牛FOXP2基因的单等位基因表达和DNA甲基化状态分析

doi:10.11843/j.issn.0366-6964.2025.09.020

摘要/Abstract

摘要： 旨在研究叉头框P2(forkhead box P2，FOXP2)基因在牛组织和胎盘中的单等位基因表达和DNA甲基化状态。本研究采集了2个不同时期(出生0 d犊牛和4~5岁成年牛)的健康雌性荷斯坦奶牛的组织和自然分娩的足月胎盘，通过荧光定量PCR方法分析FOXP2基因牛6个组织(心脏、肝脏、脾脏、肺脏、肾脏和大脑)和胎盘中的表达；应用基于单核苷酸多态性(single nucleotide polymorphism，SNP)的RT-PCR产物直接测序的方法分析FOXP2基因在牛组织和胎盘中单等位基因表达状态；采用亚硫酸盐测序法对牛组织、精子和胎盘的DNA甲基化状态进行分析。结果表明，FOXP2基因在成年牛6个组织(心脏、肝脏、脾脏、肺脏、肾脏和大脑)中的表达水平均高于犊牛组织。基于FOXP2基因3'非编码区的1个SNP位点(g.53771685G＞A)，发现FOXP2基因在犊牛的6个组织中为单等位基因表达；而在成年牛中仅大脑组织中为单等位基因表达，其余的5个组织(心脏、肝脏、脾脏、肺脏和肾脏)中为双等位基因表达；在胎盘中，FOXP2基因呈现为母源等位基因表达。FOXP2基因X1剪接体的启动子区和5'端在犊牛和成年牛组织、胎盘和精子均处于低甲基化状态。以上研究结果表明，FOXP2基因在牛胎盘中为母源等位基因表达的印记基因，而其在组织中的单等位基因表达呈现生长时期和组织特异性，基因启动子和5'端处于低甲基化状态，提示DNA甲基化未参与调控牛FOXP2基因的单等位基因表达。

关键词: 叉头框P2(FOXP2)基因, 等位基因表达, DNA甲基化, 牛

Abstract: The aim of this study was to investigate the monoallelic expressions and DNA methylation status of the forkhead box P2 (FOXP2) gene in bovine tissues and placentas. The bovine tissues of two growth periods (calves of 0 d old and adult cows of 4-5 years old) and full-term placentas naturally delivered were collected. The expression levels of the FOXP2 gene were analyzed in bovine 6 tissues (heart, liver, spleen, lung, kidney and brain) and placentas by qRT-PCR. The monoallelic expressions of the FOXP2 gene were analyzed using the method of direct sequencing of RT-PCR products based on the single nucleotide polymorphism (SNP). The analysis of DNA methylation status in bovine tissues, placentas and sperms was conducted using sulphite sequencing method. The results showed that the expression of FOXP2 gene in all 6 tissues (heart, liver, spleen, lung, kidney and brain) of adult bovine was higher than that of calf tissues. Based on the SNP site of g.53771685G＞A situated in the 3' non-coding region of the FOXP2 gene, monoallelic expression of FOXP2 was observed in 6 tissues of calves. However, in adult cattle, FOXP2 showed monoallelic expression only in brain tissue and biallelic expression in other 5 tissues (heart, liver, spleen, lung and kidney). The FOXP2 gene was mathernally expressed in the placenta. Hypomethylation was showed at the promoter and the 5' end of FOXP2 X1 splice in bovine tissues, placentas and sperms. These results indicated that the FOXP2 gene is an imprinted gene and the maternal allele is expressed in bovine placenta, and the monoallelic expression in bovine tissues presents growth period- and tissue-specific. Hypomethylation is in the promoter and 5' regions of FOXP2 gene, indicating that DNA methylation is not involved in the regulating monoallelic expression of bovine FOXP2 gene.

Key words: forkhead box P2(FOXP2) gene, allelic expression, DNA methylation, bovine

中图分类号:

郑云畅, 侯睿霖, 梁晓贺, 杨利丹, 张银蛟, 霍浩楠, 陈玮娜, 张萃, 李世杰. 牛FOXP2基因的单等位基因表达和DNA甲基化状态分析[J]. 畜牧兽医学报, 2025, 56(9): 4369-4378.

ZHENG Yunchang, HOU Ruilin, LIANG Xiaohe, YANG Lidan, ZHANG Yinjiao, HUO Haonan, CHEN Weina, ZHANG Cui, LI Shijie. Analysis of Monoallelic Expression and DNA Methylation Status of Bovine FOXP2 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4369-4378.

