{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:23:19Z","timestamp":1760059399434,"version":"build-2065373602"},"reference-count":43,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,6,11]],"date-time":"2025-06-11T00:00:00Z","timestamp":1749600000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"The medical health science and technology project of Shandong Province","award":["202402010752","6001\/601004001"],"award-info":[{"award-number":["202402010752","6001\/601004001"]}]},{"name":"Jining Medical University scientific research start-up funds for young doctor","award":["202402010752","6001\/601004001"],"award-info":[{"award-number":["202402010752","6001\/601004001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Genomic imprinting is critical for mammalian development, but its regulation varies across species. The insulin-like growth factor 2 receptor (IGF2R), which is a maternally expressed imprinted gene critical for cell proliferation and differentiation, as well as embryonic and placental development, is classically regulated by differentially methylated regions (DMRs) and lncRNA-Airn in mice. However, studies on this in equus are scarce, especially in terms of mechanistic studies. In the present study, heart, liver, spleen, lung, kidney, brain, and muscle samples were obtained from horses, donkeys, and hybrids, and gene expression and imprinting state were tested to investigate the imprinting regulation of Igf2r in these animals. Bisulfite sequencing combined with an allele-specific expression analysis revealed a tissue-specific loss of imprinting in the mule liver and hybrid brain tissues. Strikingly, we found that the maternal-specific expression of equine Igf2r did not rely on the canonical DMRs or lncRNA-Airn. Surprisingly, DNA methylation of a specific region called CpG island 2 (CpGI2) in the Igf2r promoter showed cis-acting inheritance, meaning that the DNA methylation patterns of the parental alleles are retained in hybrid tissues. Notably, the DNA methylation of CpGI2 correlated negatively with Igf2r expression in the spleen (R2 = 0.8797, p = 6.46 \u00d7 10\u22126), lung (R2 = 0.8569, p = 1.57 \u00d7 10\u22125), and kidney (R2 = 0.8650, p = 3.85 \u00d7 10\u22126). Our findings suggest that imprinting may work differently in other species. This study provides a framework for understanding imprinting diversity in hybrids and shows that equine hybrids can be used to study how epigenetic inheritance works.<\/jats:p>","DOI":"10.3390\/biology14060678","type":"journal-article","created":{"date-parts":[[2025,6,11]],"date-time":"2025-06-11T08:18:06Z","timestamp":1749629886000},"page":"678","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["DNA Methylation of Igf2r Promoter CpG Island 2 Governs Cis-Acting Inheritance and Gene Dosage in Equine Hybrids"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4828-2097","authenticated-orcid":false,"given":"Xisheng","family":"Wang","sequence":"first","affiliation":[{"name":"Lin He\u2019s Academician Workstation of New Medicine and Clinical Translation, Jining Medical University, Jining 272067, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yingchao","family":"Shen","sequence":"additional","affiliation":[{"name":"Anchee (Shandong) Animal Nutrition Research Academy Co., Ltd., Jinan 250104, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hong","family":"Ren","sequence":"additional","affiliation":[{"name":"Vocational and Technical College, Inner Mongolia Agricultural University, Hohhot 010018, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Minna","family":"Yi","sequence":"additional","affiliation":[{"name":"Equus Research Center, Inner Mongolia Agricultural University, Hohhot 010018, China"},{"name":"College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9381-2694","authenticated-orcid":false,"given":"Gerelchimeg","family":"Bou","sequence":"additional","affiliation":[{"name":"Equus Research Center, Inner Mongolia Agricultural University, Hohhot 010018, China"},{"name":"College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Schuff, M., Strong, A.D., Welborn, L.K., and Ziermann-Canabarro, J.M. (2024). Imprinting as Basis for Complex Evolutionary Novelties in Eutherians. Biology, 13.","DOI":"10.3390\/biology13090682"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Barlow, D.P., and Bartolomei, M.S. (2014). Genomic imprinting in mammals. Cold Spring Harb. Perspect. Biol., 6.","DOI":"10.1101\/cshperspect.a018382"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"548","DOI":"10.1038\/308548a0","article-title":"Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis","volume":"308","author":"Surani","year":"1984","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"374","DOI":"10.1038\/311374a0","article-title":"Role of paternal and maternal genomes in mouse development","volume":"311","author":"Barton","year":"1984","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1419","DOI":"10.1101\/gr.140236.112","article-title":"Genome-wide genetic variations are highly correlated with proximal DNA methylation patterns","volume":"22","author":"Qu","year":"2012","journal-title":"Genome Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1313","DOI":"10.1093\/nar\/gkaa1203","article-title":"A comprehensive epigenome atlas reveals DNA methylation regulating skeletal muscle development","volume":"49","author":"Yang","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Vuu, Y.M., Roberts, C.T., and Rastegar, M. (2023). MeCP2 Is an Epigenetic Factor That Links DNA Methylation with Brain Metabolism. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24044218"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Hughes, J., Surakhy, M., Can, S., Ducker, M., Davies, N., Szele, F., B\u00fchnemann, C., Carter, E., Trikin, R., and Crump, M.P. (2019). Maternal transmission of an Igf2r domain 11: IGF2 binding mutant allele (Igf2rI1565A) results in partial lethality, overgrowth and intestinal adenoma progression. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-47827-9"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.devcel.2021.12.005","article-title":"The imprinted Igf2-Igf2r axis is critical for matching placental microvasculature expansion to fetal growth","volume":"57","author":"Sandovici","year":"2022","journal-title":"Dev. Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1007\/s11033-022-07937-6","article-title":"Lower expression of the equine maternally imprinted gene IGF2R is related to the slow proliferation of hinny embryonic fibroblast","volume":"50","author":"Wang","year":"2023","journal-title":"Mol. Biol. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Gicquel, C., Weiss, J., Amiel, J., Gaston, V., Le Bouc, Y., and Scott, C.D. (2004). Epigenetic abnormalities of the mannose-6-phosphate\/ IGF2 receptor gene are uncommon in human overgrowth syndromes. J. Med. Genet., 41.","DOI":"10.1136\/jmg.2003.010488"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1159\/000090833","article-title":"Cross-species clues of an epigenetic imprinting regulatory code for the IGF2R gene","volume":"113","author":"Vu","year":"2006","journal-title":"Cytogenet. Genome Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"745","DOI":"10.1038\/39631","article-title":"Imprinted expression of the Igf2r gene depends on an intronic CpG island","volume":"389","author":"Wutz","year":"1997","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"810","DOI":"10.1038\/415810a","article-title":"The non-coding Air RNA is required for silencing autosomal imprinted genes","volume":"415","author":"Sleutels","year":"2002","journal-title":"Nature"},{"key":"ref_15","first-page":"251","article-title":"Making sense of imprinting the mouse and human IGF2R loci","volume":"214","author":"Wutz","year":"1998","journal-title":"Novartis Found. Symp."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1016\/j.ygeno.2008.08.004","article-title":"Identification of the human homolog of the imprinted mouse Air non-coding RNA","volume":"92","author":"Yotova","year":"2008","journal-title":"Genomics"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Dini, P., Kalbfleisch, T., Uribe-Salazar, J.M., Carossino, M., Ali, H.E., Loux, S.C., Esteller-Vico, A., Norris, J.K., Anand, L., and Scoggin, K.E. (2021). Parental bias in expression and interaction of genes in the equine placenta. Proc. Natl. Acad. Sci. USA, 118.","DOI":"10.1073\/pnas.2006474118"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"10705","DOI":"10.1073\/pnas.1308998110","article-title":"Paternally expressed genes predominate in the placenta","volume":"110","author":"Wang","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"103458","DOI":"10.1016\/j.jevs.2021.103458","article-title":"A Fast PCR Test for the Simultaneous Identification of Species and Gender in Horses, Donkeys, Mules and Hinnies","volume":"102","author":"Wang","year":"2021","journal-title":"J. Equine Vet. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1016\/0167-4781(85)90040-5","article-title":"DNA methylation is not increased in mouse-human somatic cell hybrids","volume":"824","author":"Feinstein","year":"1985","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Scott, E.Y., Mansour, T., Bellone, R.R., Brown, C.T., Mienaltowski, M.J., Penedo, M.C., Ross, P.J., Valberg, S.J., Murray, J.D., and Finno, C.J. (2017). Identification of long non-coding RNA in the horse transcriptome. BMC Genom., 18.","DOI":"10.1186\/s12864-017-3884-2"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"667","DOI":"10.1534\/genetics.113.158980","article-title":"Inheritance Patterns and Stability of DNA Methylation Variation in Maize Near-Isogenic Lines","volume":"196","author":"Li","year":"2014","journal-title":"Genetics"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2511","DOI":"10.1093\/hmg\/ddi255","article-title":"Neuron-specific relaxation of Igf2r imprinting is associated with neuron-specific histone modifications and lack of its antisense transcript Air","volume":"14","author":"Yamasaki","year":"2005","journal-title":"Hum. Mol. Genet."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"789","DOI":"10.2217\/epi.16.8","article-title":"Imprinted genes and imprinting control regions show predominant intermediate methylation in adult somatic tissues","volume":"8","author":"Pervjakova","year":"2016","journal-title":"Epigenomics"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1592","DOI":"10.1101\/gr.112680.110","article-title":"DNA methylation profiles of human active and inactive X chromosomes","volume":"21","author":"Sharp","year":"2011","journal-title":"Genome Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1159\/000015641","article-title":"DNA methylation and chromosome instability in lymphoblastoid cell lines","volume":"90","author":"Vilain","year":"2000","journal-title":"Cytogenet. Cell Genet."