Gy and Embryology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary; [email protected] (J.V.); [email protected] (E.K.); [email protected] (R.T.); [email protected] (C.M.); [email protected] (L.D.) Division of Healthcare Biology and Central Electron Microscope Laboratory, Healthcare School, University of P s, 7624 P s, Hungary; [email protected] Institute of Biochemistry and Healthcare Chemistry, Healthcare College, University of P s, 7624 P s, Hungary; [email protected] Correspondence: [email protected] Shared senior authors.Citation: V J.; Kiss, K.; Karanyicz, E.; Tak s, R.; Matta, C.; Ducza, L.; Rauch, T.A.; Z y, R. Evaluation of Gene 1-Methyladenosine Endogenous Metabolite expression Patterns of Epigenetic Enzymes Dnmt3a, Tet1 and Ogt in Murine Chondrogenic Models. Cells 2021, ten, 2678. https://doi.org/10.3390/cells10102678 Academic Editor: Giorgio Malpeli Received: eight September 2021 Accepted: 2 October 2021 Published: 6 OctoberAbstract: We investigated the gene expression pattern of selected enzymes involved in DNA methylation plus the effects on the DNA methylation inhibitor 5-azacytidine for the duration of in vitro and in vivo cartilage formation. Based on the information of a PCR array performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expressions of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase three), and Ogt (O-linked N-acetylglucosamine transferase) have been further examined with RT-qPCR in murine cell line-based and major chondrifying micromass cultures. We located quite powerful but progressively decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation in conjunction with powerful signals inside the cartilaginous embryonic skeleton employing certain RNA probes for in situ hybridization on frozen sections of 15-day-old mouse embryos. Dnmt3a and Ogt expressions did not show considerable alterations with RT-qPCR and gave weak in situ hybridization signals. The DNA methylation inhibitor 5-azacytidine lowered cartilage-specific gene expression and cartilage formation when applied during the early stages of chondrogenesis. In contrast, it had a stimulatory impact when added to differentiated chondrocytes, and quantitative methylation-specific PCR proved that the DNA methylation pattern of important chondrogenic marker genes was altered by the remedy. Our outcomes indicate that the DNA demethylation inducing Tet1 plays a significant role throughout chondrogenesis, and inhibition of DNA methylation exerts distinct effects in diverse phases of in vitro cartilage formation. Keyword phrases: chondrogenesis; chondrocyte; cell differentiation; C3H10T1/2; micromass culture; mouse embryo; DNA methylation; Fulvestrant Protocol 5-azacytidinePublisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.1. Introduction Epigenetics refers to those reversible and heritable biological processes that regulate gene expression without alteration from the principal DNA sequence [1]. Epigenetic regulation is accountable for cell-specific gene expression and for the inheritance of these distinctive expression patterns to daughter cells [2]. The key target of epigenetic processes will be the DNA-histone complex termed chromatin. The accessibility of a certain DNA segment for transcription elements is influenced by distinctive epigenetic marks, such as DNA methylation or histone modifications [3]. DNA methylation causes gene repression or silencing by adding a methyl gro.