参考文献

[1] MACDONALD W A, MANN M R W. Long noncoding RNA functionality in imprinted domain regulation[J]. PLoS Genet, 2020, 16(8): e1008930.
[2] THAMBAN T, AGARWAAL V, KHOSLA S. Role of genomic imprinting in mammalian development[J]. J Biosci, 2020, 45: 20.
[3] BUTLER M G. Imprinting disorders in humans:a review[J]. Curr Opin Pediatr, 2020, 32(6): 719-729.
[4] ELBRACHT M, MACKAY D, BEGEMANN M, et al. Disturbed genomic imprinting and its relevance for human reproduction:causes and clinical consequences[J]. Hum Reprod Update, 2020, 26(2): 197-213.
[5] WEINBERG-SHUKORN A, BEN-YAIR R, TAKAHASHI N, et al. Balanced gene dosage control rather than parental origin underpins genomic imprinting[J]. Nat Commun, 2022, 13(1): 4391.
[6] GLASER J, IRANZO J, BORENSZTEIN M, et al. The imprinted Zdbf2 gene finely tunes control of feeding and growth in neonates[J]. Elife, 2022, 11: e65641.
[7] SAPEHIA D, MAHAJAN A, SINGH P, et al. Enrichment of trimethyl histone 3 lysine 4 in the Dlk1 and Grb10 genes affects pregnancy outcomes due to dietary manipulation of excess folic acid and low vitamin B12[J]. Biol Res, 2024, 57(1): 85.
[8] ANGIOLINI E, SANDOVICI I, COAN P M, et al. Deletion of the imprinted Phlda2 gene increases placental passive permeability in the mouse[J]. Genes (Basel), 2021, 12(5): 639.
[9] LIU S, YU Y, ZHANG S, et al. Epigenomics and genotype-phenotype association analyses reveal conserved genetic architecture of complex traits in cattle and human[J]. BMC Biol, 2020, 18(1): 80.
[10] 王丁香，赵红波.牛印记基因的研究进展[J]. 中国牛业科学，2022, 48(6): 80-84.
WANG D X, ZHAO H B. Research advances on imprinted genes in cattle[J]. China Cattle Science, 2022, 48(6): 80-84.(in Chinese)
[11] BENVENUTO M, PALUMBO P, DI MURO E, et al. Identification of a novel FOXP1 variant in a patient with hypotonia，intellectual disability，and severe speech impairment[J]. Genes (Basel), 2023, 14(10): 1958.
[12] PERUMAL C M, THULO M, BUTHELEZI S, et al. Unraveling the interplay between the leucine zipper and forkhead domains of FOXP2:Implications for DNA binding，stability and dynamics[J]. Proteins, 2024, 92(10): 1177-1189.
[13] CHE F, LI C, ZHANG L, et al. Novel FOXP2 variant associated with speech and language dysfunction in a Chinese family and literature review[J]. J Appl Genet, 2024, 65(2): 367-373.
[14] TURNER S J, HILDEBRAND M S, BLOCK S, et al. Small intragenic deletion in FOXP2 associated with childhood apraxia of speech and dysarthria[J]. Am J Med Genet A, 2013, 161A(9): 2321-2326.
[15] SU W, HU S, ZHOU L, et al. FOXP2 inhibits the aggressiveness of lung cancer cells by blocking TGFβ signaling[J]. Oncol Lett, 2024, 27(5): 227.
[16] YANG F, XIAO Z, ZHANG S. FOXP2 regulates thyroid cancer cell proliferation and apoptosis via transcriptional activation of RPS6KA6[J]. Exp Ther Med, 2022, 23(6): 434.
[17] THOMAS A C, FROST J M, ISHIDA M, et al. The speech gene FOXP2 is not imprinted[J]. J Med Genet, 2012, 49(11): 669-670.
[18] ADEGBOLA A A, COX G F, BRADSHAW E M, et al. Monoallelic expression of the human FOXP2 speech gene[J]. Proc Natl Acad Sci U S A, 2015, 112(22): 6848-6854.
[19] REGMI S, GIHA L, ALI A, et al. Methylation is maintained specifically at imprinting control regions but not other DMRs associated with imprinted genes in mice bearing a mutation in the Dnmt1 intrinsically disordered domain[J]. Front Cell Dev Biol, 2023, 11: 1192789.
[20] BARLOW D P, BARTOLOMEI M S. Genomic imprinting in mammals[J]. Cold Spring Harb Perspect Biol, 2014, 6(2): a018382.
