Search

Embryonic stem cells (ESCs) present a characteristic pluripotency heterogeneity correspondent to specific metastates. We recently demonstrated that retinoic acid (RA) induces an increase in a specific 2C-like metastate marked by target genes specific to the two-cell embryo stage in preimplantation. Prame (Preferentially expressed antigen in melanoma) is one of the principal actors of the pluripotency stage with a specific role in RA responsiveness. Additionally, PRAME is overexpressed in a variety of cancers, but its molecular functions are poorly understood. To further investigate Prame’s downstream targets, we used a chromatin immunoprecipitation sequencing (ChIP-seq) assay in RA-enriched 2C-like metastates and identified two specific target genes, Cdk8 and Cdkn2d, bound by Prame. These two targets, involved in cancer dedifferentiation and pluripotency, have been further validated in RA-resistant ESCs. Here, we observed for the first time that Prame controls the Cdk8 and Cdkn2d genes in ESCs after RA treatment, shedding light on the regulatory network behind the establishment of naïve pluripotency.

Embryonic stem cells (ESCs) present a characteristic pluripotency heterogeneity correspondent to specific metastates. We recently demonstrated that retinoic acid (RA) induces an increase in a specific 2C-like metastate marked by target genes specific to the two-cell embryo stage in preimplantation. Prame (Preferentially expressed antigen in melanoma) is one of the principal actors of the pluripotency stage with a specific role in RA responsiveness. Additionally, PRAME is overexpressed in a variety of cancers, but its molecular functions are poorly understood. To further investigate Prame's downstream targets, we used a chromatin immunoprecipitation sequencing (ChIP-seq) assay in RA-enriched 2C-like metastates and identified two specific target genes

Preeclampsia is a leading cause of perinatal maternal-foetal mortality and morbidity. This study aims to identify the key microRNAs (miRNA) in preeclampsia and uncover their potential functions. We downloaded the miRNA expression profile of GSE119799 for plasma and GSE177049 for the placenta. Each dataset consisted of five patients (PE) and five controls (N). From a technical point of view, we analysed the counts per million (CPM) for both datasets, highlighting 358 miRNAs in common, 78 unique for plasma and 298 unique for placenta. At the same time, we performed an expression differential analysis (|logFC| ≥ 1|and FDR ≤ 0.05) to evaluate the biological impact of the miRNAs. This approach allowed us to highlight 321 miRNAs in common between plasma and placenta, within which four were upregulated in plasma. Furthermore, the same analysis revealed five miRNAs expressed exclusively in plasma; these were also upregulated. In conclusion, the in-depth bioinformatics analysis conducted during our study will allow us, on the one hand, to verify the targets of each of the nine identified miRNAs; on the other hand, to use them both as new non-invasive biomarkers and as therapeutic targets for the development of personalised treatments.

YB-1 is a multifunctional protein overexpressed in many types of cancer. It is a crucial oncoprotein that regulates cancer cell progression and proliferation. Ubiquitously expressed in human cells, YB-1 protein functions are strictly dependent on its subcellular localization. In the cytoplasm, where YB-1 is primarily localized, it regulates mRNA translation and stability. However, in response to stress stimuli and activation of PI3K and RSK signaling, YB-1 moves to the nucleus acting as a prosurvival factor. YB-1 is reported to regulate many cellular signaling pathways in different types of malignancies. Furthermore, several observations also suggest that YB-1 is a sensor of oxidative stress and DNA damage. Here we show that YB-1 reduces PTEN intracellular levels thus leading to PI3K/Akt pathway activation. Remarkably, PTEN reduction mediated by YB-1 overexpression can be observed in human immortalized keratinocytes and HEK293T cells and cannot be reversed by proteasome inhibition. Real-time PCR data indicate that YB-1 silencing up-regulates the PTEN mRNA level. Collectively, these observations indicate that YB-1 negatively controls PTEN at the transcript level and its overexpression could confer survival and proliferative advantage to PTEN proficient cancer cells.

The identification of the molecular mechanisms controlling early cell fate decisions in mammals is of paramount importance as the ability to determine specific lineage differentiation represents a significant opportunity for new therapies. Pancreatic Progenitor Cells (PPCs) constitute a regenerative reserve essential for the maintenance and regeneration of the pancreas. Besides, PPCs represent an excellent model for understanding pathological pancreatic cellular remodeling. Given the lack of valid markers of early endoderm, the identification of new ones is of fundamental importance. Both products of the Ink4a/Arf locus, in addition to being critical cell-cycle regulators, appear to be involved in several disease pathologies. Moreover, the locus’ expression is epigenetically regulated in ES reprogramming processes, thus constituting the ideal candidates to modulate PPCs homeostasis. In this study, starting from mouse embryonic stem cells (mESCs), we analyzed the early stages of pancreatic commitment. By inducing mESCs commitment to the pancreatic lineage, we observed that both products of the Cdkn2a locus, Ink4a and Arf, mark a naïve pancreatic cellular state that resembled PPC-like specification. Treatment with epi-drugs suggests a role for chromatin remodeling in the CDKN2a (Cycline Dependent Kinase Inhibitor 2A) locus regulation in line with previous observations in other cellular systems. Our data considerably improve the comprehension of pancreatic cellular ontogeny, which could be critical for implementing pluripotent stem cells programming and reprogramming toward pancreatic lineage commitment.