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1184","DOI":"10.1242\/dev.088849","article-title":"Imprinted Igf2r silencing depends on continuous Airn lncRNA expression and is not restricted to a developmental window","volume":"140","author":"Santoro","year":"2013","journal-title":"Development"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Springer, M.S., Foley, N.M., Brady, P.L., Gatesy, J., and Murphy, W.J. (2019). Evolutionary Models for the Diversification of Placental Mammals Across the KPg Boundary. Front. Genet., 10.","DOI":"10.3389\/fgene.2019.01241"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1905","DOI":"10.1093\/molbev\/msaa334","article-title":"Demographic History and Genomic Response to Environmental Changes in a Rapid Radiation of Wild Rats","volume":"38","author":"Ge","year":"2021","journal-title":"Mol. Biol. Evol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Marra, N.J., Richards, V.P., Early, A., Bogdanowicz, S.M., Bitar, P.D.P., Stanhope, M.J., and Shivji, M.S. (2017). Comparative transcriptomics of elasmobranchs and teleosts highlight important processes in adaptive immunity and regional endothermy. BMC Genom., 18.","DOI":"10.1186\/s12864-016-3411-x"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1038\/3833","article-title":"Genomic imprinting is disrupted in interspecific Peromyscus hybrids","volume":"20","author":"Vrana","year":"1998","journal-title":"Nat. Genet."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Brown, A.P., Parameswaran, S., Cai, L., Elston, S., Pham, C., Barski, A., Weirauch, M.T., and Ji, H. (2023). TET1 regulates responses to house dust mite by altering chromatin accessibility, DNA methylation, and gene expression in airway epithelial cells. Res. Sq.","DOI":"10.21203\/rs.3.rs-3726852\/v1"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3429","DOI":"10.1093\/plcell\/koad160","article-title":"Asymmetric variation in DNA methylation during domestication and de-domestication of rice","volume":"35","author":"Cao","year":"2023","journal-title":"Plant Cell"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1393","DOI":"10.1101\/gr.257576.119","article-title":"Distinct contributions of DNA methylation and histone acetylation to the genomic occupancy of transcription factors","volume":"30","author":"Cusack","year":"2020","journal-title":"Genome Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1038\/ng0993-74","article-title":"The insulin-like growth factor type-2 receptor gene is imprinted in the mouse but not in humans","volume":"5","author":"Kalscheuer","year":"1993","journal-title":"Nat. Genet."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1126\/science.aaj2114","article-title":"DNA sequence-dependent epigenetic inheritance of gene silencing and histone H3K9 methylation","volume":"356","author":"Wang","year":"2017","journal-title":"Science"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Xiong, X., Geden, C.J., Tan, Y.J., Zhang, Y., Zhang, D.P., Werren, J.H., and Wang, X. (2024). Genome Structure, Evolution, and Host Shift of. Biol. Basel, 13.","DOI":"10.3390\/biology13110952"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1807","DOI":"10.1242\/dev.129.8.1807","article-title":"Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells","volume":"129","author":"Lee","year":"2002","journal-title":"Development"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"6415","DOI":"10.1073\/pnas.77.11.6415","article-title":"Mitochondrial DNA polymorphism: Evidence that variants detected by restriction enzymes differ in nucleotide sequence rather than in methylation","volume":"77","author":"Castora","year":"1980","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"de Oliveira, N.F.P., Persuhn, D.C., and Dos Santos, M. (2024). Can Global DNA Methylation Be Influenced by Polymorphisms in Genes Involved in Epigenetic Mechanisms? A Review. Genes, 15.","DOI":"10.3390\/genes15121504"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1507","DOI":"10.1016\/j.cell.2013.11.039","article-title":"Interactome Maps of Mouse Gene Regulatory Domains Reveal Basic Principles of Transcriptional Regulation","volume":"155","author":"Tang","year":"2013","journal-title":"Cell"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Xu, D.H., Zhang, C.R., Bi, X.M., Xu, J.K., Guo, S.N., Li, P.H., Shen, Y.T., Cai, J.L., Zhang, N.H., and Tian, G.H. (2024). Mapping enhancer and chromatin accessibility landscapes charts the regulatory network of Alzheimer\u2019s disease. Comput. Biol. Med., 168.","DOI":"10.1016\/j.compbiomed.2023.107802"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Qian, J., and Liu, S.X. (2024). CRISPR\/dCas9-Tet1-Mediated DNA Methylation Editing. Bio-Protocol, 14.","DOI":"10.21769\/BioProtoc.4976"}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/6\/678\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:50:01Z","timestamp":1760032201000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/6\/678"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,11]]},"references-count":43,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2025,6]]}},"alternative-id":["biology14060678"],"URL":"https:\/\/doi.org\/10.3390\/biology14060678","relation":{},"ISSN":["2079-7737"],"issn-type":[{"type":"electronic","value":"2079-7737"}],"subject":[],"published":{"date-parts":[[2025,6,11]]}}}