[21] MAS-PARES B, CARRERAS-BADOSA G, GOMEZ-VILARRUBLA A, et al. Sex dimorphic associations of Prader-Willi imprinted gene expressions in umbilical cord with prenatal and postnatal growth in healthy infants[J]. World J Pediatr, 2025, 21(1): 100-112.
[22] KANG J, LI Q, LIU J, et al. Exploring the cellular and molecular basis of murine cardiac development through spatiotemporal transcriptome sequencing[J]. Gigascience, 2025, 14: giaf012.
[23] WEINBERG-SHUKRON A, YOUNGSON N A, FERGUSON-SMITH A C, et al. Epigenetic control and genomic imprinting dynamics of the Dlk1-Dio3 domain[J]. Front Cell Dev Biol, 2023, 11: 1328806.
[24] CROCCO P, DE RANGO F, BRUNO F, et al. Genetic variability of FOXP2 and its targets CNTNAP2 and PRNP in frontotemporal dementia：A pilot study in a southern Italian population[J]. Heliyon, 2024, 10(11): e31624.
[25] YIN M, YU W, LI W, et al. DNA methylation and gene expression changes in mouse pre- and post-implantation embryos generated by intracytoplasmic sperm injection with artificial oocyte activation[J]. Reprod Biol Endocrinol, 2021, 19(1): 163.
[26] ZHANG Y, ZHANG C, CHEN W, et al. The landscape of allelic expression and DNA methylation at the bovine SGCE/PEG10 locus[J]. Anim Genet, 2024, 55(3): 452-456.
[27] SCHUFF M, STRONG A D, WELBORN L K, et al. Imprinting as basis for complex evolutionary novelties in Eutherians[J]. Biology (Basel), 2024, 13(9): 682.
[28] ISLES A R. The contribution of imprinted genes to neurodevelopmental and neuropsychiatric disorders[J]. Transl Psychiatry, 2022, 12(1): 210.
[29] CINDROVA-DAVIES T, SFERRUZZI-PERRI A N. Human placental development and function[J]. Semin Cell Dev Biol, 2022, 131: 66-77.
[30] HARA S, MATSUHISA F, KITAJIMA S, et al. Identification of responsible sequences which mutations cause maternal H19-ICR hypermethylation with Beckwith-Wiedemann syndrome-like overgrowth[J]. Commun Biol, 2024, 7(1): 1605.
[31] DASKEVICIUTE D, CHAPPELL-MAOR L, SAINTY B, et al. Non-canonical imprinting，manifesting as post-fertilization placenta-specific parent-of-origin dependent methylation，is not conserved in humans[J]. Hum Mol Genet, 2025, 17: ddaf009.
[32] RICHARD ALBER J, KOBAYASHI T, INOUE A, et al. Conservation and divergence of canonical and non-canonical imprinting in murids[J]. Genome Biol, 2023, 24(1): 48.
[33] KANEKO-ISHINO T, ISHINO F. The evolutionary advantage in mammals of the complementary monoallelic expression mechanism of genomic imprinting and its emergence from a defense against the insertion into the host genome[J]. Front Genet, 2022, 13: 832983.
[34] DENG Q, DU Y, WANG Z, et al. Identification and validation of a DNA methylation driven gene based prognostic model for clear cell renal cell carcinoma[J]. BMC Genomics, 2023, 24(1): 307.
[35] BRENET F, MOH M, FUNK P, et al. DNA methylation of the first exon is tightly linked to transcriptional silencing[J]. PLoS One, 2011, 6(1): e14524.
[36] 刘晓倩，靳兰杰，董艳秋，等.DNA甲基化调控牛AQP1基因的胎盘特异性印记[J].畜牧兽医学报，2021, 52(8): 2181-2189.
LIU X Q, JIN L J, DONG Y Q, et al. DNA methylation regulate the genomic imprinting of AQP1 gene specific in bovine placenta[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(8): 2181-2189.(in Chinese)
[37] CHEN Z, ZHANG Y. Maternal H3K27me3-dependent autosomal and X chromosome imprinting[J]. Nat Rev Genet, 2020, 21(9): 555-571.
[38] HANNA C W, KELSEY G. Features and mechanisms of canonical and noncanonical genomic imprinting[J]. Genes Dev, 2021, 35(11-12): 821-834.