5-Aza-CdR Delivers a Gene Body Blow
Authors: Sivakanthan Kasinathan, Steven Henikoff
Journal: Cancer Cell (2014)

In this issue of Cancer Cell, Yang et al. describe a causal relationship between gene body methylation and gene expression and a role for genic methylation in response to clinical DNA methylation inhibitors, which suggests that the mechanism of action of these inhibitors includes gene body hypomethylation-induced downregulation of cancer-associated genes. In this issue of Cancer Cell, Yang et al. describe a causal relationship between gene body methylation and gene expression and a role for genic methylation in response to clinical DNA methylation inhibitors, which suggests that the mechanism of action of these inhibitors includes gene body hypomethylation-induced downregulation of cancer-associated genes. DNA cytosine methylation is an ancient regulatory mechanism present in diverse phylogenies, including vertebrates, plants, and some fungi (Jones, 2012Jones P.A. Nat. Rev. Genet. 2012; 13: 484-492Crossref PubMed Scopus (3797) Google Scholar). Cytosine methylation of CG dinucleotides by DNA methyltransferases (DNMTs) in promoters is associated with gene silencing, e.g., in X chromosome inactivation and imprinting (Jones, 2012Jones P.A. Nat. Rev. Genet. 2012; 13: 484-492Crossref PubMed Scopus (3797) Google Scholar), and promoter hypermethylation is thought to contribute to aberrant regulatory programs in cancer (Shenker and Flanagan, 2012Shenker N. Flanagan J.M. Br. J. Cancer. 2012; 106: 248-253Crossref PubMed Scopus (133) Google Scholar). Within the last decade, so-called “epigenetic” drugs have come to the fore with U.S. Food and Drug Administration approval of cytidine analog DNMT inhibitors such as 5-aza-2-deoxycytidine (5-Aza-CdR) for myelodysplastic syndrome and acute myeloid leukemia. Excitingly, studies have also hinted at the efficacy of this mode of intervention in solid tumors (Shenker and Flanagan, 2012Shenker N. Flanagan J.M. Br. J. Cancer. 2012; 106: 248-253Crossref PubMed Scopus (133) Google Scholar), expanding the utility of these chemotherapeutics beyond hematologic malignancies. Although promoter methylation, which maintains a “closed” chromatin state that impairs transcriptional initiation (Jones, 2012Jones P.A. Nat. Rev. Genet. 2012; 13: 484-492Crossref PubMed Scopus (3797) Google Scholar), has been well studied, less is known about the role of methylation in gene bodies. Intriguingly, unlike at promoters where methylation is associated with gene repression, genic methylation is positively correlated with gene expression (Figure 1A) (Maunakea et al., 2010Maunakea A.K. Nagarajan R.P. Bilenky M. Ballinger T.J. D’Souza C. Fouse S.D. Johnson B.E. Hong C. Nielsen C. Zhao Y. et al.Nature. 2010; 466: 253-257Crossref PubMed Scopus (1274) Google Scholar, Varley et al., 2013Varley K.E. Gertz J. Bowling K.M. Parker S.L. Reddy T.E. Pauli-Behn F. Cross M.K. Williams B.A. Stamatoyannopoulos J.A. Crawford G.E. et al.Genome Res. 2013; 23: 555-567Crossref PubMed Scopus (507) Google Scholar). It is thought that DNA methylation inhibitors cause promoter hypomethylation and subsequent gene reactivation (Shenker and Flanagan, 2012Shenker N. Flanagan J.M. Br. J. Cancer. 2012; 106: 248-253Crossref PubMed Scopus (133) Google Scholar). However, the effect of these drugs on gene body methylation has not been extensively studied. In this issue of Cancer Cell, the work of Yang et al., 2014Yang X. Han H. De Carvalho D.D. Lay F.D. Jones P.A. Liang G. Cancer Cell. 2014; 26 (this issue): 577-590Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar suggests a causal relationship between gene body methylation and gene expression and expands the understanding of the mechanism of action of cytidine analog cancer chemotherapeutics. They assayed genome-wide methylation at various time points after short treatment of a dividing colon cancer cell line with 5-Aza-CdR, allowing interrogation of remethylation kinetics and the durability of methylation changes upon drug withdrawal (Figure 1A). A clustering approach used to classify genomic regions into groups with high and low remethylation rates revealed that gene bodies were rapidly remethylated compared to promoters (Figure 1B). Furthermore, gene body remethylation was correlated with gene expression. Exploiting DNMT knockout cell lines, the authors determined that DNMT3B is required for rapid remethylation of gene bodies following 5-Aza-CdR treatment (Figure 1A). Both DNA methylation and gene expression were attenuated in DNMT3B deficient cells following 5-Aza-CdR treatment, suggesting that DNMT inhibitor-induced gene body demethylation downregulates gene expression. This causal relationship between 5-Aza-CdR-induced genic hypomethylation and downregulation of gene expression is particularly important in the context of the observation that c-MYC-regulated genes were overrepresented in the rapidly remethylated gene set (Figure 1B). Slowly remethylated regions tended to be de novo methylated in primary tumors and associated with tumor suppressor genes. This raises the possibility that methylation inhibitors buffer against c-MYC-associated gene expression changes characteristic of many cancers. Moreover, the observed attenuation of ostensible housekeeping