Trichostatin A ([R-(E,E)]-7-[4-(dimethylamino) phenyl]-N-hydroxy- 4,6-dimethyl- 7-oxo-2,4-heptadienamide, TSA) affects chromatin state through its potent histone deacetylase inhibitory activity. Interfering with the removal of acetyl groups from lysine residues in histones is one of many epigenetic regulatory processes that control gene expression. Histone deacetylase inhibition drives cells toward the differentiation stage, favoring the activation of specific genes. In this paper, we investigated the effects of TSA on H3 and H4 lysine acetylome and methylome profiling in mice embryonic stem cells (ES14), treated with trichostatin A (TSA) by using a new, untargeted approach, consisting of trypsin-limited proteolysis experiments coupled with MALDI-MS and LC-MS/MS analyses. The method was firstly set up on standard chicken core histones to probe the optimized conditions in terms of enzyme:substrate (E:S) ratio and time of proteolysis and, then, applied to investigate the global variations of the acetylation and methylation state of lysine residues of H3 and H4 histone in the embryonic stem cells (ES14) stimulated by TSA and addressed to differentiation. The proposed strategy was found in its simplicity to be extremely effective in achieving the identification and relative quantification of some of the most significant epigenetic modifications, such as acetylation and lysine methylation. Therefore, we believe that it can be used with equal success in wider studies concerning the characterization of all epigenetic modifications.

Water reabsorption along the collecting duct is dependent on the function of aquaporin 2 (AQP2). Currently, information on microRNA (miRNA)-mediated, post-transcriptional regulation of AQP2, which may influence water reabsorption, is limited. In mice, ablation of the Dicer enzyme (crucial for miRNA maturation) in AQP2-expressing cells induces nephrogenic diabetes insipidus (NDI) with dysregulation of the miRNA profile. A major finding is the identification of miRNAs associated with NDI through mediating epigenetic control of AQP2. This study offers novel targets for AQP2 regulation and potential treatment for governing renal water reabsorption.Background MicroRNAs (miRNAs), formed by cleavage of pre-microRNA by the endoribonuclease Dicer, are critical modulators of cell function by post-transcriptionally regulating gene expression.Methods Selective ablation of Dicer in AQP2-expressing cells (DicerAQP2Cre+ mice) was used to investigate the role of miRNAs in the kidney collecting duct of mice.Results The mice had severe polyuria and nephrogenic diabetes insipidus, potentially due to greatly reduced AQP2 and AQP4 levels. Although epithelial sodium channel levels were decreased in cortex and increased in inner medulla, amiloride-sensitive sodium reabsorption was equivalent in DicerAQP2Cre+ mice and controls. Small-RNA sequencing and proteomic analysis revealed 31 and 178 significantly regulated miRNAs and proteins, respectively. Integrated bioinformatic analysis of the miRNAome and proteome suggested alterations in the epigenetic machinery and various transcription factors regulating AQP2 expression in DicerAQP2Cre+ mice. The expression profile and function of three miRNAs (miR-7688-5p, miR-8114, and miR-409-3p) whose predicted targets were involved in epigenetic control (Phf2, Kdm5c, and Kdm4a) or transcriptional regulation (GATA3, GATA2, and ELF3) of AQP2 were validated. Luciferase assays could not demonstrate direct interaction of AQP2 or the three potential transcription factors with miR-7688-5p, miR-8114, and miR-409textendash3p. However, transfection of respective miRNA mimics reduced AQP2 expression. Chromatin immunoprecipitation assays demonstrated decreased Phf2 and significantly increased Kdm5c interactions at the Aqp2 gene promoter in DicerAQP2Cre+ mice, resulting in decreased RNA Pol II association.Conclusions Novel evidence indicates miRNA-mediated epigenetic regulation of AQP2 expression.

