DNA hypermethylation of alternatively spliced and repeat sequences in humans.

DNA methylation is presently accepted as a tentative regulatory parameter in splicing. Recently, we reported significant methylation differences among various exonic splicing-enhancing elements and alternative splicing events, based on CpG methylation data from the Human Epigenome Project for chromosomes 6, 20 and 22. Presently, using a different computational approach and the same database, we report: (a) significant increase of hypermethylation in intronic and exonic sequences close to acceptor sites, relative to overall introns and exons, respectively (1,973 CpGs examined); (b) frequent CpGs, mostly hypomethylated, in donors and infrequent CpGs mostly hypermethylated, in acceptors; and (c) hypermethylation in cassette exons which are occasionally spliced and have weaker average splicing potential, relative to constitutive exons (p < 0.0001). CpGs are hypomethylated in non-coding exons (only 16 % hypermethylation). Single-exon genes, similarly to first exons, frequently contain hypomethylated CpGs, while in internal and last exons CpGs are more frequently hypermethylated. Methylation is also more frequent in strange introns and splice sites processed by the minor spliceosome, e.g., ATAC, (p < 0.0001 in all cases), but not in sites of incomplete processing, e.g., retained introns or bleeding exons, (p = 0.706 and p = 0.313, respectively). Most Alus, which are known to contribute to transcript presentation, are heavily methylated, in contrast with other Alus, e.g., AluJo and mammalian interspersed repetitive elements which have been previously associated with alternative expression. These results elucidate the role of intragenic methylation in association with alternative splicing and facilitate the evaluation of genomic variations/polymorphisms and the development of tools for the prediction of alternative splicing events.

Epigenetic mechanisms in metal toxicity.

The true understanding of epigenetics evolved over time as our knowledge on DNA methylation and chromatin modifications and their effects on gene expression increased. The current flurry of research on epigenetics and the increasing documentation of the effects of various environmental factors on DNA methylation, chromatin modification, as well as on the expression of small non-coding RNAs (ncRNAs) have expanded the scope of research on the etiology of various diseases including cancer. The current review briefly discusses various molecular mechanisms of epigenetic regulation of gene expression, and expands the discussion with examples of heavy metal-induced alterations of gene expression and the associated epigenetic changes.

In silico structural analysis of sequences containing 5-hydroxymethylcytosine reveals its potential as binding regulator for development, ageing and cancer-related transcription factors.

The presence of 5-hydroxymethyl cytosine in DNA has been previously associated with ageing. Using in silico analysis of normal liver samples we presently observed that in 5-hydroxymethyl cytosine sequences, DNA methylation is dependent on the co-presence of G-quadruplexes and palindromes. This association exhibits discrete patterns depending on G-quadruplex and palindrome densities. DNase-Seq data show that 5-hydroxymethyl cytosine sequences are common among liver nucleosomes (p < 2.2x10-16) and threefold more frequent than nucleosome sequences. Nucleosomes lacking palindromes and potential G-quadruplexes are rare in vivo (1%) and nucleosome occupancy potential decreases with increasing G-quadruplexes. Palindrome distribution is similar to that previously reported in nucleosomes. In low and mixed complexity sequences 5-hydroxymethyl cytosine is frequently located next to three elements: G-quadruplexes or imperfect G-quadruplexes with CpGs, or unstable hairpin loops (TCCCAY6TGGGA) mostly located in antisense strands or finally A-/T-rich segments near these motifs. The high frequencies and selective distribution of pentamer sequences (including TCCCA, TGGGA) probably indicate the positive contribution of 5-hydroxymethyl cytosine to stabilize the formation of structures unstable in the absence of this cytosine modification. Common motifs identified in all total 5-hydroxymethyl cytosine-containing sequences exhibit high homology to recognition sites of several transcription factor families: homeobox, factors involved in growth, mortality/ageing, cancer, neuronal function, vision, and reproduction. We conclude that cytosine hydroxymethylation could play a role in the recognition of sequences with G-quadruplexes/palindromes by forming epigenetically regulated DNA 'springs' and governing expansions or compressions recognized by different transcription factors or stabilizing nucleosomes. The balance of these epigenetic elements is lost in hepatocellular carcinoma.

