Silencing of multiple cancer-related genes is associated with de novo methylation of linked CpG islands. Additionally, bivalent histone modification profiles characterized by the juxtaposition of active and inactive histone marks have been observed in genes that become hypermethylated in cancer. It is unknown how these ambiguous epigenetic states are maintained and how they interrelate with adjacent genomic regions with different epigenetic landscapes. Here, we present the analysis of a set of neighboring genes, including many frequently silenced in colon cancer cells, in a chromosomal region at 5q35.2 spanning 1.25 Mb. Promoter DNA methylation occurs only at genes maintained at a low transcriptional state and is characterized by the presence of bivalent histone marks, namely trimethylation of lysines 4 and 27 in histone 3. Chemically induced hyperacetylation and DNA demethylation lead to up-regulation of silenced genes in this locus yet do not resolve bivalent domains into a domain-wide active chromatin conformation. In contrast, active genes in the region become down-regulated after drug treatment, accompanied by a partial loss of chromatin domain boundaries and spreading of the inactive histone mark trimethylated lysine 27 in histone 3. Our results demonstrate that bivalent domains mark the promoters of genes that will become DNA methylated in adult tumor cells to enforce transcriptional silence. These bivalent domains not only remain upon drug induced gene reactivation, but also spread over adjacent CpG islands. These results may have important implications in understanding and managing epigenetic therapies of cancer.colorectal cancer ͉ DNA methylation ͉ epigenetic memory ͉ transcriptional silencing I t is now clear that epigenetic events, in cooperation with genetic events, are involved in every step of tumorigenesis and play a critical role in the disruption of key cellular pathways deregulated in human cancers (1, 2).De novo methylation of CpG islands is associated with the transcriptional silencing of many cancer-related genes (1, 2). The promoter regions of silenced genes, including those with promoter DNA methylation, contain specific histone modifications, which are a signature of transcriptional inactivation (3). Additionally, the DNA methylation mark itself can be read by specific proteins that can alter chromatin structure (4). Thus, a cross-talk exists between DNA methylation and histone modifications to orchestrate transcriptional silencing.A growing body of evidence suggests that the two main cell memory systems implicated in the maintenance of a stem cell state, Trithorax (Trx) and Polycomb Group (PcG) proteins, may be involved in tumor-associated aberrant gene silencing and promoter DNA methylation (5, 6). In this context, the active mark, methylated lysine 4, together with the silent mark, methylated lysine 27, have been found to coexist over the promoter regions of DNA methylated genes in human cancer cells (7). This epigenetic landscape is similar to the bivalent domains characterized by th...

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family. Demethylation of Alu elements occurs in aging and cancer processes and has been associated with gene reactivation and genomic instability. By targeting the unmethylated SmaI site within the Alu sequence as a surrogate marker, we have quantified and identified unmethylated Alu elements on the genomic scale. Normal colon epithelial cells contain in average 25 486 ± 10 157 unmethylated Alu's per haploid genome, while in tumor cells this figure is 41 995 ± 17 187 (P = 0.004). There is an inverse relationship in Alu families with respect to their age and methylation status: the youngest elements exhibit the highest prevalence of the SmaI site (AluY: 42%; AluS: 18%, AluJ: 5%) but the lower rates of unmethylation (AluY: 1.65%; AluS: 3.1%, AluJ: 12%). Data are consistent with a stronger silencing pressure on the youngest repetitive elements, which are closer to genes. Further insights into the functional implications of atypical unmethylation states in Alu elements will surely contribute to decipher genomic organization and gene regulation in complex organisms.

BackgroundMisregulation of the PTGS (prostaglandin endoperoxide synthase, also known as cyclooxygenase or COX) pathway may lead to the accumulation of pro-inflammatory signals, which constitutes a hallmark of cancer. To get insight into the role of this signaling pathway in colorectal cancer (CRC), we have characterized the transcriptional and epigenetic landscapes of the PTGS pathway genes in normal and cancer cells.ResultsData from four independent series of CRC patients (502 tumors including adenomas and carcinomas and 222 adjacent normal tissues) and two series of colon mucosae from 69 healthy donors have been included in the study. Gene expression was analyzed by real-time PCR and Affymetrix U219 arrays. DNA methylation was analyzed by bisulfite sequencing, dissociation curves, and HumanMethylation450K arrays. Most CRC patients show selective transcriptional deregulation of the enzymes involved in the synthesis of prostanoids and their receptors in both tumor and its adjacent mucosa. DNA methylation alterations exclusively affect the tumor tissue (both adenomas and carcinomas), redirecting the transcriptional deregulation to activation of prostaglandin E2 (PGE2) function and blockade of other biologically active prostaglandins. In particular, PTGIS, PTGER3, PTGFR, and AKR1B1 were hypermethylated in more than 40 % of all analyzed tumors.ConclusionsThe transcriptional and epigenetic profiling of the PTGS pathway provides important clues on the biology of the tumor and its microenvironment. This analysis renders candidate markers with potential clinical applicability in risk assessment and early diagnosis and for the design of new therapeutic strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0110-4) contains supplementary material, which is available to authorized users.