Background: The ARF tumour suppressor plays a well-established role as a tumour suppressor, halting cell growth by both p53-dependent and independent pathways in several cellular stress response circuits. However, data collected in recent years challenged the traditional role of this protein as a tumour suppressor. Cancer cells expressing high ARF levels showed that its expression, far from being dispensable, is required to guarantee tumour cell survival. In particular, ARF can promote autophagy, a self-digestion pathway that helps cells cope with stressful growth conditions arising during both physiological and pathological processes. Methods: We previously showed that ARF is regulated through the activation of the protein kinase C (PKC)-dependent pathway and that an ARF phospho-mimetic mutant on the threonine residue 8, ARF-T8D, sustains cell proliferation in HeLa cells. We now explored the role of ARF phosphorylation in both basal and starvation-induced autophagy by analysing autophagic flux in cells transfected with either WT and ARF phosphorylation mutants by immunoblot and immunofluorescence. Results: Here, we show that endogenous ARF expression in HeLa cells is required for starvation-induced autophagy. Further, we provide evidence that the hyper-expression of ARF-T8D appears to inhibit autophagy in both HeLa and lung cancer cells H1299. This effect is due to the cells’ inability to elicit autophagosomes formation upon T8D expression. Conclusions: Our results lead to the hypothesis that ARF phosphorylation could be a mechanism through which the protein promotes or counteracts autophagy. Several observations underline how autophagy could serve a dual role in cancer progression, either protecting healthy cells from damage or aiding cancerous cells to survive. Our results indicate that ARF phosphorylation controls protein’s ability to promote or counteract autophagy, providing evidence of the dual role played by ARF in cancer progression.

The extracellular microenvironment proved to exert a potent regulatory effect over different aspects of Embryonic Stem Cells (ESCs) behavior. In particular, the employment of engineered culture surfaces aimed at modulating ESC self-organization resulted effective in directing ESCs toward specific fate decision. ESCs fluctuate among different levels of functional potency and in this context the Zscan4 gene marks the so-called "metastate," a cellular state in which ESCs retain both self-renewal and pluripotency capabilities. Here we investigated the impact of topographic cues on ESCs pluripotency, differentiation and organization capabilities. To this aim, we engineered culturing platforms of nanograted surfaces with different features size and we investigated their impact on ESCs multicellular organization and Zscan4 gene expression. We showed that the morphology of ESC-derived aggregates and Zscan4 expression are strictly intertwined. Our data suggest that ESC Zscan4 metastate can be promoted if the adhesive surface conditions guide cellular self-aggregation into 3D dome-like structure, in which both cell-material interactions and cell-cell contact are supportive for Zscan4 expression.

Background Chronic pancreatitis presents a high risk of inflammation-related progression to pancreatic cancer. Pancreatic cancer is the fourth leading cause of cancer-related death worldwide. The high mortality rate is directly related to the difficulty in promptly diagnosing the disease, which often presents as overt and advanced. Hence, early diagnosis for pancreatic cancer becomes crucial, propelling research into the molecular and epigenetic landscape of the disease. Main body Recent studies have shown that cell-free DNA methylation profiles from inflammatory diseases or cancer can vary, thus opening a new venue for the development of biomarkers for early diagnosis. In particular, cell-free DNA methylation could be employed in the identification of pre-neoplastic signatures in individuals with suspected pancreatic conditions, representing a specific and non-invasive method of early diagnosis of pancreatic cancer. In this review, we describe the molecular determinants of pancreatic cancer and how these are related to chronic pancreatitis. We will then present an overview of differential methylated genes in the two conditions, highlighting their diagnostic or prognostic potential. Conclusion Exploiting the relation between abnormally methylated cell-free DNA and pre-neoplastic lesions or chronic pancreatitis may become a game-changing approach for the development of tools for the early diagnosis of pancreatic cancer. Electronic supplementary material The online version of this article (10.1186/s13148-019-0728-8) contains supplementary material, which is available to authorized users.

Epigenetic mechanisms can mediate gene-environment interactions relevant for complex disorders. The BDNF gene is crucial for development and brain plasticity, is sensitive to environmental stressors, such as hypoxia, and harbors the functional SNP rs6265 (Val(66)Met), which creates or abolishes a CpG dinucleotide for DNA methylation. We found that methylation at the BDNF rs6265 Val allele in peripheral blood of healthy subjects is associated with hypoxia-related early life events (hOCs) and intermediate phenotypes for schizophrenia in a distinctive manner, depending on rs6265 genotype: in ValVal individuals increased methylation is associated with exposure to hOCs and impaired working memory (WM) accuracy, while the opposite is true for ValMet subjects. Also, rs6265 methylation and hOCs interact in modulating WM-related prefrontal activity, another intermediate phenotype for schizophrenia, with an analogous opposite direction in the 2 genotypes. Consistently, rs6265 methylation has a different association with schizophrenia risk in ValVals and ValMets. The relationships of methylation with BDNF levels and of genotype with BHLHB2 binding likely contribute to these opposite effects of methylation. We conclude that BDNF rs6265 methylation interacts with genotype to bridge early environmental exposures to adult phenotypes, relevant for schizophrenia. The study of epigenetic changes in regions containing genetic variation relevant for human diseases may have beneficial implications for the understanding of how genes are actually translated into phenotypes.

[This corrects the article DOI: 10.1371/journal.pgen.0030110.].