SpliceIT: a hybrid method for splice signal identification based on probabilistic and biological inference.

Splice sites define the boundaries of exonic regions and dictate protein synthesis and function. The splicing mechanism involves complex interactions among positional and compositional features of different lengths. Computational modeling of the underlying constructive information is especially challenging, in order to decipher splicing-inducing elements and alternative splicing factors. SpliceIT (Splice Identification Technique) introduces a hybrid method for splice site prediction that couples probabilistic modeling with discriminative computational or experimental features inferred from published studies in two subsequent classification steps. The first step is undertaken by a Gaussian support vector machine (SVM) trained on the probabilistic profile that is extracted using two alternative position-dependent feature selection methods. In the second step, the extracted predictions are combined with known species-specific regulatory elements, in order to induce a tree-based modeling. The performance evaluation on human and Arabidopsis thaliana splice site datasets shows that SpliceIT is highly accurate compared to current state-of-the-art predictors in terms of the maximum sensitivity, specificity tradeoff without compromising space complexity and in a time-effective way. The source code and supplementary material are available at: http://www.med.auth.gr/research/spliceit/.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection: Triggering a Lethal Fight to Keep Control of the Ten-Eleven Translocase (TET)-Associated DNA Demethylation?

The extended and diverse interference of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in multiple host functions and the diverse associated symptoms implicate its involvement in fundamental cellular regulatory processes. The activity of ten-eleven translocase 2 (TET2) responsible for selective DNA demethylation, has been recently identified as a regulator of endogenous virus inactivation and viral invasion, possibly by proteasomal deregulation of the TET2/TET3 activities. In a recent report, we presented a detailed list of factors that can be affected by TET activity, including recognition of zinc finger protein binding sites and bimodal promoters, by enhancing the flexibility of adjacent sequences. In this review, we summarize the TET-associated processes and factors that could account for SARS-CoV-2 diverse symptoms. Moreover, we provide a correlation for the observed virus-induced symptoms that have been previously associated with TET activities by in vitro and in vitro studies. These include early hypoxia, neuronal regulation, smell and taste development, liver, intestinal, and cardiomyocyte differentiation. Finally, we propose that the high mortality of SARS-CoV-2 among adult patients, the different clinical symptoms of adults compared to children, the higher risk of patients with metabolic deregulation, and the low mortality rates among women can all be accounted for by the complex balance of the three enzymes with TET activity, which is developmentally regulated. This activity is age-dependent, related to telomere homeostasis and integrity, and associated with X chromosome inactivation via (de)regulation of the responsible XIST gene expression.

Li-Fraumeni and Li-Fraumeni-like syndrome mutations in p53 are associated with exonic methylation and splicing regulatory elements.

Mutations in codon 133 of p53, which cause the loss of the Delta 133 isoform(s) expression, are very frequent in the Li-Fraumeni (LF) and Li-Fraumeni-like (LFL) syndromes. In sporadic cancers, silent p53 mutations are correlated with exonic splicing enhancers (ESEs) and exonic methylated sites. The present study shows that mutations in splice sites are also very frequent in LF/LFL syndromes, while missense mutations are less common compared to other familial or sporadic cancers (P = 0 in both cases). Furthermore, it is shown that the codons at which LF/LFL germline missense mutations occur, correlate with CpG-containing ESEs (r = 0.181, P = 0.014) which are all methylated in p53. While both silent and LF/LFL missense mutations correlate with SC35 motifs, only the latter are associated with SRp55. On the contrary, only silent mutations in sporadic cancers correlate with SF2/ASF motifs in p53. Moreover, 12.1% of LF/LFL missense mutations involve the formation of potential splice sites of considerable splicing scores. Finally, mutations that are not at, or adjacent to CpGs (+/-1 codon, 34% of all LF/LFL mutated sites), introduce considerable changes of the ESE scores (>1.3 score change). The above data verify that LF/LFL missense mutations probably result also in splicing deregulation, in addition to any changes of the protein function and are mostly associated with alterations of the exonic methylation landscape. Some of the ESEs affected in LF/LFL syndromes are also genetically unstable in sporadic cancers but non-CpG cytosine instability, which is predominantly associated with specific ESEs, is only common in sporadic cancers.

