: heart (43.1%), liver (46.2%), spleen (48.4%), lung (45.6%), kidney (48.5%), and brain (44.3%), respectively. In most tissues of the newborn cloned pigs, MKRN3 was expressed as the allele of paternal origin, and the level of MKRN3-DMR methylation was similar to that of the wild-type crossbred pigs. While MKRN3-DMR methylation assay results showed that the methylation levels were 79.0% and 80.1% in spleen and spleen of NT201 and NT207, respectively, SNP sequencing results also indicated that the imprinted state was no longer maintained in the two cloned pig spleens. It is suggested that MKRN3 imprinting status was abnormal in cloned pig spleen tissues. In summary, MKRN3 was maternally imprinted and paternally expressed in liver, spleen, lung, kidney and brain tissues of wild-type pigs, and was in a non-imprinted state in the heart; MKRN3 imprinted expression and methylation status were abnormal in some tissues of the cloned pigs. The expression of MKRN3 was regulated by the DMR of the promoter.

Key words: DNA methylation, porcine, DMR, genomic imprinting

CLC Number:

S828.2

Nanzhu CHEN, Junliang LI, Dawei YU, Xinyi ZHOU, Jing WANG, Huiying ZOU, Weihua DU. Analysis of Imprinted Expression and DNA Methylation Status of the Porcine MKRN3 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3853-3863.