The ARF protein functions as an important sensor of hyper-proliferative stimuli restricting cell proliferation through both p53-dependent and -independent pathways. Although to date the majority of studies on ARF have focused on its anti-proliferative role, few studies have addressed whether ARF may also have pro-survival functions. Here we show for the first time that during the process of adhesion and spreading ARF re-localizes to sites of active actin polymerization and to focal adhesion points where it interacts with the phosphorylated focal adhesion kinase. In line with its recruitment to focal adhesions, we observe that hampering ARF function in cancer cells leads to gross defects in cytoskeleton organization resulting in apoptosis through a mechanism dependent on the Death-Associated Protein Kinase. Our data uncover a novel function for p14ARF in protecting cells from anoikis that may reflect its role in anchorage independence, a hallmark of malignant tumor cells.Oncogene advance online publication, 24 April 2017; doi:10.1038/onc.2017.104.

In this study we assessed the BDNF promoter IV methylation state of a large genomic region surrounding promoter IV and evaluated BDNF transcript IV expression from prefrontal cortex and striatum of 15 schizophrenic and 15 control subjects. In prefrontal cortex, a single CpG site at -93, appeared to be undermethylated in patients׳group. BDNF mRNA levels in frontal cortex and striatum were variable among individuals but did not associate with disease.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancer types world-wide. Its high mortality is related to the difficulty in the diagnosis, which often occurs when the disease is already advanced. As of today, no early diagnostic tests are available, while only a limited number of prognostic tests have reached clinical practice. The main reason is the lack of reliable biomarkers that are able to capture the early development or the progression of the disease. Hence, the discovery of biomarkers for early diagnosis or prognosis of PDAC remains, de facto, an unmet need. An increasing number of studies has shown that cell-free DNA (cfDNA) methylation analysis represents a promising non-invasive approach for the discovery of biomarkers with diagnostic or prognostic potential. In particular, cfDNA methylation could be utilized for the identification of disease-specific signatures in pre-neoplastic lesions or chronic pancreatitis (CP), representing a sensitive and non-invasive method of early diagnosis of PDAC. In this review, we will discuss the advantages and pitfalls of cfDNA methylation studies. Further, we will present the current advances in the discovery of pancreatic cancer biomarkers with early diagnostic or prognostic potential, focusing on pancreas-specific (e.g., CUX2 or REG1A) or abnormal (e.g., ADAMTS1 or BNC1) cfDNA methylation signatures in high risk pre-neoplastic conditions and PDAC.

Gastric cancer (GC) remains one of the major causes of cancer-related mortality worldwide. As for other types of cancers, several limitations to the success of current therapeutic GC treatments may be due to cancer drug resistance that leads to tumor recurrence and metastasis. Increasing evidence suggests that cancer stem cells (CSCs) are among the major causative factors of cancer treatment failure. The research of molecular CSC mechanisms and the regulation of their properties have been intensively studied. To date, molecular gastric cancer stem cell (GCSC) characterization remains largely incomplete. Among the GCSC-targeting approaches to overcome tumor progression, recent studies have focused their attention on microRNA (miRNA). The miRNAs are short non-coding RNAs which play an important role in the regulation of numerous cellular processes through the modulation of their target gene expression. In this review, we summarize and discuss recent findings on the role of miRNAs in GCSC regulation. In addition, we perform a meta-analysis aimed to identify novel miRNAs involved in GCSC homeostasis.

Cationic antimicrobial peptides (AMPs) possess fast and broad-spectrum activity against both Gram-negative and Gram-positive bacteria, as well as fungi. It has become increasingly evident that many AMPs, including those that derive from fragments of host proteins, are multifunctional and able to mediate various immunomodulatory functions and angiogenesis. Among these, synthetic apolipoprotein-derived peptides are safe and well tolerated in humans and have emerged as promising candidates in the treatment of various inflammatory conditions. Here, we report the characterization of a new AMP corresponding to residues 133-150 of human apolipoprotein E. Our results show that this peptide, produced either by chemical synthesis or by recombinant techniques in Escherichia coli, possesses a broad-spectrum antibacterial activity. As shown for several other AMPs, ApoE (133-150) is structured in the presence of TFE and of membrane-mimicking agents, like SDS, or bacterial surface lipopolysaccharide (LPS), and an anionic polysaccharide, alginate, which mimics anionic capsular exo-polysaccharides of several pathogenic microorganisms. Noteworthy, ApoE (133-150) is not toxic toward several human cell lines and triggers a significant innate immune response, assessed either as decreased expression levels of proinflammatory cytokines in differentiated THP-1 monocytic cells or by the induction of chemokines released from PBMCs. This novel bioactive AMP also showed a significant anti-inflammatory effect on human keratinocytes, suggesting its potential use as a model for designing new immunomodulatory therapeutics.