MeinteR: A framework to prioritize DNA methylation aberrations based on conformational and cis-regulatory element enrichment.

DNA methylation studies have been reformed with the advent of single-base resolution arrays and bisulfite sequencing methods, enabling deeper investigation of methylation-mediated mechanisms. In addition to these advancements, numerous bioinformatics tools address important computational challenges, covering DNA methylation calling up to multi-modal interpretative analyses. However, contrary to the analytical frameworks that detect driver mutational signatures, the identification of putatively actionable epigenetic events remains an unmet need. The present work describes a novel computational framework, called MeinteR, that prioritizes critical DNA methylation events based on the following hypothesis: critical aberrations of DNA methylation more likely occur on a genomic substrate that is enriched in cis-acting regulatory elements with distinct structural characteristics, rather than in genomic "deserts". In this context, the framework incorporates functional cis-elements, e.g. transcription factor binding sites, tentative splice sites, as well as conformational features, such as G-quadruplexes and palindromes, to identify critical epigenetic aberrations with potential implications on transcriptional regulation. The evaluation on multiple, public cancer datasets revealed significant associations between the highest-ranking loci with gene expression and known driver genes, enabling for the first time the computational identification of high impact epigenetic changes based on high-throughput DNA methylation data.

OPG/RANK/RANKL signaling axis in patients with type I diabetes: Associations with parathormone and vitamin D.

BACKGROUND: Type 1 diabetes (T1D) has been associated with a higher fracture risk due to alterations in bone structure and metabolism. On the other hand, the important role of the RANKL/OPG/RANK signaling axis in bone physiology is well established. The aim of this study was to evaluate the levels of receptor activator of nuclear factor kappa-B ligand (RANKL), receptor activator of nuclear factor kappa-B (RANK) and plasma osteoprotegerin (OPG) levels, in T1D youngsters and to investigate factors that could influence the OPG/RANK/RANKL signaling axis such as 25-hydroxy vitamin D [25(OH) D], parathormone (PTH) and age. METHODS: Serum RANKL, RANK, 25(OH) D, PTH levels and plasma OPG levels, were measured in 71 youngsters with T1D and 50 healthy controls matched for age and gender. RESULTS: Plasma OPG levels were significantly lower (p = 0.025) in T1D patients compared to controls. Serum RANKL levels were significantly higher (p = 0.037), while no differences were observed in serum RANK levels (p = 0.946) between the two groups. Serum 25(OH) D levels found significantly decreased (p < 0.001) while serum PTH levels were significantly elevated (p < 0.001) in T1D patients than in controls. CONCLUSIONS: Our results demonstrated that OPG and RANKL may be promising biomarkers for T1D patients. However, their circulating levels were associated with several factors including PTH, 25(OH) D and therefore, may represent an integrative biomarker for a variety of endocrine signaling disturbances observed in T1D.

Age-dependent methylation in epigenetic clock CpGs is associated with G-quadruplex, co-transcriptionally formed RNA structures and tentative splice sites.

Horvath's epigenetic clock consists of 353 CpGs whose methylation levels can accurately predict the age of individuals. Using bioinformatics analysis, we investigated the conformation, energy characteristics and presence of tentative splice sites of the sequences surrounding the epigenetic clock CpGs, in relation to the median methylation changes in different ages, the presence of CpG islands and their position in genes. Common characteristics in the 100 nt sequences surrounding the epigenetic clock CpGs are G-quadruplexes and/or tentative splice site motifs. Median methylation increases significantly in sequences which adopt less stable structures during transcription. Methylation is higher when CpGs overlap with G-quadruplexes than when they precede them. Median methylation in epigenetic clock CpGs is higher in sequences expressed as single products rather than in multiple products and those containing single donors and multiple acceptors. Age-related methylation variation is significant in sequences without G-quadruplexes, particularly those producing low stability nascent RNA and those with splice sites. CpGs in sequences close to transcription start sites and those which are possibly never expressed (hypothetical proteins) undergo similar extent of age-related median methylation decrease and increase. Preservation of methylation is observed in CpG islands without G-quadruplexes, contrary to CpGs far from CpG islands (open sea). Sequences containing G-quadruplexes and RNA pseudoknots, determining the recognition by H3K27 histone methyltransferase, are hypomethylated. The presented structural DNA and co-transcriptional RNA analysis of epigenetic clock sequences, foreshadows the association of age-related methylation changes with the principle biological processes of DNA and histone methylation, splicing and chromatin silencing.

