CpG site hypomethylation at ETS1‑binding region regulates DLK1 expression in Chinese patients with Tetralogy of Fallot

Tian,Guixiang; He,Lili; Gu,Ruoyi; Sun,Jingwei; Chen,Weicheng; Qian,Yanyan; Ma,Xiaojing; Yan,Weili; Zhao,Zhenshan; Xu,Ziqing; Suo,Meijiao; Sheng,Wei; Huang,Guoying

doi:10.3892/mmr.2022.12609

CpG site hypomethylation at ETS1‑binding region regulates DLK1 expression in Chinese patients with Tetralogy of Fallot

Authors:
- Guixiang Tian
- Lili He
- Ruoyi Gu
- Jingwei Sun
- Weicheng Chen
- Yanyan Qian
- Xiaojing Ma
- Weili Yan
- Zhenshan Zhao
- Ziqing Xu
- Meijiao Suo
- Wei Sheng
- Guoying Huang
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Affiliations: Department of Ultrasound, Cardiovascular Center, Pediatrics Research Institute, National Children's Medical Center, Children's Hospital of Fudan University, Shanghai 201102, P.R. China, Pediatrics Department, Bengbu First People's Hospital, Bengbu, Anhui 233000, P.R. China
Published online on: January 20, 2022 https://doi.org/10.3892/mmr.2022.12609
Article Number: 93
Copyright: © Tian et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart malformation accounting for ~10% of cases. Although the pathogenesis of TOF is complex and largely unknown, epigenetics plays a huge role, specifically DNA methylation. The protein δ like non‑canonical Notch ligand 1 (DLK1) gene encodes a non‑canonical ligand of the Notch signaling pathway, which is involved in heart development. However, the epigenetic mechanism of DLK1 in the pathogenesis of TOF is yet to be elucidated. Therefore, the present study aimed to clarify its specific mechanism. In this study, immunohistochemistry was used to detect the protein expression of DLK1 and the methylation status of the DLK1 promoter was measured via bisulfite sequencing PCR. Dual‑luciferase reporter assays were performed to examine the influence of transcription factor ETS proto‑oncogene 1 (ETS1) on DLK1 gene expression. The electrophoretic mobility shift assay and chromatin immunoprecipitation assay, both in vivo and in vitro, were used to verify the binding of the ETS1 transcription factor to the DLK1 promoter as well as the influence of methylation status of DLK1 promoter on this binding affinity. The expression of DLK1 in the right ventricular outflow tract was significantly lower in patients with Tetralogy of Fallot (TOF) than that in controls (P<0.001). Moreover, the methylation level of CpG site 10 and CpG site 11 in the DLK1_R region was significantly decreased in TOF cases compared with controls (P<0.01). The integral methylation levels of DLK1_R and the methylation status of the CpG site 11 were both positively associated with DLK1 protein expression in TOF cases. ETS1 was found to inhibit DLK1 transcriptional activity by binding to the CpG site 11 and this affinity could be influenced by the methylation level of the DLK1 promoter. These findings demonstrated that the hypomethylation of the DLK1 promoter could increase the binding affinity of ETS1 transcription factor, which in turn inhibited DLK1 gene transcriptional activity and contributed to the development of TOF.