Hfq is an RNA-binding protein known to regulate a variety of cellular processes by interacting with small RNAs (sRNAs) and mRNAs in prokaryotes. Stenotrophomonas maltophilia is an important opportunistic pathogen affecting primarily hospitalized and immunocompromised hosts. We constructed an hfq deletion mutant (Δ hfq ) of S. maltophilia and compared the behaviors of wild-type and Δ hfq S. maltophilia cells in a variety of assays. This revealed that S. maltophilia Hfq plays a role in biofilm formation and cell motility, as well as susceptibility to antimicrobial agents. Moreover, Hfq is crucial for adhesion to bronchial epithelial cells and is required for the replication of S. maltophilia in macrophages. Differential RNA sequencing analysis (dRNA-seq) of RNA isolated from S. maltophilia wild-type and Δ hfq strains showed that Hfq regulates the expression of genes encoding flagellar and fimbrial components, transmembrane proteins, and enzymes involved in different metabolic pathways. Moreover, we analyzed the expression of several sRNAs identified by dRNA-seq in wild-type and Δ hfq S. maltophilia cells grown in different conditions on Northern blots. The accumulation of two sRNAs was strongly reduced in the absence of Hfq. Furthermore, based on our dRNA-seq analysis we provide a genome-wide map of transcriptional start sites in S. maltophilia .

Inducible nitric oxide synthase (iNOS) expression is altered in gastrointestinal diseases. Helicobacter pylori (Hp) infection may have a critical role in iNOS disregulation. We undertook this study to investigate possible chromatin changes occurring early during iNOS gene activation as a direct consequence of Hp-gastric cells interaction. We show that Hp infection is followed by different expression and chromatin modifications in gastric cells including (1) activation of iNOS gene expression, (2) chromatin changes at iNOS promoter including decreased H3K9 methylation and increased H3 acetylation and H3K4 methylation levels, (3) selective release of methyl-CpG-binding protein 2 from the iNOS promoter. Moreover, we show that Hp-induced activation of iNOS is delayed, but not eliminated, by the treatment with LSD1 inhibitors. Our data suggest a role for specific chromatin-based mechanisms in the control of human iNOS gene expression upon Hp exposure.

Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci.

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.

In order to supplement the cytopathological assessment of thyroid tumors, there is a need for new markers to correctly diagnose malignant thyroid lesions and avoid unnecessary and potentially harmful therapies for patients. The immunohistochemical expression of galectin-3 is currently considered to be the most accurate stand-alone marker for thyroid cancer diagnosis. The aim of this study was to establish whether the methylation state of the galectin-3 gene is a candidate molecular marker for thyroid malignancy. Thyroid specimens from 50 patients were analyzed, including 5 normal thyroid, 3 goiters, 39 papillary and 3 anaplastic thyroid carcinoma cases. High-resolution methylation analyses was performed to investigate the methylation state of a large genomic region (from −89 to +408) encompassing the galectin-3 transcriptional start site. Within this region, 5 CpG sites (nucleotide positions +134, +137, +142, +147 and +156) were observed to be differentially methylated among the samples and were further analyzed by the quantitative pyrosequencing technique. The hypomethylation of the +134, +137, +142, +147 and +156 CpG sites was observed to be markedly associated with cancer. Although the methylation degree of each single site was highly variable in non-neoplastic tissues, the average methylation state of the 5 CpG sites clearly distinguished cancer from the nonneoplastic thyroid tissues.

One of the most fascinating aspects of the field of epigenetics is the emerging ability of environmental factors to trigger epigenetic changes in eukaryotic cells, thus contributing to transient or stable, and potentially heritable, changes in gene expression program in the absence of alteration in DNA sequence. Epigenetic response may result in cell adaptation to environmental stimuli or, in some instances, may contribute to generation or progression of different kind of diseases. A paradigmatic case of disease that is accompanied by multiple epigenetic alterations is gastric cancer, among other relevant examples. In turn, Helicobacter pylori (Hp) infection has been associated as a leading cause of gastric cancer. One possible hypothesis is that Hp-gastric cell interaction initiates an epigenetic reprogramming of host cell genome that may favor tumorigenesis. Accordingly, an abundance of experimental evidence indicates that several epigenetic alterations underlie the gastric cancerogenesis process and that these alterations represent one of the major hallmarks of gastric cancer. However, several critical questions remain unanswered: Does Hp directly provoke epigenetic alterations? Which mechanisms underlie these phenomena? Based on currently available data, it is often arduous to discriminate between the epigenetic modifications directly triggered by Hp-gastric cell interaction and those alterations that are mediated by inflammation process or by many other molecular and genetic events occurring during the gastric cancer progression. We will review our present knowledge of epigenetic modifications and alterations proven to occur in host cells as a direct consequence of Hp infection.