Figures/Tables 8

Table 1

Table 2

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

References 36

1	LIAO J , SONG S M , GUSSCOTT S , et al. Establishment of paternal methylation imprint at the H19/Igf2 imprinting control region[J]. Sci Adv, 2023, 9 (36): eadi2050. doi: 10.1126/sciadv.adi2050
2	PLASSCHAERT R N , BARTOLOMEI M S . Genomic imprinting in development, growth, behavior and stem cells[J]. Development, 2014, 141 (9): 1805- 1813. doi: 10.1242/dev.101428
3	CARRION S A , MICHAL J J , JIANG Z H . Imprinted genes: genomic conservation, transcriptomic dynamics and phenomic significance in health and diseases[J]. Int J Biol Sci, 2023, 19 (10): 3128- 3142. doi: 10.7150/ijbs.83712
4	MONK D , MACKAY D J G , EGGERMANN T , et al. Genomic imprinting disorders: lessons on how genome, epigenome and environment interact[J]. Nat Rev Genet, 2019, 20 (4): 235- 248. doi: 10.1038/s41576-018-0092-0
5	PENG X Q , LUO H Z , KONG X Y , et al. Metrics for evaluating differentially methylated region sets predicted from BS-seq data[J]. Brief Bioinform, 2022, 23 (1): bbab475. doi: 10.1093/bib/bbab475
6	MCEWEN K R , FERGUSON-SMITH A C . Distinguishing epigenetic marks of developmental and imprinting regulation[J]. Epigenetics Chromatin, 2010, 3 (1): 2. doi: 10.1186/1756-8935-3-2
7	ARNAUD P . Genomic imprinting in germ cells: imprints are under control[J]. Reproduction, 2010, 140 (3): 411- 423. doi: 10.1530/REP-10-0173
8	ZALETAEV D V , NEMTSOVA M V , STRELNIKOV V V . Epigenetic regulation disturbances on gene expression in imprinting diseases[J]. Mol Biol (Mosk), 2022, 56 (1): 1- 28. doi: 10.1134/S0026893321050149
9	LUO X M , ZHANG T J , ZHAI Y X , et al. Effects of DNA methylation on TFs in human embryonic stem cells[J]. Front Genet, 2021, 12, 639461. doi: 10.3389/fgene.2021.639461
10	STEINHOFF C , PAULSEN M , KIELBASA S , et al. Expression profile and transcription factor binding site exploration of imprinted genes in human and mouse[J]. BMC Genomics, 2009, 10, 144. doi: 10.1186/1471-2164-10-144
11	ECKHARDT F , LEWIN J , CORTESE R , et al. DNA methylation profiling of human chromosomes 6, 20 and 22[J]. Nat Genet, 2006, 38 (12): 1378- 1385. doi: 10.1038/ng1909
12	SINGH A , RAPPOLEE D A , RUDEN D M . Epigenetic reprogramming in Mice and Humans: from fertilization to primordial germ cell development[J]. Cells, 2023, 12 (14): 1874. doi: 10.3390/cells12141874
13	FANIS P , MORROU M , TOMAZOU M , et al. Methylation status of hypothalamic Mkrn3 promoter across puberty[J]. Front Endocrinol (Lausanne), 2023, 13, 1075341. doi: 10.3389/fendo.2022.1075341
14	LI J L , CHEN W N , LI D J , et al. Conservation of imprinting and methylation of MKRN3, MAGEL2 and NDN genes in cattle[J]. Animals (Basel), 2021, 11 (7): 1985.
15	张永杰. 多浪羊MKRN3、NDN、MAGEL2基因的印记和甲基化状态分析[D]. 阿拉尔市: 塔里木大学, 2023.
	ZHANG Y J. Analysis of imprinting and methylation status of MKRN3, NDN and MAGEL2 genes in Dolomite sheep[D]. Alaer: Tarim University, 2023. (in Chinese)
16	LI C Y , LU W L , YANG L G , et al. MKRN3 regulates the epigenetic switch of mammalian puberty via ubiquitination of MBD3[J]. Natl Sci Rev, 2020, 7 (3): 671- 685. doi: 10.1093/nsr/nwaa023
17	LI C Y , HAN T T , LI Q R , et al. MKRN3-mediated ubiquitination of Poly(A)-binding proteins modulates the stability and translation of GNRH1 mRNA in mammalian puberty[J]. Nucleic Acids Res, 2021, 49 (7): 3796- 3813. doi: 10.1093/nar/gkab155
18	KARAMI K , ZEREHDARAN S , JAVADMANESH A . Differential expression of RNAseq imprinted genes from bovine females before and after puberty[J]. Biochem Genet, 2023, 61 (6): 2633- 2649. doi: 10.1007/s10528-023-10395-9
19	NIEMANN H , TIAN X C , KING W A , et al. Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning[J]. Reproduction, 2008, 135 (2): 151- 163. doi: 10.1530/REP-07-0397
20	RUAN D G , PENG J Y , WANG X S , et al. XIST derepression in active X chromosome hinders pig somatic cell nuclear transfer[J]. Stem Cell Rep, 2018, 10 (2): 494- 508. doi: 10.1016/j.stemcr.2017.12.015
21	OKAE H , MATOBA S , NAGASHIMA T , et al. RNA sequencing-based identification of aberrant imprinting in cloned mice[J]. Hum Mol Genet, 2014, 23 (4): 992- 1001. doi: 10.1093/hmg/ddt495
22	LIAO Z D , ZHANG J X , SUN S Y , et al. Reprogramming mechanism dissection and trophoblast replacement application in monkey somatic cell nuclear transfer[J]. Nat Commun, 2024, 15 (1): 5. doi: 10.1038/s41467-023-43985-7
23	LI J L , YU D W , WANG J , et al. Identification of the porcine IG-DMR and abnormal imprinting of DLK1-DIO3 in cloned pigs[J]. Front Cell Dev Biol, 2022, 10, 964045. doi: 10.3389/fcell.2022.964045
24	ZAITOUN I , KHATIB H . Assessment of genomic imprinting of SLC38A4, NNAT, NAP1L5, and H19 in cattle[J]. BMC Genet, 2006, 7, 49.
25	KADIFE E , HARPER A , DE ALWIS N , et al. SLC38A4 amino acid transporter expression is significantly lower in early preterm intrauterine growth restriction complicated placentas[J]. Int J Mol Sci, 2022, 24 (1): 403. doi: 10.3390/ijms24010403
26	XIE Z , ZHANG W , ZHANG Y . Loss of Slc38a4 imprinting is a major cause of mouse placenta hyperplasia in somatic cell nuclear transferred embryos at late gestation[J]. Cell Rep, 2022, 38 (8): 110407. doi: 10.1016/j.celrep.2022.110407
27	HALUŠKOVÁ J , HOLEČKOVÁ B , STANIČOVÁ J . DNA methylation studies in cattle[J]. J Appl Genet, 2021, 62 (1): 121- 136. doi: 10.1007/s13353-020-00604-1
28	IWASAKI Y , AKSU C , REYES M , et al. The long-range interaction between two GNAS imprinting control regions delineates pseudohypoparathyroidism type 1B pathogenesis[J]. J Clin Invest, 2023, 133 (8): e167953. doi: 10.1172/JCI167953
29	LAU J C , HANEL M L , WEVRICK R . Tissue-specific and imprinted epigenetic modifications of the human NDN gene[J]. Nucleic Acids Res, 2004, 32 (11): 3376- 3382. doi: 10.1093/nar/gkh671
30	BARLOW D P , BARTOLOMEI M S . Genomic imprinting in mammals[J]. Cold Spring Harb Perspect Biol, 2014, 6 (2): a018382. doi: 10.1101/cshperspect.a018382
31	LIANG D , AYGVN N , MATOBA N , et al. Inference of putative cell-type-specific imprinted regulatory elements and genes during human neuronal differentiation[J]. Hum Mol Genet, 2023, 32 (3): 402- 416. doi: 10.1093/hmg/ddac207
32	JOZI M , JAFARPOUR F , MORADI R , et al. Induced DNA hypomethylation by folic acid deprivation in bovine fibroblast donor cells improves reprogramming of somatic cell nuclear transfer embryos[J]. Sci Rep, 2020, 10 (1): 5076. doi: 10.1038/s41598-020-61797-3
33	GOLDKAMP A K , LI Y H , RIVERA R M , et al. Differentially expressed tRNA-derived fragments in bovine fetuses with assisted reproduction induced congenital overgrowth syndrome[J]. Front Genet, 2022, 13, 1055343. doi: 10.3389/fgene.2022.1055343
34	VARGAS L N , CAIXETA F M C , DODE M A N , et al. DNA methylation profile of single in vitro matured bovine oocytes[J]. Mol Reprod Dev, 2023, 90 (4): 227- 235. doi: 10.1002/mrd.23679
35	INOUE K , OGONUKI N , KAMIMURA S , et al. Loss of H3K27me3 imprinting in the Sfmbt2 miRNA cluster causes enlargement of cloned mouse placentas[J]. Nat Commun, 2020, 11 (1): 2150. doi: 10.1038/s41467-020-16044-8
36	CHU C , ZHANG W H , KANG Y , et al. Analysis of developmental imprinting dynamics in primates using SNP-free methods to identify imprinting defects in cloned placenta[J]. Dev Cell, 2021, 56 (20): 2826- 2840.e7. doi: 10.1016/j.devcel.2021.09.012