Aberrant p16 promoter methylation among Greek lung cancer patients and smokers: correlation with smoking.

Genetic and environmental factors (dietary and smoking) influence lung cancer epidemiology and induce epigenetic modifications that should be assessed in individual populations. We analyzed p16 methylation among Greek non-small cell lung carcinoma patients and smokers using two-stage methylation-specific polymerase chain reaction. One hundred and fifty specimens from cancerous and adjacent non-cancerous tissue, bronchial washings and sputum from patients and 48 specimens, mostly sputum, from disease-free smokers were included. p16 methylation was very frequent in biopsies (82.85%) and bronchial washings (non-small cell lung carcinoma, 80.35%; small cell lung carcinoma, 16.66%) from patients, but also in adjacent non-cancerous tissue (45.71%). Concordance of p16 methylation and positivity by cytological examination was 51.78%. Methylation was also observed in sputum from asymptomatic cytology-negative smokers (22.5%) and chronic obstructive pulmonary disease patients (three of eight). Among disease-free individuals, methylation correlated only with heavy smoking (>50 pack-years, P<0.001) and differed among male and female disease-free smokers. In summary, p16 methylation is very frequent among non-small cell lung carcinoma patients, and correlates with heavy cigarette consumption only in disease-free smokers.

Increased Δ133p53 mRNA in lung carcinoma corresponds with reduction of p21 expression.

Modification of p53 expression levels and its principle apoptosis and cell cycle regulatory partners, mouse double minute 2 homolog (MDM­2) and p21, has been previously reported in various types of cancer. In the current study, the expression of Δ133p53 isoforms was investigated in lung carcinomas with respect to the expression of the aforementioned genes. The expression of p53 full­length transcript and Δ133p53 isoforms α, ß and γ transcripts, MDM­2 and p21 transcripts were determined by reverse transcription­quantitative polymerase chain reaction, in total RNA isolated from 17 lung carcinoma specimens and 17 corresponding adjacent non­cancerous tissues. RNA expression analysis was performed according to the Pfaffl equation and Rest tool using ß­actin as a reference gene. Detection of the above proteins was additionally performed by western blotting. Significant overexpression of the Δ133p53 mRNAs was observed in cancerous as compared with adjacent non­cancerous tissues (3.94­fold), whereas full­length p53 and MDM­2 expression exhibited a smaller, however significant, increase. The expression of the p21 transcript was significantly reduced in cancerous specimens. Δ133p53 and p21 expression levels varied in parallel, however were not significantly correlated. p53 full­length protein expression observed by western blot analysis strongly varied from the Δ133p53 isoforms, however MDM­2 protein isoforms were not detectable and p21 protein was more abundant in non­cancerous tissues. In conclusion, Δ133p53 mRNA levels is suggested as a potentially useful marker of malignancy in lung cancer. The absence of Δ133p53 protein in lung carcinomas, which overexpress Δ133p53 transcripts, may indicate the role of the latter in post­transcriptional regulation through RNA interference in the cell cycle and apoptosis.

Elevated plasma levels of miR-29a are associated with hemolysis in patients with hypertrophic cardiomyopathy.