Background Alterations of DNA methylation and expression of suicide-related genes occurring in specific brain's areas have been associated to suicidal behavior. In the BDNF pathway, TrkB gene in frontal cortex and hippocampus, and BDNF gene in Wernicke area have been found hypermethylated and down-regulated in suicide subjects as compared to controls. In this work we investigated whether epigenetic modifications of TrkB gene occur in Wernicke area of 18 suicide subjects as compared to 18 controls. Methods MassArray analysis was performed to determine the methylation degree of TrkB promoter in post-mortem samples. TrkB full length and TrkB-T1 mRNA levels were assessed by quantitative RT-PCR. Geometric averaging of four internal control genes was calculated for normalization of results. Results We found that TrkB and TrkB-T1 expression and promoter methylation in Wernicke area did not correlate with suicidal behavior whereas, in the same samples, the BDNF promoter IV was significantly hypermethylated in suicide with respect of controls. Limitation Data from a single brain's area in this study's sample. Conclusions Our data show that no correlation exists between TrkB gene methylation and suicide in Wernicke area, confirming that expression and methylation state of suicide-related genes, even belonging to the same pathway, may be specific for brain area.

COX-2 expression is altered in gastrointestinal diseases. Helicobacter pylori (Hp) infection may have a critical role in COX-2 disregulation. We undertook this study to investigate possible chromatin and DNA methylation changes occurring early during COX-2 gene activation as a direct consequence of Hp-gastric cells interaction. We show that Hp infection is followed by different expression, chromatin and DNA methylation changes including: (i) biphasic activation of COX-2 gene; (ii) rapid remodulation of HDACs expression and activity, increased acetylation and release of HDAC from COX-2 promoter; (iii) transient gradual increase of H3 acetylation and H3K4 dimethylation and decrease of H3K9 dimethylation; (iv) late and long-lasting increase of H3K27 trimethylation; (v) rapid cyclical DNA methylation/demethylation events at 8 specific CpG sites (-176, -136, +25, +36, +57, +82, +198, +231) surrounding the COX-2 gene transcriptional start site. Our data indicate that specific chromatin and DNA methylation changes occur at COX-2 gene in the first phases of Hp exposure in cultured gastric cells as a primary response to host-parasite interaction.

Background The release of LPS by bacteria stimulates both immune and specific epithelial cell types to release inflammatory mediators. It is known that LPS induces the release of IL-8 by intestinal mucosal cells. Because it is now emerging that bacteria may induce alteration of epigenetic patterns in host cells, we have investigated whether LPS-induced IL-8 activation in human intestinal epithelial cells involves changes of histone modifications and/or DNA methylation at IL-8 gene regulatory region. Results RT-PCR analysis showed that IL-8 mRNA levels rapidly increase after exposure of HT-29 cells to LPS. DNA demethylating agents had no effects on IL-8 expression, suggesting that DNA methylation was not involved in IL-8 gene regulation. Consistently we found that 5 CpG sites located around IL-8 transcription start site (-83, -7, +73, +119, +191) were unmethylated on both lower and upper strand either in LPS treated or in untreated HT-29 cells, as well as in normal intestinal mucosa. Conversely, pretreatment of HT-29 cells with deacetylase inhibitors strengthened the LPS-mediated IL-8 activation. Inhibitors of histone deacetylases could induce IL-8 mRNA expression also in the absence of LPS, suggesting that chromatin modifications could be involved in IL-8 gene regulation. Chromatin immunoprecipitation analyses showed that, concurrently with IL-8 activation, transient specific changes in H3 acetylation and H3K4, H3K9 and H3K27 methylation occurred at IL-8 gene promoter during LPS stimulation. Changes of H3-acetyl, H3K4me2 and H3K9me2 levels occurred early, transiently and corresponded to transcriptional activity, while changes of H3K27me3 levels at IL-8 gene occurred later and were long lasting. Conclusion The results showed that specific chromatin modifications occurring at IL-8 gene, including histone H3 acetylation and methylation, mark LPS-mediated IL-8 activation in intestinal epithelial cells while it is unlikely that DNA methylation of IL-8 promoter is directly involved in IL-8 gene regulation in these cells.

We have identified a human gene encoding a novel MBD2-interacting protein (MBDin) that contains an N-terminal GTP-binding site, a putative nuclear export signal (NES), and a C-terminal acidic region. MBDin cDNA was isolated through a two-hybrid interaction screening using the methyl-CpG-binding protein MBD2 as bait. The presence of the C-terminal 46-amino-acid region of MBD2 and both the presence of the acidic C-terminal 128-amino-acid region and the integrity of the GTP-binding site of MBDin were required for the interaction. Interaction between MBD2 and MBDin in mammalian cells was confirmed by immunoprecipitation experiments. Fluorescence imaging experiments demonstrated that MBDin mainly localizes in the cytoplasm but accumulates in the nucleus upon disruption of the NES or treatment with leptomycin B, an inhibitor of NES-mediated transport. We also found that MBDin partially colocalizes with MBD2 at foci of heavily methylated satellite DNA. An MBD2 deletion mutant lacking the C-terminal region maintained its subnuclear localization but failed to recruit MBDin at hypermethylated foci. Functional analyses demonstrated that MBDin relieves MBD2-mediated transcriptional repression both when Gal4 chimeric constructs and when in vitro-methylated promoter-reporter plasmids were used in transcriptional assays. Southern blotting and bisulfite analysis showed that transcriptional reactivation occurred without changes of the promoter methylation pattern. Our findings suggest the existence of factors that could be targeted on methylated DNA by methyl-CpG-binding proteins reactivating transcription even prior to demethylation.

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments., Author Summary Genomic DNA can be modified by cytosine methylation. This epigenetic modification is layered on the primary genetic information and can silence the affected gene. Epigenetic modification has been implicated in cancer and aging. To date, the primary cause and the mechanism leading to DNA methylation are not known. By using a sophisticated genetic system, we have induced a single break in the double helix of the genomes of mouse or human cells. This rupture was repaired by a very precise mechanism: the damaged chromosome pairs and retrieves genetic information from an undamaged and homologous DNA partner. This homology-directed repair was marked in half of the repaired molecules by de novo methylation of cytosines flanking the cut. As a direct consequence, the gene in these repaired molecules was silenced. In the remaining molecules, the recombinant DNA was undermethylated and expressed the reconstituted gene. Since homology-directed repair may duplicate or delete genetic information, epigenetic modification of repaired DNA represents a powerful evolutionary force. If the expression of the repaired gene is harmful, only cells inheriting the silenced copy will survive. Conversely, if the function of the repaired gene is beneficial, cells inheriting the under-methylated copy will have a selective advantage.

Re-induction of fetal genes and/or re-expression of postnatal genes represent hallmarks of pathological cardiac remodeling, and are considered important in the progression of the normal heart towards heart failure (HF). Whether epigenetic modifications are involved in these processes is currently under investigation. Here we hypothesized that histone chromatin modifications may underlie changes in the gene expression program during pressure overload-induced HF. We evaluated chromatin marks at the promoter regions of the sarcoplasmic reticulum Ca(2+)ATPase (SERCA-2A) and β-myosin-heavy chain (β-MHC) genes (Atp2a2 and Myh7, respectively) in murine hearts after one or eight weeks of pressure overload induced by transverse aortic constriction (TAC). As expected, all TAC hearts displayed a significant reduction in SERCA-2A and a significant induction of β-MHC mRNA levels. Interestingly, opposite histone H3 modifications were identified in the promoter regions of these genes after TAC, including H3 dimethylation (me2) at lysine (K) 4 (H3K4me2) and K9 (H3K9me2), H3 trimethylation (me3) at K27 (H3K27me3) and dimethylation (me2) at K36 (H3K36me2). Consistently, a significant reduction of lysine-specific demethylase KDM2A could be found after eight weeks of TAC at the Atp2a2 promoter. Moreover, opposite changes in the recruitment of DNA methylation machinery components (DNA methyltransferases DNMT1 and DNMT3b, and methyl CpG binding protein 2 MeCp2) were found at the Atp2a2 or Myh7 promoters after TAC. Taken together, these results suggest that epigenetic modifications may underlie gene expression reprogramming in the adult murine heart under conditions of pressure overload, and might be involved in the progression of the normal heart towards HF.

Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene.

The PATZ1 gene encoding a POZ/AT-hook/Kruppel zinc finger (PATZ) transcription factor, is considered a cancer-related gene because of its loss or misexpression in human neoplasias. As for other POZ/domain and Kruppel zinc finger (POK) family members, the transcriptional activity of PATZ is due to the POZ-mediated oligomer formation, suggesting that it might be not a typical transactivator but an architectural transcription factor, thus functioning either as activator or as repressor depending on the presence of proteins able to interact with it. Therefore, to better elucidate PATZ function, we searched for its molecular partners. By yeast two-hybrid screenings, we found a specific interaction between PATZ and BCL6, a human oncogene that plays a key role in germinal center (GC) derived neoplasias. We demonstrate that PATZ and BCL6 interact in germinal center-derived B lymphoma cells, through the POZ domain of PATZ. Moreover, we show that PATZ is able to bind the BCL6 regulatory region, where BCL6 itself acts as a negative regulator, and to contribute to negatively modulate its activity. Consistently, disruption of one or both Patz1 alleles in mice causes focal expansion of thymus B cells, in which BCL6 is up-regulated. This phenotype was almost completely rescued by crossing Patz1(+/-) with Bcl6(+/-) mice, indicating a key role for Bcl6 expression in its development. Finally, a significant number of Patz1 knock-out mice (both heterozygous and homozygous) also develop BCL6-expressing lymphomas. Therefore, the disruption of one or both Patz1 alleles may favor lymphomagenesis by activating the BCL6 pathway.

