{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:32:20Z","timestamp":1760146340936,"version":"build-2065373602"},"reference-count":46,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2024,10,24]],"date-time":"2024-10-24T00:00:00Z","timestamp":1729728000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Taysha Gene Therapies, Inc."},{"name":"Foundation for Prader\u2013Willi Research"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Background\/objectives: Euchromatic histone lysine methyltransferase 2 (EHMT2, also known as G9a) is a mammalian histone methyltransferase that catalyzes the dimethylation of histone 3 lysine 9 (H3K9). On human chromosome 15, the parental-specific expression of Prader\u2013Willi Syndrome (PWS)-related genes, such as SNRPN and SNORD116, are regulated through the genetic imprinting of the PWS imprinting center (PWS-IC). On the paternal allele, PWS genes are expressed whereas the epigenetic maternal silencing of PWS genes is controlled by the EHMT2-mediated methylation of H3K9 in PWS-IC. Here, we measured the effects of RNA interference of EHMT2 on the maternal expression of genes deficient in PWS in mouse model and patient iPSC-derived cells. Methods: We used small interfering RNA (siRNA) oligonucleotides and lentiviral short harpin RNA (shRNA) to reduce Ehtm2\/EHMT2 expression in mouse Snord116 deletion primary neurons, PWS patient-derived induced pluripotent stem cell (iPSC) line and PWS iPSC-derived neurons. We then measured the expression of transcript or protein (if relevant) of PWS genes normally silenced on the maternal allele. Results: With an approximate reduction of 90% in EHMT2 mRNA and more than 80% of the EHMT2 protein, we demonstrated close to a 2-fold increase in the expression of maternal transcripts for SNRPN and SNORD116 in PWS iPSCs treated with siEHMT2 compared to PWS iPSC siControl. A similar increase in SNORD116 and SNRPN RNA expression was observed in PWS iPSC-derived neurons treated with shEHMT2. Conclusions: RNAi reduction in EHMT2 activates maternally silenced PWS genes. Further studies are needed to determine whether the increase is therapeutically relevant. This study confirms the role of EHMT2 in the epigenetic regulation of PWS genes.<\/jats:p>","DOI":"10.3390\/genes15111366","type":"journal-article","created":{"date-parts":[[2024,10,24]],"date-time":"2024-10-24T04:11:38Z","timestamp":1729743098000},"page":"1366","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["RNAi Knockdown of EHMT2 in Maternal Expression of Prader\u2013Willi Syndrome Genes"],"prefix":"10.3390","volume":"15","author":[{"given":"Violeta","family":"Zaric","sequence":"first","affiliation":[{"name":"Department of Psychiatry, UT Southwestern Medical Center, Dallas, TX 75390, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hye Ri","family":"Kang","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, UT Southwestern Medical Center, Dallas, TX 75390, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4092-0779","authenticated-orcid":false,"given":"Volodymyr","family":"Rybalchenko","sequence":"additional","affiliation":[{"name":"Department of Psychiatry, UT Southwestern Medical Center, Dallas, TX 75390, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jeffrey M.","family":"Zigman","sequence":"additional","affiliation":[{"name":"Department of Psychiatry, UT Southwestern Medical Center, Dallas, TX 75390, USA"},{"name":"Department of Pediatrics, UT Southwestern Medical Center, Dallas, TX 75390, USA"},{"name":"O\u2019Donnell Brain Institute, UT Southwestern Medical Center, Dallas, TX 75390, USA"},{"name":"Center for Hypothalamic Research, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX 75390, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6240-8621","authenticated-orcid":false,"given":"Steven J.","family":"Gray","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, UT Southwestern Medical Center, Dallas, TX 75390, USA"},{"name":"O\u2019Donnell Brain Institute, UT Southwestern Medical Center, Dallas, TX 75390, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5857-6489","authenticated-orcid":false,"given":"Ryan K.","family":"Butler","sequence":"additional","affiliation":[{"name":"Department of Psychiatry, UT Southwestern Medical Center, Dallas, TX 75390, USA"},{"name":"Department of Pediatrics, UT Southwestern Medical Center, Dallas, TX 75390, USA"},{"name":"O\u2019Donnell Brain Institute, UT Southwestern Medical Center, Dallas, TX 75390, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,10,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"207","DOI":"10.2174\/1573396315666190716120925","article-title":"Prader-Willi Syndrome\u2014Clinical Genetics, Diagnosis and Treatment Approaches: An Update","volume":"15","author":"Butler","year":"2019","journal-title":"Curr. Pediatr. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1038\/gim.0b013e31822bead0","article-title":"Prader-Willi syndrome","volume":"14","author":"Cassidy","year":"2012","journal-title":"Genet. Med."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Godler, D.E., and Butler, M.G. (2021). Special Issue: Genetics of Prader-Willi Syndrome. Genes, 12.","DOI":"10.3390\/genes12091429"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1136\/jmedgenet-2018-105301","article-title":"Molecular genetic classification in Prader-Willi syndrome: A multisite cohort study","volume":"56","author":"Butler","year":"2019","journal-title":"J. Med. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1002\/1096-8628(200022)97:2<136::AID-AJMG5>3.0.CO;2-V","article-title":"Prader-Willi and Angelman syndromes: Sister imprinted disorders","volume":"97","author":"Cassidy","year":"2000","journal-title":"Am. J. Med. Genet."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"893","DOI":"10.1093\/hmg\/3.6.893","article-title":"Detection of aberrant DNA methylation in unique Prader-Willi syndrome patients and its diagnostic implications","volume":"3","author":"Buiting","year":"1994","journal-title":"Hum. Mol. Genet."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3257","DOI":"10.1093\/hmg\/ddp263","article-title":"A deletion of the HBII-85 class of small nucleolar RNAs (snoRNAs) is associated with hyperphagia, obesity and hypogonadism","volume":"18","author":"Purmann","year":"2009","journal-title":"Hum. Mol. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1038\/ng.158","article-title":"Prader-Willi phenotype caused by paternal deficiency for the HBII-85 C\/D box small nucleolar RNA cluster","volume":"40","author":"Sahoo","year":"2008","journal-title":"Nat. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"452","DOI":"10.1038\/ng0496-452","article-title":"Exclusion of SNRPN as a major determinant of Prader-Willi syndrome by a translocation breakpoint","volume":"12","author":"Schulze","year":"1996","journal-title":"Nat. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1196","DOI":"10.1038\/ejhg.2010.102","article-title":"Paternally inherited microdeletion at 15q11.2 confirms a significant role for the SNORD116 C\/D box snoRNA cluster in Prader-Willi syndrome","volume":"18","author":"Duker","year":"2010","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Grootjen, L.N., Juriaans, A.F., Kerkhof, G.F., and Hokken-Koelega, A.C.S. (2022). Atypical 15q11.2-q13 Deletions and the Prader-Willi Phenotype. J. Clin. Med., 11.","DOI":"10.3390\/jcm11154636"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"416","DOI":"10.1016\/j.ejmg.2017.05.003","article-title":"A novel deletion of SNURF\/SNRPN exon 1 in a patient with Prader-Willi-like phenotype","volume":"60","author":"Cao","year":"2017","journal-title":"Eur. J. Med. Genet."},{"key":"ref_13","first-page":"4225092","article-title":"An Atypical 15q11.2 Microdeletion Not Involving SNORD116 Resulting in Prader-Willi Syndrome","volume":"2023","author":"Crenshaw","year":"2023","journal-title":"Case Rep. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1958","DOI":"10.1086\/302917","article-title":"Parent-of-origin specific histone acetylation and reactivation of a key imprinted gene locus in Prader-Willi syndrome","volume":"66","author":"Saitoh","year":"2000","journal-title":"Am. J. Hum. Genet."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1093\/hmg\/10.6.645","article-title":"Association of acetylated histones with paternally expressed genes in the Prader-Willi deletion region","volume":"10","author":"Francke","year":"2001","journal-title":"Hum. Mol. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4674","DOI":"10.1093\/hmg\/ddu187","article-title":"Reactivation of maternal SNORD116 cluster via SETDB1 knockdown in Prader-Willi syndrome iPSCs","volume":"23","author":"Cruvinel","year":"2014","journal-title":"Hum. Mol. Genet."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1016\/S0188-4409(02)00366-1","article-title":"DNA methylation, chromatin boundaries, and mechanisms of genomic imprinting","volume":"33","year":"2002","journal-title":"Arch. Med. Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"544","DOI":"10.1007\/s00335-009-9223-4","article-title":"Chromatin mechanisms in genomic imprinting","volume":"20","author":"Kacem","year":"2009","journal-title":"Mamm. Genome"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3121","DOI":"10.1111\/j.1742-4658.2006.05343.x","article-title":"The epigenetic magic of histone lysine methylation","volume":"273","author":"Jenuwein","year":"2006","journal-title":"FEBS J."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1186\/s13395-016-0093-7","article-title":"The lysine methyltransferase Ehmt2\/G9a is dispensable for skeletal muscle development and regeneration","volume":"6","author":"Zhang","year":"2016","journal-title":"Skelet. Muscle"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Liang, L., Gu, X., Zhao, J.Y., Wu, S., Miao, X., Xiao, J., Mo, K., Zhang, J., Lutz, B.M., and Bekker, A. (2016). G9a participates in nerve injury-induced Kcna2 downregulation in primary sensory neurons. Sci. Rep., 6.","DOI":"10.1038\/srep37704"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e2004515","DOI":"10.1002\/advs.202004515","article-title":"Downregulation of a Dorsal Root Ganglion-Specifically Enriched Long Noncoding RNA is Required for Neuropathic Pain by Negatively Regulating RALY-Triggered Ehmt2 Expression","volume":"8","author":"Pan","year":"2021","journal-title":"Adv. Sci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"866","DOI":"10.1038\/aps.2017.190","article-title":"Inhibition of the G9a\/GLP histone methyltransferase complex modulates anxiety-related behavior in mice","volume":"39","author":"Wang","year":"2018","journal-title":"Acta Pharmacol. Sin."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1038\/nm.4257","article-title":"Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader-Willi syndrome","volume":"23","author":"Kim","year":"2017","journal-title":"Nat. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1038\/35053110","article-title":"Role for a bidentate ribonuclease in the initiation step of RNA interference","volume":"409","author":"Bernstein","year":"2001","journal-title":"Nature"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"14385","DOI":"10.1073\/pnas.0405913101","article-title":"Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity","volume":"101","author":"Rand","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1146\/annurev-biophys-083012-130404","article-title":"Molecular mechanisms of RNA interference","volume":"42","author":"Wilson","year":"2013","journal-title":"Annu. Rev. Biophys."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1016\/j.omtn.2023.07.018","article-title":"25 years of maturation: A systematic review of RNAi in the clinic","volume":"33","author":"Corydon","year":"2023","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"424","DOI":"10.1007\/s00335-005-2460-2","article-title":"Lack of Pwcr1\/MBII-85 snoRNA is critical for neonatal lethality in Prader-Willi syndrome mouse models","volume":"16","author":"Ding","year":"2005","journal-title":"Mamm. Genome"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1644","DOI":"10.1002\/oby.23766","article-title":"Effects of thermoneutrality on food intake, body weight, and body composition in a Prader-Willi syndrome mouse model","volume":"31","author":"Lawrence","year":"2023","journal-title":"Obesity"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2364","DOI":"10.1093\/hmg\/ddt628","article-title":"Imprinted expression of UBE3A in non-neuronal cells from a Prader-Willi syndrome patient with an atypical deletion","volume":"23","author":"Hsiao","year":"2014","journal-title":"Hum. Mol. Genet."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1038\/nbt.1529","article-title":"Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling","volume":"27","author":"Chambers","year":"2009","journal-title":"Nat. Biotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1741","DOI":"10.1038\/nprot.2012.099","article-title":"Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex","volume":"7","author":"Beaudoin","year":"2012","journal-title":"Nat. Protoc."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Moutin, E., Hemonnot, A.L., Seube, V., Linck, N., Rassendren, F., Perroy, J., and Compan, V. (2020). Procedures for Culturing and Genetically Manipulating Murine Hippocampal Postnatal Neurons. Front. Synaptic Neurosci., 12.","DOI":"10.3389\/fnsyn.2020.00019"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e9","DOI":"10.1093\/nar\/gks797","article-title":"siSPOTR: A tool for designing highly specific and potent siRNAs for human and mouse","volume":"41","author":"Boudreau","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"3285","DOI":"10.1093\/hmg\/ddaa210","article-title":"Specific ZNF274 binding interference at SNORD116 activates the maternal transcripts in Prader-Willi syndrome neurons","volume":"29","author":"Langouet","year":"2020","journal-title":"Hum. Mol. Genet."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"14311","DOI":"10.1073\/pnas.250426397","article-title":"Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization","volume":"97","author":"Cavaille","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1038\/ng0598-25","article-title":"A mouse model for Prader-Willi syndrome imprinting-centre mutations","volume":"19","author":"Yang","year":"1998","journal-title":"Nat. Genet."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1093\/hmg\/ddx420","article-title":"Zinc finger protein 274 regulates imprinted expression of transcripts in Prader-Willi syndrome neurons","volume":"27","author":"Langouet","year":"2018","journal-title":"Hum. Mol. Genet."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"107639","DOI":"10.1016\/j.celrep.2020.107639","article-title":"The Histone Methyltransferase G9a Controls Axon Growth by Targeting the RhoA Signaling Pathway","volume":"31","author":"Wilson","year":"2020","journal-title":"Cell Rep."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"816","DOI":"10.1016\/j.molcel.2021.12.037","article-title":"SETDB1\/NSD-dependent H3K9me3\/H3K36me3 dual heterochromatin maintains gene expression profiles by bookmarking poised enhancers","volume":"82","author":"Barral","year":"2022","journal-title":"Mol. Cell"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1038\/s41467-023-44578-0","article-title":"Cell-type differential targeting of SETDB1 prevents aberrant CTCF binding, chromatin looping, and cis-regulatory interactions","volume":"15","author":"Tam","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Weirich, S., Khella, M.S., and Jeltsch, A. (2021). Structure, Activity and Function of the Suv39h1 and Suv39h2 Protein Lysine Methyltransferases. Life, 11.","DOI":"10.3390\/life11070703"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1038\/nsmb.1384","article-title":"The ankyrin repeats of G9a and GLP histone methyltransferases are mono- and dimethyllysine binding modules","volume":"15","author":"Collins","year":"2008","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"25309","DOI":"10.1074\/jbc.M101914200","article-title":"Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3","volume":"276","author":"Tachibana","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Able, A.A., Richard, A.J., and Stephens, J.M. (2023). TNFalpha Effects on Adipocytes Are Influenced by the Presence of Lysine Methyltransferases, G9a (EHMT2) and GLP (EHMT1). Biology, 12.","DOI":"10.3390\/biology12050674"}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/15\/11\/1366\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:19:17Z","timestamp":1760113157000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/15\/11\/1366"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,10,24]]},"references-count":46,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2024,11]]}},"alternative-id":["genes15111366"],"URL":"https:\/\/doi.org\/10.3390\/genes15111366","relation":{},"ISSN":["2073-4425"],"issn-type":[{"type":"electronic","value":"2073-4425"}],"subject":[],"published":{"date-parts":[[2024,10,24]]}}}