BACKGROUND: miR-29a is a small non-coding RNA that is known to repress collagen synthesis. Interestingly, elevated plasma miR-29a was reported to correlate with pronounced myocardial fibrosis in patients with hypertrophic cardiomyopathy. The objective of this study was to elucidate the origin of plasma miR-29a, and evaluate its significance as a biomarker. METHODS: miR-29a expression was evaluated in plasma (n=50) and myocardial samples (n=4) from patients with hypertrophic cardiomyopathy using RT-qPCR. RESULTS: Although miR-29a was highly expressed in the myocardium, miR-29a plasma levels did not show any correlation with serum troponin I levels (rs=-0.12, p=0.43), and the heart does not release significant amounts of miR-29a into the circulation via exosome secretion. Conversely, miR-29a was present in red blood cells, and plasma levels correlated significantly with markers of hemolysis: lactic dehydrogenase (rs=0.36, p=0.01) and the absorbance of oxyhemoglobin at 414nm (rs=0.39, p=0.006). Furthermore, the association between serum haptoglobin and the maximal blood flow velocity in the left ventricle outflow tract (rs=-0.42, p=0.008) indicated that intravascular hemolysis is a manifestation of the disease. CONCLUSIONS: miR-29a is highly expressed in myocardial tissue from patients with hypertrophic cardiomyopathy. In contrast, plasma miR-29a is primarily of nonmyocardial origin and is correlated significantly with the extent of hemolysis observed in these patients.

Association of two synonymous splicing-associated CpG single nucleotide polymorphisms in calpain 10 and solute carrier family 2 member 2 with type 2 diabetes.

Coding synonymous single nucleotide polymorphisms (SNPs) have attracted little attention until recently. However, such SNPs located in epigenetic, CpG sites modifying exonic splicing enhancers (ESEs) can be informative with regards to the recently verified association of intragenic methylation and splicing. The present study describes the association of type 2 diabetes (T2D) with the exonic, synonymous, epigenetic SNPs, rs3749166 in calpain 10 (CAPN10) glucose transporter (GLUT4) translocator and rs5404 in solute carrier family 2, member 2 (SLC2A2), also termed GLUT2, which, according to prior bioinformatic analysis, strongly modify the splicing potential of glucose transport-associated genes. Previous association studies reveal that only rs5404 exhibits a strong negative T2D association, while data on the CAPN10 polymorphism are contradictory. In the present study DNA from blood samples of 99 Greek non-diabetic control subjects and 71 T2D patients was analyzed. In addition, relevant publicly available cases (40) resulting from examination of 110 Personal Genome Project data files were analyzed. The frequency of the rs3749166 A allele, was similar in the patients and non-diabetic control subjects. However, AG heterozygotes were more frequent among patients (73.24% for Greek patients and 54.55% for corresponding non-diabetic control subjects; P=0.0262; total cases, 52.99 and 75.00%, respectively; P=0.0039). The rs5404 T allele was only observed in CT heterozygotes (Greek non-diabetic control subjects, 39.39% and Greek patients, 22.54%; P=0.0205; total cases, 34.69 and 21.28%, respectively; P=0.0258). Notably, only one genotype, heterozygous AG/CC, was T2D-associated (Greek non-diabetic control subjects, 29.29% and Greek patients, 56.33%; P=0.004; total cases, 32.84 and 56.58%, respectively; P=0.0008). Furthermore, AG/CC was strongly associated with very high (≥8.5%) glycosylated plasma hemoglobin levels among patients (P=0.0002 for all cases). These results reveal the complex heterozygotic SNP association with T2D, and indicate possible synergies of these epigenetic, splicing-regulatory, synonymous SNPs, which modify the splicing potential of two alternative glucose transport-associated genes.

Methylation and repeats in silent and nonsense mutations of p53.