One of the most fascinating aspects of the field of epigenetics is the emerging ability of environmental factors to trigger epigenetic changes in eukaryotic cells, thus contributing to transient or stable, and potentially heritable, changes in gene expression program in the absence of alteration in DNA sequence. Epigenetic response may result in cell adaptation to environmental stimuli or, in some instances, may contribute to generation or progression of different kind of diseases. A paradigmatic case of disease that is accompanied by multiple epigenetic alterations is gastric cancer, among other relevant examples. In turn, Helicobacter pylori (Hp) infection has been associated as a leading cause of gastric cancer. One possible hypothesis is that Hp-gastric cell interaction initiates an epigenetic reprogramming of host cell genome that may favor tumorigenesis. Accordingly, an abundance of experimental evidence indicates that several epigenetic alterations underlie the gastric cancerogenesis process and that these alterations represent one of the major hallmarks of gastric cancer. However, several critical questions remain unanswered: Does Hp directly provoke epigenetic alterations? Which mechanisms underlie these phenomena? Based on currently available data, it is often arduous to discriminate between the epigenetic modifications directly triggered by Hp-gastric cell interaction and those alterations that are mediated by inflammation process or by many other molecular and genetic events occurring during the gastric cancer progression. We will review our present knowledge of epigenetic modifications and alterations proven to occur in host cells as a direct consequence of Hp infection.

We have recently reported that a novel MBD2 interactor (MBDin) has the capacity to reactivate transcription from MBD2-repressed methylated promoters even in the absence of demethylation events. Here we show that another unrelated protein, TACC3, displays a similar activity on methylated genes. In addition the data reported here provide possible molecular mechanisms for the observed phenomenon. Immunoprecipitation experiments showed that MBD2/TACC3 form a complex in vivo with the histone acetyltransferase pCAF. MBD2 could also associate with HDAC2, a component of MeCP1 repression complex. However, we found that the complexes formed by MBD2 with TACC3/pCAF and with HDAC2 were mutually exclusive. Moreover, HAT enzymatic assays demonstrated that HAT activity associates with MBD2 in vivo and that such association significantly increased when TACC3 was over-expressed. Overall our findings suggest that TACC3 can be recruited by MBD2 on methylated promoters and is able to reactivate transcription possibly by favoring the formation of an HAT-containing MBD2 complex and, thus, switching the repression potential of MBD2 in activation even prior to eventual demethylation.

The three-way connection between DNA methylation, chromatin configuration, and transcriptional regulation is under increasing attention, but the fine rules governing the epi-genetic control are still poorly understood. In several studies, the authors have concluded that the methylation status of CpG sites could be critical for the binding of factors to DNA and, consequently, for chromatin conformation. We tested the possibility that a novel technical approach combining chromatin immunoprecipitation and bisulfite genomic sequencing analysis (ChIP-BA) could provide useful information on the role of specific CpG methylation patterns in driving the association in vivo of proteins to given genomic regions. Our results show that ChIP-BA permits the establishment in vivo of the methylation patterns required for the binding of a methyl-CpG binding protein and, in addition, can potentially identify methylation patterns that do not allow a protein to bind specific genomic regions. Possible fields of application are discussed. We believe that wide use of ChIP-BA could make possible the exploration of a novel aspect of the intricate epigenetic web.

Increasing evidence points to a role for dysfunctional glutamate N-methyl-D-aspartate receptor (NMDAR) neurotransmission in schizophrenia. D-aspartate is an atypical amino acid that activates NMDARs through binding to the glutamate site on GluN2 subunits. D-aspartate is present in high amounts in the embryonic brain of mammals and rapidly decreases after birth, due to the activity of the enzyme D-aspartate oxidase (DDO). The agonistic activity exerted by D-aspartate on NMDARs and its neurodevelopmental occurrence make this D-amino acid a potential mediator for some of the NMDAR-related alterations observed in schizophrenia. Consistently, substantial reductions of D-aspartate and NMDA were recently observed in the postmortem prefrontal cortex of schizophrenic patients. Here we show that DDO mRNA expression is increased in prefrontal samples of schizophrenic patients, thus suggesting a plausible molecular event responsible for the D-aspartate imbalance previously described. To investigate whether altered D-aspartate levels can modulate schizophrenia-relevant circuits and behaviors, we also measured the psychotomimetic effects produced by the NMDAR antagonist, phencyclidine, in Ddo knockout mice (Ddo(-)(/-)), an animal model characterized by tonically increased D-aspartate levels since perinatal life. We show that Ddo(-/-) mice display a significant reduction in motor hyperactivity and prepulse inhibition deficit induced by phencyclidine, compared with controls. Furthermore, we reveal that increased levels of D-aspartate in Ddo(-/-) animals can significantly inhibit functional circuits activated by phencyclidine, and affect the development of cortico-hippocampal connectivity networks potentially involved in schizophrenia. Collectively, the present results suggest that altered D-aspartate levels can influence neurodevelopmental brain processes relevant to schizophrenia.