引物名称 Primer name	引物序列(5′→3′) Primer sequence	退火温度/℃ Tm	用途 Application	片段长度/bp Length
esm-MKRN3-snp1F	CAGTGTCGGAAGGAACAATGGA	58	SNP检测	1 131
esm-MKRN3-snp1R	ACGAAAGCATTGCCCCATGATA	58	SNP检测	1 131
esm-MKRN3-snp2F	GGAGCATGCCATTGAGTTTGTTC	59	SNP检测	1 067
esm-MKRN3-snp2R	GGATGTTTTGTGACTTTTTAGCACAGTG	59	SNP检测	1 067
esm-MKRN3-snp3F	AGAAGCTGCTGAAACCATTTGT	58	SNP检测	929
esm-MKRN3-snp3R	CAAAGAATGTGTATCTGAGTCCAGG	58	SNP检测	929
cDNA-SNP1F	CTGTCGATACTCCCATGACCT	58	MKRN3表达	169
cDNA-SNP1R	CAGAGGAAAAGAGCGTGAGGA	58	MKRN3表达	169
MKRN3-WF1	GTTAGTAGGGAAAGTTAGGA	50	DMR甲基化	692
MKRN3-WR1	AAATAAACCAAAACTTAATCTCC	50	DMR甲基化	692
MKRN3-NF1	GATATAAAATGGAGTGAATAAATAAAG	55	DMR甲基化	465
MKRN3-NR1	TCCATAACAACTTCTACTCCCC	55	DMR甲基化	465

步骤 Procedure	时间/min Time	温度/℃ Temperature
变性 Denaturation	5	95
转化 Conversion	25	60
变性 Denaturation	5	95
转化 Conversion	85	60
变性 Denaturation	5	95
转化 Conversion	175	60
保存 Preservation	+∞	20

