DMSO is a strong inducer of DNA hydroxymethylation in pre-osteoblastic MC3T3-E1 cells

Abstract

Artificial induction of active DNA demethylation appears to be a possible and useful strategy in molecular biology research and therapy development. Dimethyl sulfoxide (DMSO) was shown to cause phenotypic changes in embryonic stem cells altering the genome-wide DNA methylation profiles. Here we report that DMSO increases global and gene-specific DNA hydroxymethylation levels in pre-osteoblastic MC3T3-E1 cells. After 1 day, DMSO increased the expression of genes involved in DNA hydroxymethylation (TET) and nucleotide excision repair (GADD45) and decreased the expression of genes related to DNA methylation (Dnmt1, Dnmt3b, Hells). Already 12 hours after seeding, before first replication, DMSO increased the expression of the pro-apoptotic gene Fas and of the early osteoblastic factor Dlx5, which proved to be Tet1 dependent. At this time an increase of 5-methyl-cytosine hydroxylation (5-hmC) with a concomitant loss of methyl-cytosines on Fas and Dlx5 promoters as well as an increase in global 5-hmC and loss in global DNA methylation was observed. Time course-staining of nuclei suggested euchromatic localization of DMSO induced 5-hmC. As consequence of induced Fas expression, caspase 3/7 and 8 activities were increased indicating apoptosis. After 5 days, the effect of DMSO on promoter- and global methylation as well as on gene expression of Fas and Dlx5 and on caspases activities was reduced or reversed indicating down-regulation of apoptosis. At this time, up regulation of genes important for matrix synthesis suggests that DMSO via hydroxymethylation of the Fas promoter initially stimulates apoptosis in a subpopulation of the heterogeneous MC3T3-E1 cell line, leaving a cell population of extra-cellular matrix producing osteoblasts.

Anabolic and Antiresorptive Modulation of Bone Homeostasis by the Epigenetic Modulator Sulforaphane, a Naturally Occurring Isothiocyanate

“…Depletion of Tet1 but not Tet2 significantly reduces the cytostatic effect of DL-SFN (Fig. 9C) in MC3T3-E1 cells, concurring with our previous results with DMSO (30).…”

“…Effects of SFN-induced Active DNA Demethylation in MC3T3-E1 Osteoblasts-The results presented here are consistent with the idea that Tet-dependent hydroxymethylation may compromise cell survival as shown previously by our group (30). To test this hypothesis, we treated MC3T3-E1 cells with non-silencing RNA (negative control) or siRNA against either Tet1 or Tet2 (Fig.…”

“…SFN Induces Extrinsic Apoptosis in Cells of the Osteoblastic Lineage-As we have shown previously, DMSO induces the extrinsic pathway of apoptosis (30), suggesting that the growthsuppressive action of SFN (as reflected by a decrease in cell proliferation and metabolic activity; see Fig. 1) may be caused by programmed cell death in both MC3T3-E1 osteoblasts and MLO-Y4 osteocytes.…”

“…SFN Induces Global DNA Hydroxymethylation Changes-As we have shown previously, DMSO dramatically induces active DNA demethylation in preosteoblastic MC3T3-E1 cells within less than 1 day of treatment (Ͻ16 h) through formation of 5hmC (30). Due to the structural and biological similarities between DMSO and SFN, we analyzed whether SFN alters the level of active DNA demethylation in cells of the osteogenic linage.…”

“…9D; GenBank TM accession number CH466550). After DNA purification, hydroxymethylated DNA fragments were extracted by immunoprecipitation as described previously (30). In brief, hydroxymethylated DNA of three independent biological experiments was captured by a specific hydroxymethylcytosine antibody (Active Motif catalog number 39769) coupled to magnetic beads to separate the 5hmC-containing DNA fragments.…”