functions upon 5-Aza-CdR treatment might manifest as inhibition of cell growth, consistent with an association between genic methylation and constitutive expression (Coleman-Derr and Zilberman, 2012Coleman-Derr D. Zilberman D. Cold Spring Harb. Symp. Quant. Biol. 2012; 77: 147-154Crossref PubMed Scopus (41) Google Scholar). By examining histone modifications and distribution of the H2A.Z histone variant in regions exhibiting rapid and slow remethylation kinetics, Yang et al., 2014Yang X. Han H. De Carvalho D.D. Lay F.D. Jones P.A. Liang G. Cancer Cell. 2014; 26 (this issue): 577-590Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar defined the interplay between 5-Aza-CdR treatment, gene body methylation, and chromatin signatures. H3K36m3 is enriched in actively transcribed genic regions where it is thought to suppress histone turnover to maintain transcriptional fidelity, whereas H3K27m3 is a mark of Polycomb-associated silencing. Additionally, the distribution of H2A.Z, which is thought to destabilize nucleosomes, is strikingly anticorrelated with DNA methylation at promoters in vertebrates and plants (Conerly et al., 2010Conerly M.L. Teves S.S. Diolaiti D. Ulrich M. Eisenman R.N. Henikoff S. Genome Res. 2010; 20: 1383-1390Crossref PubMed Scopus (100) Google Scholar, Zilberman et al., 2008Zilberman D. Coleman-Derr D. Ballinger T. Henikoff S. Nature. 2008; 456: 125-129Crossref PubMed Scopus (426) Google Scholar). Upon DNMT depletion, gene bodies of rapidly remethylated genes became slightly enriched for H2A.Z, while H3K36m3 was enriched independent of methylation level, possibly implicating this mark in rapid remethylation. Slowly remethylated regions were strongly enriched for H3K27m3 and H2A.Z, consistent with antagonism between these chromatin features and DNA methylation. Additionally, analysis of selected genes showed that gene body demethylation caused by 5-Aza-CdR was associated with H2A.Z deposition, a modest depletion of H3K36m3, and increased chromatin accessibility (Figure 1B). Collectively, these results suggest that genic demethylation by cytidine analogs may influence transcription by modulating nucleosome stability (Figure 1B) and that remethylation after drug withdrawal is influenced by chromatin context. There are many hypotheses regarding the function of gene body methylation. For example, genic methylation might regulate alternative promoters (Maunakea et al., 2010Maunakea A.K. Nagarajan R.P. Bilenky M. Ballinger T.J. D’Souza C. Fouse S.D. Johnson B.E. Hong C. Nielsen C. Zhao Y. et al.Nature. 2010; 466: 253-257Crossref PubMed Scopus (1274) Google Scholar). Alternatively, gene body methylation might inhibit cryptic transcription initiation events. In plants, cryptic initiation was proposed to generate antisense transcripts that base pair with mRNA transcribed from the canonical transcription start site (TSS), leading to double-stranded RNA-mediated methylation and silencing of the cryptic TSS (Tran et al., 2005Tran R.K. Henikoff J.G. Zilberman D. Ditt R.F. Jacobsen S.E. Henikoff S. Curr. Biol. 2005; 15: 154-159Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) (Figure 1C). Consistent with this model, genic methylation is anticorrelated with transcriptional noise in vertebrates (Huh et al., 2013Huh I. Zeng J. Park T. Yi S.V. Epigenetics Chromatin. 2013; 6: 9Crossref PubMed Scopus (89) Google Scholar). It is attractive to hypothesize that an increase in cryptic initiation in active genes upon nucleosome destabilization by methylation inhibitors leads to DNMT3B recruitment and suppression of cryptic transcription by rapid remethylation. This work by Yang et al., 2014Yang X. Han H. De Carvalho D.D. Lay F.D. Jones P.A. Liang G. Cancer Cell. 2014; 26 (this issue): 577-590Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar also provides important insights into the pharmacology of DNA methylation inhibitors. Despite the apparent lack of specificity of methylation inhibitors, remethylation kinetics after drug withdrawal are dependent on genomic and chromatin contexts and driven by DNMT3B. Promoter demethylation appears to be sustained after drug withdrawal, while genic demethylation is comparatively short-lived, which may have implications for understanding clinical responses to methylation inhibitors. Mechanistically, DNA methylation inhibitors may normalize gene expression in cancer by reactivating silenced tumor suppressors through their action at promoters and buffer overexpression of oncogenes and metabolic genes by hypomethylating gene bodies. We thank Gabriel E. Zentner for comments on this manuscript. S.K. is supported by NIH grant 1F30CA186458 and the Micki & Robert Flowers ARCS Endowment from the Seattle Chapter of the ARCS Foundation. S.H. is supported by the Howard Hughes Medical Institute. Gene Body Methylation Can Alter Gene Expression and Is a Therapeutic Target in CancerYang et al.Cancer CellSeptember 25, 2014In BriefYang et al. show a causal relationship between DNA methylation at the gene body and gene expression; thus, the cancer therapeutic activity of DNA methylation inhibitors may be due to not only inducing the expression of aberrantly silenced genes but also to repressing the expression of aberrantly overexpressed genes. Full-Text PDF Open Archive