DNA methylation has a role in the regulation of gene expression during normal mammalian development but can also mediate epigenetic silencing of CpG island genes in cancer and other diseases. Many individual genes (including tumor suppressors) have been shown to undergo de novo methylation in specific tumor types, but the biological logic inherent in this process is not understood. To decipher this mechanism, we have adopted a new approach for detecting CpG island DNA methylation that can be used together with microarray technology. Genome-wide analysis by this technique demonstrated that tumor-specific methylated genes belong to distinct functional categories, have common sequence motifs in their promoters and are found in clusters on chromosomes. In addition, many are already repressed in normal cells. These results are consistent with the hypothesis that cancer-related de novo methylation may come about through an instructive mechanism.

Embryonic stem cells are characterized by unique epigenetic features including decondensed chromatin and hyperdynamic association of chromatin proteins with chromatin. Here we investigate the potential mechanisms that regulate chromatin plasticity in embryonic stem cells. Using epigenetic drugs and mutant embryonic stem cells lacking various chromatin proteins, we find that histone acetylation, G9a-mediated histone H3 lysine 9 (H3K9) methylation and lamin A expression, all affect chromatin protein dynamics. Histone acetylation controls, almost exclusively, euchromatin protein dynamics; lamin A expression regulates heterochromatin protein dynamics, and G9a regulates both euchromatin and heterochromatin protein dynamics. In contrast, we find that DNA methylation and nucleosome repeat length have little or no effect on chromatin-binding protein dynamics in embryonic stem cells. Altered chromatin dynamics associates with perturbed embryonic stem cell differentiation. Together, these data provide mechanistic insights into the epigenetic pathways that are responsible for chromatin plasticity in embryonic stem cells, and indicate that the genome’s epigenetic state modulates chromatin plasticity and differentiation potential of embryonic stem cells.

Corneal differentiation and maturation are associated with major changes in the expression levels of numerous genes, including those coding for the chromatin-binding high-mobility group (HMG) proteins. Here we report that HMGN1, a nucleosome-binding protein that alters the structure and activity of chromatin, affects the development of the corneal epithelium in mice. The corneal epithelium of Hmgn1 −/− mice is thin, has a reduced number of cells, is poorly stratified, is depleted of supra-basal wing cells, and its most superficial cell layer blisters. In mature Hmgn1 −/− mice, the basal cells retain the ovoid shape of immature cells, and rest directly on the basal membrane which is disorganized. Gene expression was modified in Hmgn1 −/− corneas: glutathione-S-transferase (GST)α 4and GST ω 1, epithelial layer-specific markers, were selectively reduced while E-cadherin and α-, β-, and γ-catenin, components of adherens junctions, were increased. Immunofluorescence analysis reveals a complete co-localization of HMGN1 and p63 in small clusters of basal corneal epithelial cells of wild-type mice, and an absence of p63 expressing cells in the central region of the Hmgn1 −/− cornea. We suggest that interaction of HMGN1 with chromatin modulates the fidelity of gene expression and affects corneal development and maturation.

The mechanism for demethylation of DNA in rat myoblasts has recently been studied using a new in vitro system that monitors demethylation in whole cell extracts. Previous investigations using this system had indicated that demethylation is resistant to conditions that are normally assumed to denature or digest proteins. Remarkably, it was reported that the activity appeared to be sensitive to the action of ribonuclease, suggesting a role for RNA in the demethylation of DNA. This manuscript reports that, upon further purification of the extract, demethylation activity has properties that are different. When subjected to more rigorous procedures for digestion of proteins, the demethylase activity disappears. Furthermore, RNase sensitivity of the extract disappears when a quantity of unmethylated competitor DNA is added to the reaction mix or when extracts treated with RNase are subsequently treated with protease. Although a role for RNA cannot be completely discounted, it is unlikely that this demethyl-ation reaction involves RNA cofactors or ribozyme components. These results have important implications for the mechanism of DNA demethylation and they exemplify the potential pitfalls of experiments in which new biological roles for RNA are evaluated using RNase sensitivity experiments.

The H19 imprinted gene locus is regulated by an upstream 2 kb imprinting control region (ICR) that influences allele-specific expression, DNA methylation, and replication timing. This ICR becomes de novo methylated during late spermatogenesis in the male but emerges from oogenesis in an unmethylated form, and this allele-specific pattern is then maintained throughout early development and in all tissues of the mouse. We have used a genetic approach involving transfection into embryonic stem (ES) cells in order to decipher how the maternal allele is protected from de novo methylation at the time of implantation. Our studies show that CCCTC binding factor (CTCF) boundary elements within the ICR have the ability to prevent de novo methylation on the maternal allele. Since CTCF does not recognize its binding sequence when methylated, this reaction does not occur on the paternal allele, thus preserving the gamete-derived, allele-specific pattern. These results suggest that CTCF may play a general role in the maintenance of differential methylation patterns in vivo.

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Although most mammalian genes are expressed from both alleles, there is a small group of special genes which are imprinted so that only one of the parental alleles is actually expressed in target cells. This epigenetic process involves regulation at a number of different stages of development and is very complex. In principle, imprinted gene regions must be marked in cis in the gametes using epigenetic features capable of being maintained through cell division and able to direct multigenic monoallelic expression in differentiated cells of the mature organism. The difference between alleles must be erased during early gametogenesis to allow the imprint to be reset in the mature gametes. In this review we will summarize what is currently known about the molecular mechanisms which mediate these steps.

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