[1]	Liguo GAO, Hanqin SHEN, Yiquan CHEN, Sheng CHEN, Wencheng LIN, Feng CHEN. Prokaryotic Expression of Recombinant VP6* Protein of Porcine Rotavirus and Establishment of Indirect ELISA Detection Method [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4021-4028.
[2]	PENG Ning, LIANG Yaxu, LONG Fei, YU Dongming, ZHONG Xiang. Inhibitory Effect of Resveratrol on Rotavirus-infected Porcine Intestinal Epithelial Cells IPEC-J2 [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4213-4225.
[3]	Zhentao XIA, Nan WANG, Wanjie WANG, Qilü ZHOU, Lei HUANG, Yulian MU. Characteristics Analysis of TGEV Infection Mediated by IPEC-J2 with Knockout of pAPN Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3395-3407.
[4]	Yi WANG, Juan GAO, Yuemin HU, Yuefei YANG, Bojun FAN, Huiming JU. Effect of Transient Serum Starvation on Metabolism and Autophagy of Porcine Skeletal Muscle Satellite Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3408-3417.
[5]	Lindan LÜ, Hao MU, Xia HU, Mingni LIU, Shaomei LI, Xing LI, Zhenhui SONG, Liu YANG. Establishment and Preliminary Application of RAA Assay for the Detection of Porcine Transmissible Gastroenteritis Virus based on S Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3590-3599.
[6]	Weizhe LIU, Chenggang LUO, Rong YUAN, Yijie LIAO, Yimin WEN, Ying SUN, Enbo YU, Sanjie CAO, Xiaobo HUANG. Isolation and Identification of a Highly Pathogenic Strain of Porcine Epidemic Diarrhea Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3049-3063.
[7]	Jinting LUO, Fafang XU, Lei WANG, Xuan LUO, Yuhong MA, Jianbo ZHANG, Weihua HUANG, Yuejun SHANG, Guofang WU. The Effect of RSP on Cell Proliferation and Apoptosis of Porcine Leydig Cells with Hypoxia [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2441-2450.
[8]	Dongliang LI, Guanmin ZHENG, Shuai LI, Hongsen ZHU, Chao WU. Differential Expression of Transcriptome in Jejunal of Piglets Infected with Porcine Epidemic Diarrhea Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2652-2661.
[9]	Shuqi XIAO, Jun LIU, Yingtong FENG, Yang LI, Lele XU. Effect of Antimicrobial Use in the Perinatal Period on the Performance of Sows and Piglets in PRRSV-positive Farms [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2751-2760.
[10]	ZHANG Wei, PAN Zhihao, FANG Fugui. Advances in Epigenetic Regulation of the Onset of Puberty in Female Animals [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1875-1882.
[11]	HAN Yang, GUAN Shuaiyin, LI Zhen, ZHOU Saisai, YUAN Honggen, SONG Yunfeng. Prokaryotic Expression and Protein Activity of Porcine Circovirus Type 3 Rep [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2061-2071.
[12]	MA Rumeng, ZHAO Yuliang, MA Mingshuang, GUO Guihai, LIU Xinzi, LI Jiaxuan, CUI Wen, JIANG Yanping, SHAN Zhifu, ZHOU Han, WANG Li, QIAO Xinyuan, TANG Lijie, WANG Xiaona, LI Yijing. Comparative Study on the Immune Response Induced by the Different Porcine Receptor Bacteria with Expressing the Protective Antigen S1 of Porcine Epidemic Diarrhea Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2090-2099.
[13]	WANG Jing, ZHANG Shujuan, HU Xia, LIU Xiangyang, ZHANG Xingcui, SONG Zhenhui. CD44 Regulates Na⁺/H⁺ Exchanger 3 Activity by Influencing Porcine Epidemic Diarrhea Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2176-2185.
[14]	HU Zeqi, LI Runcheng, TAN Zuming, XIE Xiuyan, WANG Jiangping, QIN Lejuan, LI Rong, GE Meng. Establishment and Preliminary Application of PEDV, PoRVA and PDCoV TaqMan Triple RT-qPCR Assay [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2267-2272.
[15]	LAN Xinrui, ZHAO Baobao, ZHANG Bihan, LIN Xiaoyu, MA Huiming, WANG Yongsheng. Effects of β-sitosterol on Porcine Oocyte Maturation and Embryonic Development in Vitro [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1629-1637.

Analysis of Imprinted Expression and DNA Methylation Status of the Porcine MKRN3 Gene

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 8

References 36

Related Articles 15

Recommended Articles

Metrics

Comments