Oncogene (2001) 20, 3156 ± 3165 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc DNA methylation, chromatin inheritance, and cancer Michael R Rountree*,1, Kurtis E Bachman1, James G Herman1 and Stephen B Baylin1 1The Johns Hopkins Oncology Center, Tumor Biology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, Maryland, MD 21231, USA Cancer is a process driven by the accumulation of sion capacity without DNA sequence alterations, are abnormalities in gene function. While many of these also central to tumor progression. The epigenetic changes are genetic, epigenetically mediated changes in control of gene function involves the formation of gene expression are being increasingly appreciated. This chromatin that modulates gene transcription. The latter process emphasizes the need to understand two key study of this level of gene control has blossomed over components of heritable, but reversible, modulation of the last decade and is a critical link to our under- gene promoter function that are closely tied to one standing of the neoplastic process. another ± formation of chromatin which modulates transcription and establishing patterns of DNA methyla- tion. The link lies ®rst in the recruitment to methylated Chromatin and transcription cytosines of a family of methyl-CpG binding domain proteins (MBDs), which are direct transcriptional Eukaryotic cells must accomplish the daunting task of repressors and can complex with transcriptional core- packaging an enormous amount of DNA into their pressors including histone deacetylases (HDACs). Ad- nucleus, while ensuring the proper expression of a ditionally, the proteins that catalyze DNA methylation, subset of genes and the silencing of other regions of the DNA methyltransferases (DNMTs), also directly repress genome. Higher eukaryotes are especially burdened in transcription and associate with HDACs. Regulation of this task due to the large amount of repetitive elements these above chromatin-DNA methylation interactions as (Alu, LINEs, SINEs, etc.) that are scattered through- a function of DNA replication timing is emerging as a out their genomes. In general, the genome is key event in the inheritance of transcriptionally repressed compartmentalized into transcriptionally competent domains of the genome. Importantly, synergy between euchromatin and transcriptionally incompetent hetero- HDAC activity and DNA methylation is operative for a chromatin. Cells accomplish this feat by packaging key epigenetic abnormality in cancer cells, transcrip- DNA into chromatin, whose basic unit is the tional silencing of tumor suppressor genes. This change nucleosome with *146 bp of DNA wrapped around has now been recognized for genes that are essential for it. The proper transcriptional status is then achieved normal regulation of virtually every major cell function through the interplay of protein complexes that including cell growth, dierentiation, apoptosis, DNA associate with, manipulate, and epigenetically modify repair, and cell ± cell, cell ± substratum interaction. this basic unit to either foster or inhibit transcription. Understanding the molecular determinants of both These epigenetic modi®cations, employing combina- normal and abnormal patterns of chromatin formation tions of factors, allow the cell to modulate the and DNA methylation thus holds great promise for our transcriptional activity of given gene promoters. In understanding of cancer and for means to better this manner, a rheostat of transcriptional activity diagnose, prevent, and treat this disease. Oncogene provides a range of gene function from high-level (2001) 20, 3156 ± 3165. expression to complete silencing, as well as setting up gene expression events to react quickly to environ- Keywords: DNA methylation; histone acetylation; mental stimuli. chromatin inheritance; cancer Two epigenetic modi®cations have emerged as critical layers of regulation that participate in this transcriptional rheostat (Figure 1). The ®rst, histone Introduction acetylation appears to be used by all eukaryotes as one layer of transcriptional control (Grunstein, 1997). The The hallmark of cancer is a progressive appearance of acetylation of the amino-terminal tails of histones H3 malignant cell behavior that is triggered by the and H4 by histone acetyltransferases (HATs) creates an evolution of altered gene function. Many of the gene accessible chromatin con®guration that facilitates changes stem from genetic abnormalities that disrupt transcriptional activity. Removal of these acetyl groups coding regions. However, it is becoming clear that by histone deacetylases (HDACs) facilitates chromatin epigenetic events, or heritable changes in gene expres- compaction that is detrimental to transcription. The