Gene Reactivation by 5-Aza-2′-Deoxycytidine–Induced Demethylation Requires SRCAP–Mediated H2A.Z Insertion to Establish Nucleosome Depleted Regions

Abstract

5-Aza-2′-deoxycytidine, approved by the FDA for the treatment of myelodysplastic syndrome (MDS), is incorporated into the DNA of dividing cells where it specifically inhibits DNA methylation by forming covalent complexes with the DNA methyltransferases (DNMTs). In an effort to study the correlations between DNA methylation, nucleosome remodeling, and gene reactivation, we investigate the integrated epigenetic events that worked coordinately to reprogram the methylated and closed promoters back to permissive chromatin configurations after 5-Aza-2′-deoxycytidine treatment. The ChIP results indicate that H2A.Z is deposited at promoter regions by the Snf2-related CBP activator protein (SRCAP) complex following DNA demethylation. According to our genome-wide expression and DNA methylation profiles, we find that the complete re-activation of silenced genes requires the insertion of the histone variant H2A.Z, which facilitates the acquisition of regions fully depleted of nucleosome as demonstrated by NOMe–seq (Nucleosome Occupancy Methylome–sequencing) assay. In contrast, SRCAP–mediated H2A.Z deposition is not required for maintaining the active status of constitutively expressed genes. By combining Hpa II digestion with NOMe–seq assay, we show that hemimethylated DNA, which is generated following drug incorporation, remains occupied by nucleosomes. Our data highlight H2A.Z as a novel and essential factor involved in 5-Aza-2′-deoxycytidine–induced gene reactivation. Furthermore, we elucidate that chromatin remodeling translates the demethylation ability of DNMT inhibitors to their downstream efficacies, suggesting future therapeutic implications for chromatin remodelers.

Histone variants in pluripotency and disease

“…Work in Arabidopsis has demonstrated the mutual antagonism between DNA methylation and H2A.Z occupancy, with this pattern also being observed in normal mammalian cells and during tumorigenesis (Zilberman et al, 2008;Conerly et al, 2010). Although the mechanism behind this interdependence is unclear, loss of H2A.Z at tumor suppressor genes could lead to DNA methylation and heritable repression, consistent with the observation that deposition of H2A.Z is required for the full reactivation of silent genes following 5-AzaC treatment (Yang et al, 2012).…”

“…Moreover, macroH2A has recently been shown to localize to the regulatory regions of pluripotency genes, with depletion resulting in a 25-fold increase in reprogramming efficiency and increased reactivation of the pluripotency genes, suggesting that macroH2A might have a more genome-wide role in repression (Pasque et al, 2012). Furthermore, 5-aza-2Ј-deoxycytidine (5-AzaC), which inhibits DNA methyltransferases, has been shown to increase reprogramming efficiency, presumably by aiding the reactivation of pluripotency genes that have been silenced by DNA methylation (De Carvalho et al, 2010), and the subsequent deposition of H2A.Z following 5-AzaC treatment was recently shown to be required for the complete reactivation of silent genes, raising the possibility of a role for H2A.Z in reprogramming (Yang et al, 2012). Overall, reactivation of the pluripotency gene network seems to require both the incorporation and loss of specific histone variants (Fig.…”

Epigenetic therapy of cancer stem and progenitor cells by targeting DNA methylation machineries

“…The authors devised a novel technique called AcceSssIble [48] . They find that approximately 2% of chromatin regions, which are demethylated after Aza-dC treatment in a colon cancer cell line, govern a feature of open chromatin [48] possibly associated with deposition of the histone variant H2A.Z [49] . Importantly, they show that of all genes reactivated by Aza-dC treatment, 90% of them possess DNA demethylation and chromatin accessibility supporting the role of the DNA methylation inhibitor as a gene reactivator.…”

Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma

“…MAPit exploits exogenous addition of DNA methyltransferases (DNMTs), to probe accessibility of DNA in chromatin (Kladde et al 1996;Xu et al 1998b;Kilgore et al 2007;Pardo et al 2009). This technique has been used to simultaneously map DNA methylation and nucleosome positions on single molecules in many gene-specific studies (Kilgore et al 2007;Wolff et al 2010;Delmas et al 2011;Pardo et al 2011a;You et al 2011;Yang et al 2012;Darst et al 2013), and more recently, genome wide (Kelly et al 2012).…”