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1	Apitz C, Webb GD and Redington AN: Tetralogy of Fallot. Lancet. 374:1462–1471. 2009. View Article : Google Scholar : PubMed/NCBI
2	Page DJ, Miossec MJ, Williams SG, Monaghan RM, Fotiou E, Cordell HJ, Sutcliffe L, Topf A, Bourgey M, Bourque G, et al: Whole exome sequencing reveals the major genetic contributors to Nonsyndromic Tetralogy of Fallot. Circ Res. 124:553–563. 2019. View Article : Google Scholar : PubMed/NCBI
3	Grunert M, Dorn C, Cui H, Dunkel I, Schulz K, Schoenhals S, Sun W, Berger F, Chen W and Sperling SR: Comparative DNA methylation and gene expression analysis identifies novel genes for structural congenital heart diseases. Cardiovasc Res. 112:464–477. 2016. View Article : Google Scholar : PubMed/NCBI
4	Finn J, Sottoriva K, Pajcini KV, Kitajewski JK, Chen C, Zhang W, Malik AB and Liu Y: Dlk1-Mediated Temporal Regulation of Notch Signaling is required for differentiation of Alveolar Type II to Type I cells during repair. Cell Rep. 26:2942–2954.e5. 2019. View Article : Google Scholar : PubMed/NCBI
5	de la Pompa JL and Epstein JA: Coordinating tissue interactions: Notch signaling in cardiac development and disease. Dev Cell. 22:244–254. 2012. View Article : Google Scholar : PubMed/NCBI
6	McCright B, Lozier J and Gridley T: A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency. Development. 129:1075–1082. 2002. View Article : Google Scholar : PubMed/NCBI
7	Digilio MC, Luca AD, Lepri F, Guida V, Ferese R, Dentici ML, Angioni A, Marino B and Dallapiccola B: JAG1 mutation in a patient with deletion 22q11.2 syndrome and tetralogy of Fallot. Am J Med Genet A. 161A:3133–3136. 2013. View Article : Google Scholar : PubMed/NCBI
8	Garg V: Notch signaling in aortic valve development and disease, Etiology and Morphogenesis of Congenital Heart Disease: From Gene Function and Cellular Interaction to Morphology. Nakanishi T, Markwald RR, Baldwin HS, Keller BB, Srivastava D and Yamagishi H: Springer; Tokyo: pp. 371–376. 2016
9	Huang CC, Kuo HM, Wu PC, Cheng SH, Chang TT, Chang YC, Kung ML, Wu DC, Chuang JH and Tai MH: Soluble delta-like 1 homolog (DLK1) stimulates angiogenesis through Notch1/Akt/eNOS signaling in endothelial cells. Angiogenesis. 21:299–312. 2018. View Article : Google Scholar : PubMed/NCBI
10	Shamis Y, Cullen DE, Liu L, Yang G, Ng SF, Xiao L, Bell FT, Ray C, Takikawa S, Moskowitz IP, et al: Maternal and zygotic Zfp57 modulate NOTCH signaling in cardiac development. Proc Natl Acad Sci USA. 112:E2020–2029. 2015. View Article : Google Scholar : PubMed/NCBI
11	Rodriguez P, Sassi Y, Troncone L, Benard L, Ishikawa K, Gordon RE, Lamas S, Laborda J, Hajjar RJ and Lebeche D: Deletion of delta-like 1 homologue accelerates fibroblast-myofibroblast differentiation and induces myocardial fibrosis. Eur Heart J. 40:967–978. 2019. View Article : Google Scholar : PubMed/NCBI
12	Zhu H, Wang G and Qian J: Transcription factors as readers and effectors of DNA methylation. Nat Rev Genet. 17:551–565. 2016. View Article : Google Scholar : PubMed/NCBI
13	Moore-Morris T, van Vliet PP, Andelfinger G and Puceat M: Role of epigenetics in cardiac development and congenital diseases. Physiol Rev. 98:2453–2475. 2018. View Article : Google Scholar : PubMed/NCBI
14	Serra-Juhé C, Cuscó I, Homs A, Flores R, Torán N and Pérez-Jurado LA: DNA methylation abnormalities in congenital heart disease. Epigenetics. 10:167–177. 2015. View Article : Google Scholar : PubMed/NCBI
15	Cao J, Wu Q, Huang Y, Wang L, Su Z and Ye H: The role of DNA methylation in syndromic and non-syndromic congenital heart disease. Clin Epigenetics. 13:932021. View Article : Google Scholar : PubMed/NCBI
16	Sheng W, Qian Y, Wang H, Ma X, Zhang P, Diao L, An Q, Chen L, Ma D and Huang G: DNA methylation status of NKX2-5, GATA4 and HAND1 in patients with tetralogy of fallot. BMC Med Genomics. 6:462013. View Article : Google Scholar : PubMed/NCBI
17	Fornes O, Castro-Mondragon JA, Khan A, van der Lee R, Zhang X, Richmond PA, Modi BP, Correard S, Gheorghe M, Baranasic D, et al: JASPAR 2020: update of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 48(D1): D87–D92. 2020.PubMed/NCBI
18	Lacazette E: A laboratory practical illustrating the use of the ChIP-qPCR method in a robust model: Estrogen receptor alpha immunoprecipitation using Mcf-7 culture cells. Biochem Mol Biol Educ. 45:152–160. 2017. View Article : Google Scholar : PubMed/NCBI
