CpG island hypermethylation and global genomic hypomethylation are common epigenetic features of cancer cells. Less attention has been focused on histone modifications in cancer cells. We characterized post-translational modifications to histone H4 in a comprehensive panel of normal tissues, cancer cell lines and primary tumors. Using immunodetection, high-performance capillary electrophoresis and mass spectrometry, we found that cancer cells had a loss of monoacetylated and trimethylated forms of histone H4. These changes appeared early and accumulated during the tumorigenic process, as we showed in a mouse model of multistage skin carcinogenesis. The losses occurred predominantly at the acetylated Lys16 and trimethylated Lys20 residues of histone H4 and were associated with the hypomethylation of DNA repetitive sequences, a well-known characteristic of cancer cells. Our data suggest that the global loss of monoacetylation and trimethylation of histone H4 is a common hallmark of human tumor cells.

Centromeres are essential for ensuring proper chromosome segregation in eukaryotes. Their definition relies on the presence of a centromere-specific H3 histone variant CenH3, known as CENP-A in mammals. Its overexpression in aggressive cancers raises questions concerning its effect on chromatin dynamics and contribution to tumorigenesis. We find that CenH3 overexpression in human cells leads to ectopic enrichment at sites of active histone turnover involving a heterotypic tetramer containing CenH3-H4 with H3.3-H4. Ectopic localization of this particle depends on the H3.3 chaperone DAXX rather than the dedicated CenH3 chaperone HJURP. This aberrant nucleosome occludes CTCF binding and has a minor effect on gene expression. Cells overexpressing CenH3 are more tolerant of DNA damage. Both the survival advantage and CTCF occlusion in these cells are dependent on DAXX. Our findings illustrate how changes in histone variant levels can disrupt chromatin dynamics and suggests a possible mechanism for cell resistance to anticancer treatments.

The present work investigates the occurrence and significance of aberrant DNA methylation patterns during early stages of atherosclerosis. To this end, we asked whether the genetically atherosclerosis-prone APOEnull mice show any changes in DNA methylation patterns before the appearance of histologically detectable vascular lesion. We exploited a combination of various techniques: DNA fingerprinting, in vitro methyl-accepting assay, 5-methylcytosine quantitation, histone posttranslational modification analysis, Southern blotting, and PCR. Our results show that alterations in DNA methylation profiles, including both hyper-and hypomethylation, were present in aortas and PBMC of 4-week-old mutant mice with no detectable atherosclerotic lesion. Sequencing and expression analysis of 60 leukocytic polymorphisms revealed that epigenetic changes involve transcribed genic sequences, as well as repeated interspersed elements. Furthermore, we showed for the first time that atherogenic lipoproteins promote global DNA hypermethylation in a human monocyte cell line. Taken together, our results unequivocally show that alterations in DNA methylation profiles are early markers of atherosclerosis in a mouse model and may play a causative role in atherogenesis.Atherosclerosis and its complications are a major cause of death and disability in the developed world. The disease is characterized by infiltration of lipid particles in the arterial wall, accompanied by the recruitment of inflammatory and immune cells, migration and proliferation of smooth muscle cells (SMC), 1 and synthesis of extracellular matrix. These processes eventually result in the gradual development of an elevated lipid-rich, fibrocellular lesion (1).In mammals, DNA methyltransferases use S-adenosyl methionine (SAM) as a methyl group donor to methylate the carbon in position 5 of cytosine residues in a CpG dinucleotide (CG) context (2). DNA methylation regulates fundamental biological phenomena such as gene expression, genome stability, mutation rate, genomic imprinting, and X chromosome inactivation (3-6). Both global and gene-specific alterations in DNA methylation are associated with abnormal phenotypes in disease (7,8). For example, cancer cells show global genomic hypomethylation and dense hypermethylation of CpG islands, which are normally unmethylated (9). The identification of cancer type-and stage-specific changes in DNA methylation has justified hopes for novel diagnostic and therapeutic avenues (10).Two general observations suggest that alterations in DNA methylation patterns are involved in atherogenesis (11-13). First, global hypomethylation and dense hypermethylation of certain CpG islands are associated with aging, a major risk factor for atherosclerosis (14). Second, hyperhomocysteinemia and the subsequent decreased production or bioavailability of SAM is associated with an increased risk of cardiovascular disease (15). Accordingly, mice with genetically reduced levels of methylenetetrahydrofolate reductase, a key enzyme in the pathway generating ...

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Cancer is as much an epigenetic disease as it is a genetic and cytogenetic disease. The discovery that drastic changes in DNA methylation and histone modifications are commonly found in human tumors has inspired various laboratories and pharmaceutical companies to develop and study epigenetic drugs. One of the most promising groups of agents is the inhibitors of histone deacetylases (HDACs), which have different biochemical and biologic properties but have a single common activity: induction of acetylation in histones, the key proteins in nucleosome and chromatin structure. One of the main mechanisms of action of HDAC inhibitors is the transcriptional reactivation of dormant tumor-suppressor genes, such as p21 WAF1 . However, their pleiotropic nature leaves open the possibility that their wellknown differentiation, cell-cycle arrest and apoptotic properties are also involved in other functions associated with HDAC inhibition. Many phase I clinical trials indicate that HDAC inhibitors appear to be well-tolerated drugs. Thus, the field is ready for rigorous biologic and clinical scrutiny to validate the therapeutic potential of these drugs. Our current data indicate that the use of HDAC inhibitors, probably in association with classical chemotherapy drugs or in combination with DNA-demethylating agents, could be promising for cancer patients.

