Simona Keller

Increased BDNF Promoter Methylation in the Wernicke Area of Suicide Subjects

CONTEXT Brain-derived neurotrophic factor (BDNF) plays a pivotal role in the pathophysiology of suicidal behavior and BDNF levels are decreased in the brain and plasma of suicide subjects. So far, the mechanisms leading to downregulation of BDNF expression are poorly understood. OBJECTIVES To test the hypothesis that alterations of DNA methylation could be involved in the dysregulation of BDNF gene expression in the brain of suicide subjects. DESIGN Three independent quantitative methylation techniques were performed on postmortem samples of brain tissue. BDNF messenger RNA levels were determined by quantitative real-time polymerase chain reaction. SETTING Academic medical center. PATIENTS OR OTHER PARTICIPANTS Forty-four suicide completers and 33 nonsuicide control subjects of white ethnicity. MAIN OUTCOME MEASURES The DNA methylation degree at BDNF promoter IV and the genome-wide DNA methylation levels in the brains Wernicke area. RESULTS Postmortem brain samples from suicide subjects showed a statistically significant increase of DNA methylation at specific CpG sites in BDNF promoter/exon IV compared with nonsuicide control subjects (P < .001). Most of the CpG sites lying in the -300/+500 region, on both strands, had low or no methylation, with the exception of a few sites located near the transcriptional start site that had differential methylation, while genome-wide methylation levels were comparable among the subjects. The mean methylation degree at the 4 CpG sites analyzed by pyrosequencing was always less than 12.9% in the 33 nonsuicide control subjects, while in 13 of 44 suicide victims (30%), the mean methylation degree ranged between 13.1% and 34.2%. Higher methylation degree corresponded to lower BDNF messenger RNA levels. CONCLUSIONS BDNF promoter/exon IV is frequently hypermethylated in the Wernicke area of the postmortem brain of suicide subjects irrespective of genome-wide methylation levels, indicating that a gene-specific increase in DNA methylation could cause or contribute to the downregulation of BDNF expression in suicide subjects. The reported data reveal a novel link between epigenetic alteration in the brain and suicidal behavior.

DNA methylation in intron 1 of the frataxin gene is related to GAA repeat length and age of onset in Friedreich ataxia patients

Background: The most frequent mutation of Friedreich ataxia (FRDA) is the abnormal expansion of a GAA repeat located within the first intron of FXN gene. It is known that the length of GAA is directly correlated with disease severity. The effect of mutation is a severe reduction of mRNA. Recently, a link among aberrant CpG methylation, chromatin organisation and GAA repeat was proposed. Methods: In this study, using pyrosequencing technology, we have performed a quantitative analysis of the methylation status of five CpG sites located within the region upstream of GAA repeat, in 67 FRDA patients. Results: We confirm previous observation about differences in the methylation degree between FRDA individuals and controls. We showed a direct correlation between CpG methylation and triplet expansion size. Significant differences were found for each CpG tested (ANOVA p<0.001). These differences were largest for CpG1 and CpG2: 84.45% and 76.80%, respectively, in FRDA patients compared to 19.65% and 23.34% in the controls. Most importantly, we found a strong inverse correlation between CpG2 methylation degree and age of onset (Spearman’s ρ  =  −0.550, p<0.001). Conclusion: Because epigenetic changes may cause or contribute to gene silencing, our data may have relevance for the therapeutic approach to FRDA. Since the analysis can be performed in peripheral blood leucocytes (PBL), evaluation of the methylation status of specific CpG sites in FRDA patients could be a convenient biomarker.

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.

Epigenetic Regulation of miR-212 Expression in Lung Cancer

Many studies have shown that microRNA expression in cancer may be regulated by epigenetic events. Recently, we found that in lung cancer miR-212 was strongly down-regulated. However, mechanisms involved in the regulation of miR-212 expression are unknown. Therefore, we addressed this point by investigating the molecular mechanisms of miR-212 silencing in lung cancer. We identified histone modifications rather than DNA hypermethylation as epigenetic events that regulate miR-212 levels in NSCLC. Moreover, we found that miR-212 silencing in vivo is closely associated with the severity of the disease.

Chromobox Protein Homologue 7 Protein, with Decreased Expression in Human Carcinomas, Positively Regulates E-Cadherin Expression by Interacting with the Histone Deacetylase 2 Protein

Chromobox protein homologue 7 (CBX7) is a chromobox family protein encoding a novel polycomb protein, the expression of which shows a progressive reduction, well related with the malignant grade of the thyroid neoplasias. Indeed, CBX7 protein levels decreased in an increasing percentage of cases going from benign adenomas to papillary, follicular, and anaplastic thyroid carcinomas. To elucidate the function of CBX7 in carcinogenesis, we searched for CBX7 interacting proteins by a proteomic analysis. By this approach, we identified several proteins. Among these proteins, we selected histone deacetylase 2 (HDAC2), which is well known to play a key role in neoplastic cell transformation and down-regulation of E-cadherin expression, the loss of which is a critical event in the epithelial-to-mesenchymal transition. We confirmed by coimmunoprecipitation that CBX7 physically interacts with the HDAC2 protein and is able to inhibit its activity. Then, we showed that both these proteins bind the E-cadherin promoter and that CBX7 up-regulates E-cadherin expression. Consistent with these data, we found a positive statistical correlation between CBX7 and E-cadherin expression in human thyroid carcinomas. Finally, we showed that the expression of CBX7 increases the acetylation status of the histones H3 and H4 on the E-cadherin promoter. Therefore, the ability of CBX7 to positively regulate E-cadherin expression by interacting with HDAC2 and inhibiting its activity on the E-cadherin promoter would account for the correlation between the loss of CBX7 expression and a highly malignant phenotype.

Brain derived neurotrophic factor (BDNF) genetic polymorphism (Val66Met) in suicide: a study of 512 cases.

Brain Derived Neurotrophic Factor (BDNF) Genetic Polymorphism (Val66Met) in Suicide: A Study of 512 Cases F. Zarrilli, A. Angiolillo, G. Castaldo, L. Chiariotti, S. Keller, S. Sacchetti, A. Marusic, T. Zagar, V. Carli, A. Roy, and M. Sarchiapone* Facolt a di Scienze MFN, Universit a del Molise, Isernia, Italy Dipartimento di Biochimica e Biotecnologie Mediche and Facolt a di Scienze Biotecnologiche, Universit a ‘‘Federico II’’, CEINGE-Biotecnologie Avanzate, Naples, Italy Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universit a ‘‘Federico II’’, CEINGE-Biotecnologie Avanzate, Naples, Italy University of Primorska, Primorska, Slovenia Department of Health Sciences, University of Molise, Campobasso, Italy Psychiatry Service, Department of Veteran Affairs, Newark, New Jersey

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.

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.