All exonic CG sequences in p53 are methylated; this epigenetic modification is correlated with frequent G:C-->A:T transitions in p53. Recent reports reveal the presence in p53 of non-CG methylation in CC and CCC sequences, complementary to sites of selective guanosine adduct formation (GG and GGG), and the association of genetic instability with methylation at repetitive sequences. We presently investigated the distribution of methylation sites and repetitive elements in silent and nonsense p53 mutations (2051) among the IARC's TP53 somatic mutation database for exons 5-8. Silent mutations are nonrandom, but mostly involve G:C-->A:T transitions (62%); in particular C-->T mutations (39% of all silent mutations) are mostly correlated with CC and CCC sequences, while G-->A mutations with GG sequences. Sequence analysis of all non-G:C-->A:T silent mutations reveals the frequent formation of new methylation sites (CG), new CCC and GGG sequences in the resulting sequence, refinement of symmetry elements at interrupted microsatellite-like sequences and formation of small repeats (55.3%). The G:C-->A:T silent mutations characterize cancers associated with cigarette smoking (e.g. bladder or lung and bronchus cancer versus colorectal cancer); on the contrary, non-G:C-->A:T silent mutations have similar frequencies in most cancers. Nonsense mutations in exons 5-8, all resulting in mutants lacking amino acids 307-393, which are crucial for p53 activity, were also analyzed. The frequency of nonsense mutations is higher at methylated sites or repeats 1-2 nucleotides removed from methylation sites. Frameshift mutations are also more frequent at repeated sequences. The frequent G:C-->A:T silent mutations could indicate that CC and CCC sequences of exons 5-8 are occasionally targets of non-CpG methylation of cytosine. This process of de novo methylation in the presence of microsatellite-like sequences and small repeats might influence the genetic stability of a variety of genes.

Salivary mRNA markers having the potential to detect oral squamous cell carcinoma segregated from oral leukoplakia with dysplasia.

BACKGROUND: In the current study the presence of extracellular IL-1B, IL-8, OAZ and SAT mRNAs in the saliva was evaluated as a tool in the early detection of oral squamous cell carcinoma. METHODS: 34 patients with primary oral squamous cell carcinoma stage T1N0M0/T2N0M0, 20 patients with oral leukoplakia and dysplasia (15 patients with mild dysplasia and 5 with severe dysplasia/in situ carcinoma) and 31 matched healthy-control subjects were included in the study. The presence of IL-1B, IL-8, OAZ and SAT mRNA was evaluated in extracellular RNA isolated from saliva samples using sequence-specific primers and real-time RT-PCR. ROC curve analysis was used to estimate the ability of the biomarkers to detect oral squamous cell carcinoma patients. RESULTS: The data reveal that the combination of these four biomarkers provides a good predictive probability of up to 80% (AUC=0.799, p=0.002) for patients with oral squamous cell carcinoma but not patients suffering from oral leukoplakia with dysplasia. Moreover, the combination of only the two biomarkers (SAT and IL-8) also raises a high predictive ability of 75.5% (AUC=0.755, p=0.007) approximately equal to the four biomarkers suggesting the use of the two biomarkers only in the prediction model for oral squamous cell carcinoma patients limiting the economic and health cost in half. CONCLUSION: SAT and IL-8 mRNAs are present in the saliva in high quality and quantity, with a good discriminatory ability for oral squamous cell carcinoma patients only but not for patients with oral leukoplakia and dysplasia an oral potentially malignant disorder.

Non-CpG cytosine methylation of p53 exon 5 in non-small cell lung carcinoma.

Non-CpG methylation of cytosine residues, a mechanism associated with regulation of gene expression, has not been investigated in human cancer until now. Analysis of the p53 exon 5 mutation spectrum in mutation databases for lung cancer reveals frequent GC>AT transitions, several of which occur at non-CpG sequences. To investigate the involvement of cytosine methylation in this mutagenesis process, we analyzed the methylation profile of p53 exon 5, in lung carcinoma. In this report, we present evidence that extensive clustered non-CpG methylation is observed in three regions of this exon, namely the sequences spanning codons 156-159, 175-179 and the 3' splice site, as well as in scattered CpA sequences. This methylation pattern was verified using direct methylation sequencing, and a two-stage methylation-specific PCR assay (MSP), designed for the detection of methylation in a GC rich region (oligo C sequence, of codons 175-179) of exon 5. The results from this MSP assay reveal that DNA from cancerous specimens was more heavily methylated in non-CpG cytosines, compared to that from non-cancerous lung tissue of cancer patients (14/19 cancerous and 6/19 non-cancerous, respectively). DNA isolated from human leucocytes and some non-cancerous specimens (2/19) was free of non-CpG methylation. Careful analysis of the mutations reported in p53 mutation databases also provides corroborating evidence that the high incidence of GC>AT mutations in the p53 gene, observed in lung cancer, might also be related to non-CpG methylation, as well as to the overall increase of methylation sites in this locus.

