Tiziana Angrisano

DNA Damage, Homology-Directed Repair, and DNA Methylation

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.

LPS-induced IL-8 activation in human intestinal epithelial cells is accompanied by specific histone H3 acetylation and methylation changes

BackgroundThe 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.ResultsRT-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.ConclusionThe 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.

MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters

ABSTRACT 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.

Chromatin and DNA methylation dynamics during retinoic acid-induced RET gene transcriptional activation in neuroblastoma cells

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.

TrkB gene expression and DNA methylation state in Wernicke area does not associate with suicidal behavior

BACKGROUND Alterations of DNA methylation and expression of suicide-related genes occurring in specific brains 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 brains area in this studys 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.

Chromatin and DNA methylation dynamics of Helicobacter pylori-induced COX-2 activation

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.

In vivo analysis of DNA methylation patterns recognized by specific proteins: coupling ChIP and bisulfite analysis

The three-way connection between DNA methylation, chromatin configuration, and transcriptional regulation is under increasing attention, but the fine rules governing the epigenetic 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.

Helicobacter pylori regulates iNOS promoter by histone modifications in human gastric epithelial cells

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.

A role for D-aspartate oxidase in schizophrenia and in schizophrenia-related symptoms induced by phencyclidine in mice

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.

Functional Characterization of the RNA Chaperone Hfq in the Opportunistic Human Pathogen Stenotrophomonas maltophilia

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 (Δhfq) of S. maltophilia and compared the behaviors of wild-type and Δhfq 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 Δhfq 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 Δhfq 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.