Raffaela Pero

Galectin-1 and galectin-3 expression in human bladder transitional-cell carcinomas

Galectin‐1 and galectin‐3 are galactoside‐binding proteins involved in different steps of tumor progression and potential targets for therapy. We have investigated the expression of these galectins in 38 human bladder transitional‐cell carcinomas of different histological grade and clinical stage and in 5 normal urothelium samples. Galectin‐1 mRNA levels were highly increased in most high‐grade tumors compared with normal bladder or low‐grade tumors. Western blot and immuno‐histochemical analysis of normal and neoplastic tissues revealed a higher content of galectin‐1 in tumors. Galectin‐3 mRNA levels were also increased in most tumors compared with normal urothelium, but levels were comparable among tumors of different histological grade. Int. J. Cancer (Pred. Oncol.) 84:39–43, 1999.

High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth.

The HMGI proteins (HMGI, HMGY and HMGI-C) have an important role in the chromatin organization and interact with different transcriptional factors. The HMGI genes are expressed at very low levels in normal adult tissues, whereas they are very abundant during embryonic development and in several experimental and human tumours. In order to isolate proteins interacting with the HMGI(Y) proteins, a yeast two-hybrid screening was performed using the HMGI(Y) protein as bait. This analysis led to the isolation of homeodomain-interacting protein kinase-2 (HIPK2), a serine/threonine nuclear kinase. HIPK2 co-immunoprecipitates with the HMGI(Y) protein in 293T cells. The interaction between HIPK2 and HMGI(Y) occurs through the PEST domain of HIPK2 and it is direct because in vitro translated HIPK2 binds HMGI(Y). We also show that HIPK2 is able to phosphorylate the HMGI(Y) protein by an in vitro kinase assay. In order to understand a possible role of HIPK2 gene in cell growth we performed a colony assay which showed an impressive HIPK2 inhibitory effect on normal thyroid cells. Flow cytometric analysis would indicate the block of cell growth at the G2/M phase of the cell cycle. Since normal thyroid cells do not express detectable HMGI(Y) protein levels, we assume that the HIPK2 inhibitory effect is independent from the interaction with the HMGI(Y) protein.

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.

PATZ1 gene has a critical role in the spermatogenesis and testicular tumours

PATZ1 is a recently discovered zinc finger protein that, due to the presence of the POZ domain, acts as a transcriptional repressor affecting the basal activity of different promoters. To gain insights into its biological role, we generated mice lacking the PATZ1 gene. Male PATZ1−/− mice were unfertile, suggesting a crucial role of this gene in spermatogenesis. Consistently, most of adult testes from these mice showed only few spermatocytes, associated with increased apoptosis, and complete absence of spermatids and spermatozoa, with the subsequent loss of tubular structure. The analysis of PATZ1 expression, by northern blot, western blot and immunohistochemistry, revealed its presence in Sertoli cells and, among the germ cells, exclusively in the spermatogonia. Since PATZ1 has been indicated as a potential tumour suppressor gene, we also looked at its expression in tumours deriving from testicular germ cells (TGCTs). Although expression of PATZ1 protein was increased in these tumours, it was delocalized in the cytoplasm, suggesting an impaired function. These results indicate that PATZ1 plays a crucial role in normal male gametogenesis and that its up‐regulation and mis‐localization could be associated to the development of TGCTs. Copyright

RNF4 is a growth inhibitor expressed in germ cells but not in human testicular tumors

The RING-finger protein RNF4 modulates both steroid-receptor-dependent and basal transcription and interacts with a variety of nuclear proteins involved in cell growth control. RNF4 is expressed at very high levels in testis and at much lower levels in several other tissues. We show that in germ cells RNF4 expression is strongly modulated during progression of spermatogonia to spermatids, with a peak in spermatocytes. Analysis of human testicular germ cell tumors shows that RNF4 is not expressed in all tumors analyzed including seminomas, the highly malignant embryonal carcinomas, yolk sac, and mixed germ cell tumors. We also show that the ectopically expressed RNF4 gene inhibits cell proliferation of both somatic and germ cell tumor-derived cells. Mutation of critical cysteine residues in the RING finger domain abolished the RNF4 growth inhibition activity. Our results suggest that the lack of RNF4 expression may play a role in the progression of testicular tumors.

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.