cell uses these HATs and HDACs as coactivators and corepressors respectively to modulate promoter activ- *Correspondence: MR Rountree ity. DNA methylation, chromatin inheritance and cancer MR Rountree et al 3157 Transcriptionally Transcriptionally Transcriptionally Incompetent Inducible Competent Heterochromatin Chromatin Euchromatin Histone Acetylation DNA Methylation Inactive X-chromosome Environmentally ActiveGenes Silenced Imprinted Genes Responsive Genes Alu, LINEs, SINEs Developmentally Pericentromeric Repeats Responsive Genes Figure 1 The transcriptional rheostat. DNA methylation and histone acetylation help to establish chromatin states that either foster or inhibit transcription. The shaded bars represent the inverse correlation between these two epigenetic modi®cations. While a cell will use histone acetylation status to modulate gene expression, DNA methylation primarily serves as a transcriptionally repressive `lock' The second epigenetic modi®cation, DNA methyla- recombination as well (Bird, 1995; Yoder et al., 1997a; tion, has a long-standing relationship with gene Walsh et al., 1998). The methylation of cytosines makes inactivity and has been implicated as a critical layer of them more susceptible to deamination, which has control for enhancing transcriptional silencing (Bird, reduced the overall frequency of CpGs in the bulk 1992; Eden and Cedar, 1994). However, the lack of such genome (Bird, 1980). Conversely, the promoter regions an epigenetic modi®cation in some favorite model of many genes generally remain free of methylation and organisms (e.g. S. cerevisiae, C. elegans) has long raised therefore less susceptible to deamination. Thus, these questions about the importance of DNA methylation promoters have retained the expected frequency of CpG for gene control in eukaryotes. Interestingly, while dinucleotides and are referred to as `CpG islands'. Drosophila was long thought not to have DNA Studies of the adenine phosphoribosyltransferase (Aprt) methylation, recent reports have identi®ed genes with locus have identi®ed a potential mechanism for how homology to the vertebrate DNA methylation machin- CpG islands remain free of methylation in embryonic ery (Tweedie et al., 1999; Hung et al., 1999; Roder et al., cells. Three groups demonstrated that the presence of 2000; Lyko et al., 2000a). In addition, trace amounts of Sp1 binding sites and presumably the trans-acting factor cytosine methylation have been detected in this that binds to these sites protects the CpG island of the organism (Gowher et al., 2000; Lyko et al., 2000b). Aprt gene from methylation (Macleod et al., 1994; In mammals, genomic methylation patterns are Brandeis et al., 1994; Mummaneni et al., 1995, 1998). established during embryogenesis through the interplay Importantly, while most CpG islands remain free of of at least three DNA methyltransferases (Dnmt1, methylation, those associated with transcriptionally Dnmt3a, and Dnmt3b; Figure 2) and presumably the silenced genes on the inactive X-chromosome and factors that associate with these enzymes to target and silenced alleles of imprinted genes are densely methylated regulate their enzymatic activity. All three enzymes are (Brandeis et al., 1993). essential for proper murine development (Li et al., 1992; Okano et al., 1999). It has been proposed that the newly identi®ed Dnmt3a and Dnmt3b enzymes act primarily as DNA methylation and histone deacetylation: partners in de novo methyltransferases to establish methylation transcriptional repression patterns during embryogenesis (Okano et al., 1999). In contrast, Dnmt1 is thought to maintain these methyla- DNA methylation appears capable of directly prevent- tion patterns during DNA replication. However, the ing the binding of some transcription factors to their strict designation of these enzymes as either de novo or DNA binding sites (Tate and Bird, 1993). However, the maintenance methyltransferases is not yet de®nitive. majority of inhibition of transcription in association The vast majority of CpG dinucleotides (*70%) in with DNA methylation appears to occur through mammalian genomes are methylated and reside within complex indirect mechanisms involving changes in repetitive elements (Yoder et al., 1997a). This methyla- chromatin formation. Initial experiments demonstrated tion is a candidate mechanism for helping to transcrip- that in vitro methylated genes transfected into cells tionally silence these elements and thus, has been were transcriptionally inactive and assumed a chroma- proposed to serve as a host defense mechanism to inhibit tin conformation devoid of nuclease sensitive sites transposition and perhaps to subdue homologous present in active genes (Keshet et al., 1986). However, Oncogene