Almost 1-2% of the human genome is located within 500 bp of either side of a transcription initiation site, whereas a far larger proportion (Ϸ25%) is potentially transcribable by elongating RNA polymerases. This observation raises the question of how the genome is packaged into chromatin to allow start sites to be recognized by the regulatory machinery at the same time as transcription initiation, but not elongation, is blocked in the 25% of intragenic DNA. We developed a chromatin scanning technique called ChAP, coupling the chromatin immunoprecipitation assay with arbitrarily primed PCR, which allows for the rapid and unbiased comparison of histone modification patterns within the eukaryotic nucleus. Methylated lysine 4 (K4) and acetylated K9͞14 of histone H3 were both highly localized to the 5 regions of transcriptionally active human genes but were greatly decreased downstream of the start sites. Our results suggest that the large transcribed regions of human genes are maintained in a deacetylated conformation in regions read by elongating polymerase. Common models depicting widespread histone acetylation and K4 methylation throughout the transcribed unit do not therefore apply to the majority of human genes.

Epigenetic reprogramming is commonly observed in cancer, and is hypothesized to involve multiple mechanisms, including DNA methylation and Polycomb repressive complexes (PRCs). Here we devise a new experimental and analytical strategy using customized highdensity tiling arrays to investigate coordinated patterns of gene expression, DNA methylation, and Polycomb marks which differentiate prostate cancer cells from their normal counterparts. Three major changes in the epigenomic landscape distinguish the two cell types. Developmentally significant genes containing CpG islands which are silenced by PRCs in the normal cells acquire DNA methylation silencing and lose their PRC marks (epigenetic switching). Because these genes are normally silent this switch does not cause de novo repression but might significantly reduce epigenetic plasticity. Two other groups of genes are silenced by either de novo DNA methylation without PRC occupancy (5mC reprogramming) or by de novo PRC occupancy without DNA methylation (PRC reprogramming). Our data suggest that the two silencing mechanisms act in parallel to reprogram the cancer epigenome and that DNA hypermethylation may replace Polycomb-based repression near key regulatory genes, possibly reducing their regulatory plasticity.spatial clustering ͉ DNA methylation ͉ MeDIP normalization ͉ Polycomb B iochemical processes including DNA cytosine methylation and histone modifications interact with each other to ensure the stability of epigenetic states. These processes are clearly altered in cancer and contribute to the establishment and maintenance of the malignant phenotype (1). Recent discoveries and technological developments have greatly expanded our understanding of the cell's epigenetic makeup, revealing a rich repertoire of histone modifications and protein complexes that regulate them (2, 3). Chromatin regulating complexes, and most notably the family of Polycomb repressive complexes (PRCs), which mediate trimethylation at H3K27, are highly active in cancer cells (4). Studies of Polycomb activity in pluripotent embryonic stem cells (ESC) (5-7) revealed broad patterns of Polycomb-based repression near key developmental regulators, many of which are known DNA hypermethylation targets in cancer (8). It is therefore pertinent to take an integrated look at both DNA methylation and histone modifications simultaneously in a coherent cell set in which the appropriate presumed normal cell counterpart is compared to its malignant state.The evidence on interaction between PRCs and the DNA methylation machinery is partial and sometimes conflicting. The correlations between ESC PRC targets and cancer hypermethylation (8), or the reported enrichment of H3K27me3 marks at specific hypermethylated CpG islands (9), have led to multiple mechanistic hypotheses on the underlying process. The lack of DNA hypermethylation at PRC occupied regions in embryonic carcinoma cells (10) and of DNA hypomethylation after knockdown of a PRC2 component (EZH2) in cancer cells (11) further demonstrate that the i...

Proper DNA methylation patterns are essential for mammalian development and differentiation. DNA methyltransferases (DNMTs) primarily establish and maintain global DNA methylation patterns; however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. We used sucrose density gradients of nucleosomes prepared by partial and maximum micrococcal nuclease digestion, coupled with Western blot analysis to probe for the interactions between DNMTs and native nucleosomes. This method allows for analysis of the in vivo interactions between the chromatin modification enzymes and their actual nucleosomal substrates in the native state. We show that little free DNA methyltransferase 3A and 3B (DNMT3A/3B) exist in the nucleus and that almost all of the cellular contents of DNMT3A/3B, but not DNMT1, are strongly anchored to a subset of nucleosomes. This binding of DNMT3A/3B does not require the presence of other well-known chromatin-modifying enzymes or proteins, such as proliferating cell nuclear antigen, heterochromatin protein 1, methyl-CpG binding protein 2, Enhancer of Zeste homolog 2, histone deacetylase 1, and UHRF1, but it does require an intact nucleosomal structure. We also show that nucleosomes containing methylated SINE and LINE elements and CpG islands are the main sites of DNMT3A/3B binding. These data suggest that inheritance of DNA methylation requires cues from the chromatin component in addition to hemimethylation.Proper DNA methylation patterns are essential for mammalian development and differentiation. More than three decades ago, de novo cytosine DNA methylation and its maintenance were proposed to exist in eukaryotic cells (29, 54); however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. DNA methyltransferases (DNMTs) DNMT1, DNMT3A, and DNMT3B primarily establish and maintain global DNA methylation patterns (39, 48). DNMT1 preferentially methylates hemimethylated DNA in vitro (7) and is tethered to replication foci during S phase (38). In contrast, DNMT3A and DNMT3B (DNMT3A/3B) have no preference for hemimethylated DNA (49) and are required for de novo methylation of genomic DNA (48). It has been thought that DNMT1 acts mainly as a "maintenance methyltransferase" during DNA synthesis and that DNMT3A and DNMT3B act as "de novo" enzymes. However, more recent studies indicate that DNMT1 may also be required for de novo methylation of genomic DNA (17,30) and that DNMT3A/3B are also required for maintenance functions (11,40,55). Furthermore, the different DNMTs cooperate in maintaining the methylation of some regions of the genome, particularly repetitive elements (40,53).Recruitment of individual DNMT enzymes to different regions of chromatin in vivo, particularly to gene regulatory regions, may require interaction with auxiliary factors (28, 36). DNMT1, which is diffusely localized throughout nuclei in non-S-phase cells (38), is targeted to replication foci by interacting with prolif...

Epigenetic silencing of tumor suppressor genes is generally thought to involve DNA cytosine methylation, covalent modifications of histones, and chromatin compaction. Here, we show that silencing of the three transcription start sites in the bidirectional MLH1 promoter CpG island in cancer cells involves distinct changes in nucleosomal occupancy. Three nucleosomes, almost completely absent from the start sites in normal cells, are present on the methylated and silenced promoter, suggesting that epigenetic silencing may be accomplished by the stable placement of nucleosomes into previously vacant positions. Activation of the promoter by demethylation with 5-aza-2'-deoxycytidine involves nucleosome eviction. Epigenetic silencing of tumor suppressor genes may involve heritable changes in nucleosome occupancy enabled by cytosine methylation.