DNA methyltransferase 1 (DNMT1) plays an essential role in murine development and is thought to be the enzyme primarily responsible for maintenance of the global methylation status of genomic DNA. However, loss of DNMT1 in human cancer cells affects only the methylation status of a limited number of pericentromeric sequences. Here we show that human cancer cells lacking DNMT1 display at least two important differences with respect to wild type cells: a profound disorganization of nuclear architecture, and an altered pattern of histone H3 modification that results in an increase in the acetylation and a decrease in the dimethylation and trimethylation of lysine 9. Additionally, this phenotype is associated with a loss of interaction of histone deacetylases (HDACs) and HP1 (heterochromatin protein 1) with histone H3 and pericentromeric repetitive sequences (satellite 2). Our data indicate that DNMT1 activity, via maintenance of the appropriate histone H3 modifications, contributes to the preservation of the correct organization of large heterochromatic regions.Methylation of CpG dinucleotides is an epigenetic phenomenon involved in regulating important intranuclear events, such as the organization of chromatin structure, transcriptional control, and replication timing (1). The process is characterized by the transfer of methyl groups to the C-5 position of cytosine and is catalyzed by members of the DNA methyltransferase (DNMT) 1 protein family. To date, five mammalian DNMTs have been identified as follows: DNMT1, DNMT3a, DNMT3b, DNMT2, and DNMT3L (1). Of these five, DNMT1, DNMT3a, and DNMT3b are known to be essential for proper development in murine knockout models (1-3). DNMT1, the most abundant DNA methyltransferase in somatic cells, has a strong preference for hemimethylated DNA and is therefore believed to be the enzyme primarily responsible for copying and maintaining methylation patterns from the parental to the daughter strand following DNA replication (1). Strikingly, significant genomic hypomethylation was only found at pericentromeric satellite 2 and 3 sequences in human cancer cells lacking DNMT1 (KO1 cells) (4). This suggests the possibility of regional specificity on the part of DNMT1 and, furthermore, that DNMT1 loss might be partially compensated by other DNMTs (4).Accumulated evidence suggests that DNA methylation status and changes in the biochemical modification of histone tails, the "histone code," can regulate the higher order organization and function of large intranuclear regions, implicating these modifications in chromosome positioning and the maintenance of specific chromatin domains within the nucleus in a wide variety of organisms (5-10). Recent data from plant cells indicate that loss of methylated CpG dinucleotides alters the methylation pattern of histone H3 (11), suggesting that DNA methylation may also function as a central signal for the regulation of chromatin structure. A reciprocal dependence may exist, because mouse embryonic stem cells lacking Suv39h histone methyltransferase...

The nucleolus is the site of ribosome synthesis in the nucleus, whose integrity is essential. Epigenetic mechanisms are thought to regulate the activity of the ribosomal RNA (rRNA) gene copies, which are part of the nucleolus. Here we show that human cells lacking DNA methyltransferase 1 (Dnmt1), but not Dnmt33b, have a loss of DNA methylation and an increase in the acetylation level of lysine 16 histone H4 at the rRNA genes. Interestingly, we observed that SirT1, a NAD+-dependent histone deacetylase with a preference for lysine 16 H4, interacts with Dnmt1; and SirT1 recruitment to the rRNA genes is abrogated in Dnmt1 knockout cells. The DNA methylation and chromatin changes at ribosomal DNA observed are associated with a structurally disorganized nucleolus, which is fragmented into small nuclear masses. Prominent nucleolar proteins, such as Fibrillarin and Ki-67, and the rRNA genes are scattered throughout the nucleus in Dnmt1 deficient cells. These findings suggest a role for Dnmt1 as an epigenetic caretaker for the maintenance of nucleolar structure.

The AT-hook has been defined as a DNA binding peptide motif that contains a glycine-arginine-proline (G-R-P) tripeptide core flanked by basic amino acids. Recent reports documented variations in the sequence of AT-hooks and revealed RNA binding activity of some canonical AT-hooks, suggesting a higher structural and functional variability of this protein domain than previously anticipated. Here we describe the discovery and characterization of the extended AT-hook peptide motif (eAT-hook), in which basic amino acids appear symmetrical mainly at a distance of 12–15 amino acids from the G-R-P core. We identified 80 human and 60 mouse eAT-hook proteins and biochemically characterized the eAT-hooks of Tip5/BAZ2A, PTOV1 and GPBP1. Microscale thermophoresis and electrophoretic mobility shift assays reveal the nucleic acid binding features of this peptide motif, and show that eAT-hooks bind RNA with one order of magnitude higher affinity than DNA. In addition, cellular localization studies suggest a role for the N-terminal eAT-hook of PTOV1 in nucleocytoplasmic shuttling. In summary, our findings classify the eAT-hook as a novel nucleic acid binding motif, which potentially mediates various RNA-dependent cellular processes.