Epigenome Chaos: Stochastic and Deterministic DNA Methylation Events Drive Cancer Evolution.

Cancer evolution is associated with genomic instability and epigenetic alterations, which contribute to the inter and intra tumor heterogeneity, making genetic markers not accurate to monitor tumor evolution. Epigenetic changes, aberrant DNA methylation and modifications of chromatin proteins, determine the "epigenome chaos", which means that the changes of epigenetic traits are randomly generated, but strongly selected by deterministic events. Disordered changes of DNA methylation profiles are the hallmarks of all cancer types, but it is not clear if aberrant methylation is the cause or the consequence of cancer evolution. Critical points to address are the profound epigenetic intra- and inter-tumor heterogeneity and the nature of the heterogeneity of the methylation patterns in each single cell in the tumor population. To analyze the methylation heterogeneity of tumors, new technological and informatic tools have been developed. This review discusses the state of the art of DNA methylation analysis and new approaches to reduce or solve the complexity of methylated alleles in DNA or cell populations.

Methylation of the Suppressor Gene p16INK4a: Mechanism and Consequences.

Tumor suppressor genes in the CDKN2A/B locus (p15INK4b, p16INK4a, and p14ARF) function as biological barriers to transformation and are the most frequently silenced or deleted genes in human cancers. This gene silencing frequently occurs due to DNA methylation of the promoter regions, although the underlying mechanism is currently unknown. We present evidence that methylation of p16INK4a promoter is associated with DNA damage caused by interference between transcription and replication processes. Inhibition of replication or transcription significantly reduces the DNA damage and CpGs methylation of the p16INK4a promoter. We conclude that de novo methylation of the promoter regions is dependent on local DNA damage. DNA methylation reduces the expression of p16INK4a and ultimately removes this barrier to oncogene-induced senescence.

RNA Stabilizes Transcription-Dependent Chromatin Loops Induced By Nuclear Hormones.

We show that transcription induced by nuclear receptors for estrogen (E2) or retinoic acid (RA) is associated with formation of chromatin loops that juxtapose the 5' end (containing the promoter) with the enhancer and the 3' polyA addition site of the target gene. We find three loop configurations which change as a function of time after induction: 1. RA or E2-induced loops which connect the 5' end, the enhancer and the 3' end of the gene, and are stabilized by RNA early after induction; 2. E2-independent loops whose stability does not require RNA; 3. Loops detected only by treatment of chromatin with RNAse H1 prior to hormonal induction. RNAse H1 digests RNA that occludes the relevant restriction sites, thus preventing detection of these loops. R-loops at the 5' and 3' ends of the RA or E2-target genes were demonstrated by immunoprecipitation with anti-DNA-RNA hybrid antibodies as well as by sensitivity to RNAse H1. The cohesin RAD21 subunit is preferentially recruited to the target sites upon RA or E2 induction of transcription. RAD21 binding to chromatin is eliminated by RNAse H1. We identified E2-induced and RNase H1-sensitive antisense RNAs located at the 5' and 3' ends of the E2-induced transcription unit which stabilize the loops and RAD21 binding to chromatin. This is the first report of chromatin loops that form after gene induction that are maintained by RNA:DNA hybrids.

Akap1 Regulates Vascular Function and Endothelial Cells Behavior.

MitoAKAPs (mitochondrial A kinase anchoring proteins), encoded by the Akap1 gene, regulate multiple cellular processes governing mitochondrial homeostasis and cell viability. Although mitochondrial alterations have been associated to endothelial dysfunction, the role of mitoAKAPs in the vasculature is currently unknown. To test this, postischemic neovascularization, vascular function, and arterial blood pressure were analyzed in Akap1 knockout mice (Akap1-/- ) and their wild-type (wt) littermates. Primary cultures of aortic endothelial cells (ECs) were also obtained from Akap1-/- and wt mice, and ECs migration, proliferation, survival, and capillary-like network formation were analyzed under different experimental conditions. After femoral artery ligation, Akap1-/- mice displayed impaired blood flow and functional recovery, reduced skeletal muscle capillary density, and Akt phosphorylation compared with wt mice. In Akap1-/- ECs, a significant enhancement of hypoxia-induced mitophagy, mitochondrial dysfunction, reactive oxygen species production, and apoptosis were observed. Consistently, capillary-like network formation, migration, proliferation, and AKT phosphorylation were reduced in Akap1-/- ECs. Alterations in Akap1-/- ECs behavior were also confirmed in Akap1-/- mice, which exhibited a selective reduction in acetylcholine-induced vasorelaxation in mesenteric arteries and a mild but significant increase in arterial blood pressure levels compared with wt. Finally, overexpression of a constitutively active Akt mutant restored vascular reactivity and ECs function in Akap1-/- conditions. These results demonstrate the important role of mitoAKAPs in the modulation of multiple ECs functions in vivo and in vitro, suggesting that mitochondria-dependent regulation of ECs might represent a novel therapeutic approach in cardiovascular diseases characterized by endothelial dysfunction.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair.

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.

DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism.

We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells.

The Management of Diabetes Mellitus in Patients with Chronic Kidney Disease: A Population-Based Study in Southern Italy.

BACKGROUND AND OBJECTIVES: Diabetes mellitus in patients with chronic kidney disease (CKD) is known as diabetic kidney disease (DKD). Pharmacological management of DKD is challenging due to reduced renal excretion of some antidiabetic drugs. The aim of this population-based study was to explore antidiabetic drug use in DKD patients from Southern Italy. METHODS: The Arianna database from Caserta Local Health Unit was used. Diabetic patients with incident CKD [first diagnosis date: index date (ID)] were identified by searching for specific ICD9-CM codes among hospital discharge diagnoses/procedures and/or indication of use associated with drug prescriptions. To evaluate any change in the use of antidiabetic drugs after the CKD diagnosis, the prevalence of antidiabetic drug use among DKD patients was calculated within 1 year prior to/after ID and after dialysis entry. A Kaplan-Meier analysis was used to assess the time to discontinuation of antidiabetic drugs after CKD diagnosis. The frequency of antidiabetic drugs contraindicated in renal disease in DKD patients was measured. RESULTS: Overall, 725 diabetic patients (mean age 72.8 ± 11.4 years) had incident CKD from 2006 to 2011. The use of combination antidiabetic drugs, biguanides and sulphonamides decreased by approximately 10, 7 and 5%, respectively, after the ID. The use of insulins increased by 10% after the ID and by 20% after entry into dialysis. The use of antidiabetic drugs not contraindicated in CKD decreased marginally after the diagnosis of CKD. CONCLUSION: In a general practice of Southern Italy the management of diabetes mellitus changed only marginally in newly diagnosed CKD patients, suggesting a therapeutic inertia on the part of prescribers.

Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene.

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15-20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression.