{"title":"Epigenome editing revealed the role of DNA methylation of T-DMR/CpG island shore on Runx2 transcription","authors":"Yutaro Kawa , Miyuki Shindo , Jun Ohgane , Masafumi Inui","doi":"10.1016/j.bbrep.2024.101733","DOIUrl":null,"url":null,"abstract":"<div><p>RUNX2 is a transcription factor crucial for bone formation. Mutant mice with varying levels of <em>Runx2</em> expression display dosage-dependent skeletal abnormalities, underscoring the importance of <em>Runx2</em> dosage control in skeletal formation. RUNX2 activity is regulated by several molecular mechanisms, including epigenetic modification such as DNA methylation. In this study, we investigated whether targeted repressive epigenome editing including hypermethylation to the Runx2-DMR/CpG island shore could influence <em>Runx2</em> expression using Cas9-based epigenome-editing tools. Through the transient introduction of CRISPRoff-v2.1 and gRNAs targeting Runx2-DMR into MC3T3-E1 cells, we successfully induced hypermethylation of the region and concurrently reduced <em>Runx2</em> expression during osteoblast differentiation. Although the epigenome editing of Runx2-DMR did not impact the expression of RUNX2 downstream target genes, these results indicate a causal relationship between the epigenetic status of the Runx2-DMR and <em>Runx2</em> transcription. Additionally, we observed that hypermethylation of the Runx2-DMR persisted for at least 24 days under growth conditions but decreased during osteogenic differentiation, highlighting an endogenous DNA demethylation activity targeting the Runx2-DMR during the differentiation process. In summary, our study underscore the usefulness of the epigenome editing technology to evaluate the function of endogenous genetic elements and revealed that the Runx2-DMR methylation is actively regulated during osteoblast differentiation, subsequently could influence <em>Runx2</em> expression.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.3000,"publicationDate":"2024-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824000979/pdfft?md5=de0799a06bd730c27be6c87410af848e&pid=1-s2.0-S2405580824000979-main.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry and Biophysics Reports","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2405580824000979","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
RUNX2 is a transcription factor crucial for bone formation. Mutant mice with varying levels of Runx2 expression display dosage-dependent skeletal abnormalities, underscoring the importance of Runx2 dosage control in skeletal formation. RUNX2 activity is regulated by several molecular mechanisms, including epigenetic modification such as DNA methylation. In this study, we investigated whether targeted repressive epigenome editing including hypermethylation to the Runx2-DMR/CpG island shore could influence Runx2 expression using Cas9-based epigenome-editing tools. Through the transient introduction of CRISPRoff-v2.1 and gRNAs targeting Runx2-DMR into MC3T3-E1 cells, we successfully induced hypermethylation of the region and concurrently reduced Runx2 expression during osteoblast differentiation. Although the epigenome editing of Runx2-DMR did not impact the expression of RUNX2 downstream target genes, these results indicate a causal relationship between the epigenetic status of the Runx2-DMR and Runx2 transcription. Additionally, we observed that hypermethylation of the Runx2-DMR persisted for at least 24 days under growth conditions but decreased during osteogenic differentiation, highlighting an endogenous DNA demethylation activity targeting the Runx2-DMR during the differentiation process. In summary, our study underscore the usefulness of the epigenome editing technology to evaluate the function of endogenous genetic elements and revealed that the Runx2-DMR methylation is actively regulated during osteoblast differentiation, subsequently could influence Runx2 expression.
期刊介绍:
Open access, online only, peer-reviewed international journal in the Life Sciences, established in 2014 Biochemistry and Biophysics Reports (BB Reports) publishes original research in all aspects of Biochemistry, Biophysics and related areas like Molecular and Cell Biology. BB Reports welcomes solid though more preliminary, descriptive and small scale results if they have the potential to stimulate and/or contribute to future research, leading to new insights or hypothesis. Primary criteria for acceptance is that the work is original, scientifically and technically sound and provides valuable knowledge to life sciences research. We strongly believe all results deserve to be published and documented for the advancement of science. BB Reports specifically appreciates receiving reports on: Negative results, Replication studies, Reanalysis of previous datasets.