Analysis of Imprinting and Methylation Status of the Bovine (Bos taurus) WWOX Gene
CHEN Wei-Na1,2,*, MA Chao3,*, LIANG Xiao-He2, ZHANG Yin-Jiao2, ZHENG Yun-Chang2, HOU Rui-Lin2, ZHANG Cui2, LI Shi-Jie2,**
1 College of Traditional Chinese Medicine, Hebei University, Baoding 071000, China; 2 College of Life Science, Hebei Agricultural University, Baoding 071001, China; 3 Baoding No.2 Hospital, Baoding 071000, China
Abstract：Imprinted genes play key roles in embryonic and placental development, animal growth, and the process of disease development. Imprinted genes are usually found in clusters on the genome. The WW domain-containing oxidoreductase (WWOX) gene is located 2.97 Mb upstream of the CDH13-GSE1 imprinted region on chromosome 18 in cattle (Bos taurus) and encodes a WW domain-containing oxidoreductase. To analyze the imprinting status of the WWOX gene in cattle, in this study, the imprinting status of the WWOX gene in tissues (heart, liver, spleen, lung, kidney and brain) and placenta of wild type and somatic cloned cattle was first analyzed using SNP-based approach. Based on a SNP locus (A/G, rs380382156) on exon 7, the WWOX gene was found to be biallelic expression in wild type bovine tissues and monoallelic expression in the placenta. WWOX gene was identified as a paternally imprinted gene in the placenta by analyzing the parental genotypes. WWOX gene also showed biallelic expression in the tissues of cloned cattle, whereas in the placenta of cloned cattle it showed biallelic expression. Methylation status was subsequently examined in the heart, lung and placenta of wild type and cloned cattle. There was a differential methylation region (DMRs) found on intron 4 of the WWOX gene in the placenta, while it was hypermethylated in the heart and lung of wild type cattle. In cloned cattle, the WWOX gene exhibited a hypermethylation state across multiple organs (heart, lung, and placenta). The absence of its differentially methylated region suggested that DNA methylation participates in regulating the placenta-specific imprinted expression of this gene in wild-type cattle, while the aberrant methylation in the placenta of cloned cattle led to the disruption of this imprinted expression. This study provides a theoretical basis for further analyzing the role of imprinted genes in epigenetic reprogramming of donor nuclear in cloned animals and improving cloning efficiency.
[1] 陈南珠, 李俊良, 余大为, 等. 2024. 猪MKRN3基因的印记表达和DNA甲基化状态分析[J]. 畜牧兽医学报, (09): 3853-3863. (Chen N Z, Li J L, Yu D W, et al. 2024. Analysis of imprinted expression and DNA methylation status of the porcine MKRN3 gene[J]. Acta Veterinaria et Zootechnica Sinica, (09): 3853-3863.) [2] 董艳秋, 靳兰杰, 杨欣怡, 等. 2023. 牛KLF14和PDE10A基因的印记鉴定和甲基化分析[J]. 农业生物技术学报, 31(04): 776-783. (Dong Y Q, Jin L J, Yang X Y, et al.2023. Imprinting identification and methylation analysis of bovine (Bos taurus) KLF14 and PDE10A genes[J]. Journal of Agricultural Biotechnology, 31(04): 776-783.) [3] Angiolini E Y, Sandovici I, Coan P M, et al.2021. Deletion of the imprinted Phlda2 gene increases placental passive permeability in the mouse[J]. Genes, 12(5): 639-639. [4] Chen Z, Zhang Y.2020. Maternal H3K27me3-dependent autosomal and X chromosome imprinting[J]. Nature Reviews. Genetics, 21(9): 555-571. [5] Gao R, Wang C, Gao Y, et al.2018. Inhibition of aberrant DNA re-methylation improves post-implantation development of somatic cell nuclear transfer embryos[J]. Cell Stem Cell, 23(3): 426-435.e5. [6] Giaccari C, Cecere F, Argenziano L, et al.2024. A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes, as well as failure of epigenetic reprogramming and zygotic genome activation in embryos[J]. Genes & Development, 38(3-4): 131-150. [7] Cindrova-Davies T, Sferruzzi-Perri A N.2022. Human placental development and function[J]. Seminars in cell & developmental biology, 131: 66-77. [8] Goissis M D, Cibelli J B.2023. Early cell specification in mammalian fertilized and somatic cell nuclear transfer embryos[J]. Methods in Molecular Biology, 2647, 59-81. [9] Huo H, Zhang C, Wang K, et al.2024. A novel imprinted locus on bovine chromosome 18 homologous with human chromosome 16q24.1[J]. Molecular Genetics and Genomics, 299(1): 40. [10] Inoue A.2023. Noncanonical imprinting: intergenerational epigenetic inheritance mediated by polycomb complexes[J]. Current Opinion in Genetics & Development, 78: 102015. [11] Kośla K, Kałuzińska Ż, Bednarek A K.2020. The WWOX gene in brain development and pathology[J]. Experimental Biology and Medicine, 245(13): 1122-1129. [12] Li J, Yu D, Wang J, et al.2022. Identification of the porcine IG-DMR and abnormal imprinting of DLK1-DIO3 in cloned pigs[J]. Frontiers in Cell and Developmental Biology, 10: 964045. [13] Liu C C, Ho P C, Lee I T, et al.2018. WWOX phosphorylation, signaling, and role in neurodegeneration[J]. Frontiers in Neuroscience, 12: 563. [14] Liu T Y, Nagarajan G, Chiang M F, et al.2022. WWOX controls cell survival, immune response and disease progression by pY33 to pS14 transition to alternate signaling partners[J]. Cells, 11(14): 2137. [15] Lobanova Y V, Zhenilo S V.2024. Genomic imprinting and random monoallelic expression[J]. Biochemistry. Biokhimiia, 89(1): 84-96. [16] Lopez S J, Segal D J, LaSalle J M.2019. UBE3A: An E3 ubiquitin ligase with genome-wide impact in neurodevelopmental disease[J]. Frontiers in Molecular Neuroscience, 11: 476. [17] Lopez-Tello J, Yong H E J, Sandovici I, et al.2023. Fetal manipulation of maternal metabolism is a critical function of the imprinted IGF2 gene[J]. Cell Metabolism, 35(7): 1195-1208.e6. [18] Luo Z, Lin C, Woodfin A R, et al.2016. Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity[J]. Genes & Development, 30(1): 92-101. [19] Monk D, Mackay D J G, Eggermann T, et al.2019. Genomic imprinting disorders: Lessons on how genome, epigenome and environment interact[J]. Nature Reviews Genetics, 20(4): 235-248. [20] Niemann H, Tian X C, King W A, et al.2008. Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning[J]. Reproduction, 135(2): 151-163. [21] Okae H, Matoba S, Nagashima T, et al.2014. RNA sequencing-based identification of aberrant imprinting in cloned mice[J]. Human Molecular Genetics, 23(4): 992-1001. [22] Raas M W D, Zijlmans D W, Vermeulen M, et al.2022. There is another: H3K27me3-mediated genomic imprinting[J].Trends in Genetics, 38(1): 82-96. [23] Sandovici I, Georgopoulou A, Pérez-García V, et al.2022. The imprinted Igf2-Igf2r axis is critical for matching placental microvasculature expansion to fetal growth[J]. Developmental Cell, 57(1): 63-79.e8. [24] Schrock M S, Huebner K.2015. WWOX: A fragile tumor suppressor[J]. Experimental Biology and Medicine, 240(3): 296-304. [25] Schuff M, Strong A D, Welborn L K, et al.2024. Imprinting as basis for complex evolutionary novelties in eutherians[J]. Biology, 13(9): 682-682. [26] Song X, Li F, Jiang Z, et al.2017. Imprinting disorder in donor cells is detrimental to the development of cloned embryos in pigs[J]. Oncotarget, 8(42): 72363-72374. [27] Tucci V.2016. Genomic imprinting: A new epigenetic perspective of sleep regulation[J]. PLOS Genetics, 12(5): e1006004. [28] Tucci V, Isles A R, Kelsey G, et al.2019. Genomic imprinting and physiological processes in mammals[J]. Cell, 176(5): 952-965. [29] Vargas L N, Silveira M M, Franco M M.2023. Epigenetic reprogramming and somatic cell nuclear transfer[J]. Methods in Molecular Biology, 2647: 37-58. [30] Zhu Z J, Teng M, Li H Z, et al.2021. Virus-encoded miR-155 ortholog in Marek's disease virus promotes cell proliferation via suppressing apoptosis by targeting tumor suppressor WWOX[J]. Veterinary Microbiology, 252: 108919.