19	Kuhnert F, Kirshner JR and Thurston G: Dll4-Notch signaling as a therapeutic target in tumor angiogenesis. Vasc Cell. 3:202011. View Article : Google Scholar : PubMed/NCBI
20	MacGrogan D, Münch J and de la Pompa JL: Notch and interacting signalling pathways in cardiac development, disease, and regeneration. Nat Rev Cardiol. 15:685–704. 2018. View Article : Google Scholar : PubMed/NCBI
21	Luxan G, D'Amato G, MacGrogan D and de la Pompa JL: Endocardial Notch signaling in cardiac development and disease. Circ Res. 118:e1–e18. 2016. View Article : Google Scholar : PubMed/NCBI
22	Chen H, Zhang W, Sun X, Yoshimoto M, Chen Z, Zhu W, Liu J, Shen Y, Yong W, Li D, et al: Fkbp1a controls ventricular myocardium trabeculation and compaction by regulating endocardial Notch1 activity. Development. 140:1946–1957. 2013. View Article : Google Scholar : PubMed/NCBI
23	van Weerd JH, Koshiba-Takeuchi K, Kwon C and Takeuchi JK: Epigenetic factors and cardiac development. Cardiovasc Res. 91:203–211. 2011. View Article : Google Scholar : PubMed/NCBI
24	Wang Z, Zhai W, Richardson JA, Olson EN, Meneses JJ, Firpo MT, Kang C, Skarnes WC and Tjian R: Polybromo protein BAF180 functions in mammalian cardiac chamber maturation. Genes Dev. 18:3106–3116. 2004. View Article : Google Scholar : PubMed/NCBI
25	Jones PA: Functions of DNA methylation: Islands, start sites, gene bodies and beyond. Nat Rev Genet. 13:484–492. 2012. View Article : Google Scholar : PubMed/NCBI
26	Zhu Y, Ye M, Xu H, Gu R, Ma X, Chen M, Li X, Sheng W and Huang G: Methylation status of CpG sites in the NOTCH4 promoter region regulates NOTCH4 expression in patients with tetralogy of Fallot. Mol Med Rep. 22:4412–4422. 2020.PubMed/NCBI
27	Xiaodi L, Ming Y, Hongfei X, Yanjie Z, Ruoyi G, Ma X, Wei S and Guoying H: DNA methylation at CpG island shore and RXRα regulate NR2F2 in heart tissues of tetralogy of Fallot patients. Biochem Biophys Res Commun. 529:1209–1215. 2020. View Article : Google Scholar : PubMed/NCBI
28	Vohra M, Adhikari P, Souza SC, Nagri SK, Umakanth S, Satyamoorthy K and Rai PS: CpG-SNP site methylation regulates allele-specific expression of MTHFD1 gene in type 2 diabetes. Lab Invest. 100:1090–1101. 2020. View Article : Google Scholar : PubMed/NCBI
29	Dayeh TA, Olsson AH, Volkov P, Almgren P, Ronn T and Ling C: Identification of CpG-SNPs associated with type 2 diabetes and differential DNA methylation in human pancreatic islets. Diabetologia. 56:1036–1046. 2013. View Article : Google Scholar : PubMed/NCBI
30	Bogdanović O and Lister R: DNA methylation and the preservation of cell identity. Curr Opin Genet Dev. 46:9–14. 2017. View Article : Google Scholar : PubMed/NCBI
31	Greenberg MVC and Bourc'his D: The diverse roles of DNA methylation in mammalian development and disease. Nat Rev Mol Cell Biol. 20:590–607. 2019. View Article : Google Scholar : PubMed/NCBI
32	Bahar Halpern K, Vana T and Walker MD: Paradoxical role of DNA methylation in activation of FoxA2 gene expression during endoderm development. J Biol Chem. 289:23882–23892. 2014. View Article : Google Scholar : PubMed/NCBI
33	Nabilsi NH, Broaddus RR and Loose DS: DNA methylation inhibits p53-mediated survivin repression. Oncogene. 28:2046–2050. 2009. View Article : Google Scholar : PubMed/NCBI
34	Jia N, Wang J, Li Q, Tao X, Chang K, Hua K, Yu Y, Wong KK and Feng W: DNA methylation promotes paired box 2 expression via myeloid zinc finger 1 in endometrial cancer. Oncotarget. 7:84785–84797. 2016. View Article : Google Scholar : PubMed/NCBI
35	Smith J, Sen S, Weeks RJ, Eccles MR and Chatterjee A: Promoter DNA Hypermethylation and Paradoxical Gene Activation. Trends Cancer. 6:392–406. 2020. View Article : Google Scholar : PubMed/NCBI
36	Ruan H, Liao Y, Ren Z, Mao L, Yao F, Yu P, Ye Y, Zhang Z, Li S, Xu H, et al: Single-cell reconstruction of differentiation trajectory reveals a critical role of ETS1 in human cardiac lineage commitment. BMC Biol. 17:892019. View Article : Google Scholar : PubMed/NCBI
37	Nie S and Bronner ME: Dual developmental role of transcriptional regulator Ets1 in Xenopus cardiac neural crest vs. heart mesoderm. Cardiovasc Res. 106:67–75. 2015. View Article : Google Scholar : PubMed/NCBI