Gene amplification is one of the most frequent manifestations of genomic instability in human tumors and plays an important role in tumor progression and acquisition of drug resistance. To better understand the factors involved in acquired resistance to cytotoxic drugs via gene amplification, we have analyzed the structure and dynamics of dihydrofolate reductase (DHFR) gene amplification in HT29 cells treated with methotrexate (MTX). Analysis of the DHFR gene amplification process shows that the amplicon exhibits a complex structure that is consistently reproduced in independent treatments. The cytogenetic manifestation of the amplification in advanced stages of the treatment may be in the form of double minutes or as a homogeneously stained region. To get insights into the mechanisms of resistance, we have also investigated the sensitization to MTX of MTX-resistant cells after drug withdrawal and reexposure to MTX. Passive loss of the DHFR amplicon by withdrawal of the drug results in MTXsensitive cells exhibiting a substantial reduction of their capacity or even an incapacity to generate resistance when submitted to a second cycle of MTX treatment. On a second round of drug administration, the resistant cells generate a different amplicon structure, suggesting that the formation of the amplicon as in the first cycle of treatment is not feasible. These results indicate that DHFR gene amplification is a ''wear and tear'' process in HT29 cells and that MTX-resistant cells may become responsive to a second round of treatment if left untreated during a sufficient period of time. [Mol Cancer Ther 2009; 8(2):424 -32]

Inactivation of specific tumor suppressor genes by transcriptional silencing associated with hypermethylation of the promoter is a common event in cancer. We have applied the amplification of intermethylated sites (AIMS) technique to a 100 human colorectal cancers and seven cell lines to identify recurrent alterations that may unveil silenced tumor suppressor genes. Bisulfite sequencing was used to confirm differential DNA methylation results. Gene expression analysis was performed by real-time RT-PCR. An AIMS band recurrently displayed in tumors but not in normal tissues was isolated and identified as part of the CpG island of the prostacyclin synthase (PTGIS) gene promoter. PTGIS promoter was hypermethylated in 43 out of 100 colorectal cancers and in all cell lines. Bisulfite sequencing and clonal analysis confirmed the results obtained by AIMS and demonstrated biallelic hypermethylation of PTGIS promoter. Hypermethylation of the PTGIS promoter was associated with diminished gene expression, that was restored after treatment with demethylating and histone deacetylases inhibitor agents. PTGIS hypermethylation was associated with aneuploidy and p53 mutations. In the adjusted model, PTGIS methylation, but not p53 mutation, maintained the association with aneuploidy. We conclude that epigenetic inactivation of the PTGIS gene is a recurrent alteration in colorectal carcinogenesis. Oncogene(2005) 24, 7320-7326.

BackgroundSkeletal muscle stem cells enable the formation, growth, maintenance, and regeneration of skeletal muscle throughout life. The regeneration process is compromised in several pathological conditions, and muscle progenitors derived from pluripotent stem cells have been suggested as a potential therapeutic source for tissue replacement. DNA methylation is an important epigenetic mechanism in the setting and maintenance of cellular identity, but its role in stem cell determination towards the myogenic lineage is unknown. Here we addressed the DNA methylation dynamics of the major genes orchestrating the myogenic determination and differentiation programs in embryonic stem (ES) cells, their Pax7-induced myogenic derivatives, and muscle stem cells in proliferating and differentiating conditions.ResultsOur data showed a common muscle-specific DNA demethylation signature required to acquire and maintain the muscle-cell identity. This specific-DNA demethylation is Pax7-mediated, and it is a prime event in muscle stem cells gene activation. Notably, downregulation of the demethylation-related enzyme Apobec2 in ES-derived myogenic precursors reduced myogenin-associated DNA demethylation and dramatically impaired the expression of differentiation markers and, ultimately, muscle differentiation.ConclusionsOur results underscore DNA demethylation as a key mechanism driving myogenesis and identify specific Pax7-mediated DNA demethylation signatures to acquire and maintain the muscle-cell identity. Additionally, we provide a panel of epigenetic markers for the efficient and safe generation of ES- and induced pluripotent stem cell (iPS)-derived myogenic progenitors for therapeutic applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0250-9) contains supplementary material, which is available to authorized users.

Skeletal muscle is the largest tissue in mammalian organisms and is a key determinant of basal metabolic rate and whole‐body energy metabolism. Histone deacetylase 11 (HDAC11) is the only member of the class IV subfamily of HDACs, and it is highly expressed in skeletal muscle, but its role in skeletal muscle physiology has never been investigated. Here, we describe for the first time the consequences of HDAC11 genetic deficiency in skeletal muscle, which results in the improvement of muscle function enhancing fatigue resistance and muscle strength. Loss of HDAC11 had no obvious impact on skeletal muscle structure but increased the number of oxidative myofibers by promoting a glycolytic‐to‐oxidative muscle fiber switch. Unexpectedly, HDAC11 was localized in muscle mitochondria and its deficiency enhanced mitochondrial content. In particular, we showed that HDAC11 depletion increased mitochondrial fatty acid β‐oxidation through activating the AMP‐activated protein kinase‐acetyl‐CoA carboxylase pathway and reducing acylcarnitine levels in vivo, thus providing a mechanistic explanation for the improved muscle strength and fatigue resistance. Overall, our data reveal a unique role of HDAC11 in the maintenance of muscle fiber‐type balance and the mitochondrial lipid oxidation. These findings shed light on the mechanisms governing muscle metabolism and may have implications for chronic muscle metabolic disease management.

Upon acute muscle damage, HDAC11 is involved in regeneration processes, both satellite and inflammatory cell‐dependent. HDAC11‐deficient myoblasts proliferate longer and present a delayed cell cycle exit, with sustained expression of proliferation genes at early differentiation. Differentiating HDAC11−/− myocytes express higher levels of myogenin, and regenerating myofibers show an increased size. Recruited macrophages secrete increased M1 pro‐inflammatory and M2 anti‐inflammatory cytokines that further enhance HDAC11 KO muscle regeneration capacity.