Epigenetically modified nucleotides in chronic heroin and cocaine treated mice.

Epigenetic changes include the addition of a methyl group to the 5' carbon of the cytosine ring, known as DNA methylation, which results in the generation of the fifth DNA base, namely 5-methylcytosine. During active or passive demethylation, an intermediate modified base is formed, 5-hydroxymethylcytosine. We have currently quantified 5-methylcytosine and 5-hydroxymethylcytosine in the liver and brain of mice treated with cocaine or heroin, using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Our results show that global 5-methylcytosine levels are not affected by heroin or cocaine administration, neither in the liver nor in the brain. However, 5-hydroxymethylcytosine levels are reduced in the liver following cocaine administration, while they are not affected by cocaine in the brain or by heroin administration in the liver and the brain. Elucidation of the epigenetic phenomena that takes place with respect to drug abuse and addiction, via quantitative analysis of different modified bases, may enable a better understanding of the underlying mechanisms and may lead to more personalized and effective treatment options.

GREMET: an integrative tool for the prediction of mutation effects on gene regulation.

The identification of thousands of mutations yearly has put new challenges to researchers who are interested in fast and effective annotation as well as the prediction of potential implications to the gene regulation mechanisms. This work presents an integrative tool, called GREMET, for the prediction of alterations in gene splicing regulation inferred by mutations of the human genome. GREMET supports the characterization of mutations either single-point or indels with respect to their effect on the splicing potential of the neighboring sequences and the binding strength of auxiliary cis-acting splicing enhancers. In addition, GREMET identifies possible consequences of mutations on the DNA methylation through the disruption or creation of CpG sequences. Besides locus-specific mutations, GREMET performs the analyses on newly identified mutations and provides an easy-to-use Web interface helping researchers to save time in routine mutation analyses. GREMET is freely accessible at: http://kedip.med.auth.gr/biotools/gremet/.

Effect of chronic heroin and cocaine administration on global DNA methylation in brain and liver.

Drug abuse is associated with epigenetic changes, such as histone modifications and DNA methylation. The purpose of the present study was to examine the effect of chronic cocaine and heroin administration on global DNA methylation in brain and liver. Male, 8 week old, C57BL/6J mice received heroin in a chronic 'intermittent' escalating dose paradigm, or cocaine in a chronic escalating dose 'binge' paradigm, which mimic the human pattern of opioid or cocaine abuse respectively. Following sacrifice, livers and brains were removed and DNA was extracted from them. The extracted DNA was hydrolyzed and 2'-deoxycytidine and 5-methyl-2'-deoxycytidine were determined by HPLC-UV. The % 5-methyl-2'-deoxycytidine content of DNA was significantly higher in the brain compared to the liver. There were no differences between the control animals and the cocaine or heroin treated animals in neither of the tissues examined, which is surprising since cocaine administration induced gross morphological changes in the liver. Moreover, there was no difference in the % 5-methyl-2'-deoxycytidine content of DNA between the cocaine and the heroin treated animals. The global DNA methylation status in the brain and liver of mice chronically treated with cocaine or heroin remains unaffected, but this finding cannot exclude the existence of anatomical region or gene-specific methylation differences. This is the first time that global DNA methylation in the liver and whole brain has been studied following chronic cocaine or heroin treatment.

Recent advances in the bioanalysis of modified nucleotides in epigenetic studies.

Epigenetic alterations, such as DNA methylation, are involved in the pathogenesis of various diseases, the toxicity of diverse agents, the process of aging, the development of stem cells and numerous other mechanisms. DNA methylation is one of the most well-studied epigenetic alterations in mammals. Nevertheless, the scientific interest is now focusing on novel modified nucleotides with potential regulatory roles, such as 5-hydroxymethylcytosine. We currently present and discuss novel bioanalytical strategies developed for the determination of various modified nucleotides in epigenetic studies.