5-azacytidine treatment reorganizes genomic histone modification patterns

Abstract

Methylation of DNA in combination with histone modifications establishes an epigenetic code that ensures the proper control of gene expression. Although DNA methyltransferases have been shown to interact with histone methyltransferases such as EZH2 (which methylates histone H3 on lysine 27) and G9a (which methylates histone H3 on lysine 9), the relationship between DNA methylation and repressive histone marks has not been fully studied. In cancer cells, promoters of genes are often aberrantly methylated. Accordingly, 5-azacytidine (a DNA demethylating drug) is used for treating patients with myelodysplastic syndrome. However, no genome-scale studies of the effects of this drug have been reported. In this work, we report the effects of 5-azacytidine on global gene expression and analyze ~24,000 human promoters using ChIP-chip to determine how 5-azacytidine treatment effects H3K27me3 and H3K9me3 levels. We found that (1) 5-azacytidine treatment results in large changes in gene regulation with distinct functional categories of genes showing increased (e.g. C2H2 zinc finger transcription factors) and decreased (e.g. genes involved in regulation of mitochondria and oxidoreductase activity) levels; (2) most genes that show altered expression are not regulated by promoters that display DNA methylation prior to the treatment; (3) certain gene classes switch their repression mark upon treatment with 5-azacytidine (from H3K27me3 to H3K9me3 and vice versa); and (4) most changes in gene expression are not due to relief of repression mediated by DNA or histone methylation.

5-Azacitidine in aggressive myelodysplastic syndromes regulates chromatin structure at PU.1 gene and cell differentiation capacity

“…41 We initially determined two sublethal concentrations of AZA (1 and 5 mM, freshly prepared every time) and used them as reported elsewhere. 34 Experimental design of AZA in vitro treatment was described previously 40 and consists of three doses of 1 or 5 mM AZA every 24 h and alternatively three doses of G-CSF 50 ng/ml (Neupogen, Amgen, Thousand Oaks, CA, USA) or granulocyte --macrophage (GM)-CSF 50 ng/ml or macrophage (M)-CSF 50 ng/ml (Sigma-Aldrich, St Louis, MO, USA, GM-CSF---cat. no.…”

“…AZA affects DNA methylation status of the derepressed genes 34 and may also involve histone modifications. 35 --38 Epigenetic therapies may not simply block the tumor cell growth 39 but induce cellular differentiation.…”

Factors affecting the persistence of drug-induced reprogramming of the cancer methylome

“…To account for differences in initial methylation at each CpG site and amount of methylation lost, we normalized CpGs by their initial and post-treatment DNA methylation levels, such that the degree of demethylation achieved after 6 d of treatment was 100%. DNA methylation changes at later time points (3,6,9, and 27 passages) were used to calculate the fractional recovery of methylation over time in the absence of drug. A self-organizing map (SOM) 21 approach was then used to cluster the CpGs into classes based on their patterns of remethylation.…”

“…Although tumor-suppressor genes are frequently silenced in cancer through hypermethylation of promoter-associated CpG Islands (CGI), 8 and DAC is capable of reversing this aberrant methylation to achieve gene reactivation, less than 10% of DACinduced transcriptional changes are accounted for by relief of promoter hypermethylation. 9,10 Cell culture studies have demonstrated that at low doses, transient exposure to DAC results in a loss of tumorigenic potential that persists for many generations following drug withdrawal, 11 suggesting that durable methylation changes underlie its antitumor activity. Leukemia cells from patients undergoing DAC treatment experience rapid remethylation at the end of each treatment cycle, as measured globally, 12 and at LINE elements.…”

Oncometabolite Tinkers with Genome Folding, Boosting Oncogene Expression

“…Mechanistically, 5-azacytidine reduces DNA methylation by inhibiting the methyl transferase DNMT1, but has additional effects on chromatin and gene regulation [8]. To further test the hypothesis that increased Clinically, high levels of 2HG may sensitise IDH-mutant tumour cells to other therapeutic approaches [1].…”