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.

Identification, characterization, and variable expression of a naturally occurring inhibitor protein of IS1106 transposase in clinical isolates of Neisseria meningitidis.

Transposition plays a role in the epidemiology and pathogenesis of Neisseria meningitidis. Insertion sequences are involved in reversible capsulation and insertional inactivation of virulence genes encoding outer membrane proteins. In this study, we have investigated and identified one way in which transposon IS1106 controls its own activity. We have characterized a naturally occurring protein (Tip) that inhibits the transposase. The inhibitor protein is a truncated version of the IS1106 transposase lacking the NH(2)-terminal DNA binding sequence, and it regulates transposition by competing with the transposase for binding to the outside ends of IS1106, as shown by gel shift and in vitro transposition assays. IS1106Tip mRNA is variably expressed among serogroup B meningococcal clinical isolates, and it is absent in most collection strains belonging to hypervirulent lineages.

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.

Expression analysis and chromosomal assignment of PRA1 and RILP genes.

PRA1 (prenylated Rab acceptor) is a general regulator of Rab proteins, while RILP (Rab interacting lysosomal protein) is a specific effector for Rab7. It has been shown that PRA1 interacts with Rab proteins and with VAMP2. Therefore PRA1 is probably an important factor for membrane traffic, linking together the function of Rab proteins and SNAREs. RILP has a key role in the control of transport to degradative compartments together with Rab7 and probably links Rab7 function to the cytoskeleton. Here we have studied by Northern blot the expression of the two genes in several different human tissues. The 0.8-kb mRNA for human PRA1 is ubiquitously expressed, while the two mRNAs for RILP are differentially expressed. In addition, we have assigned the human PRA1 gene to chromosome 19q13.13-q13.2 and the human RILP gene to chromosome 17p13.3.

Evolution and function of the neisserial dam-replacing gene.

Phase variation through slippage-like mechanisms involving homopolymeric tracts depends in part on the absence of Dam-methylase in several pathogenic isolates of Neisseria meningitidis. In Dam-defective strains drg (dam-replacing gene), flanked by pseudo-transposable small repeated elements (SREs), replaced dam. We demonstrate that drg encodes a restriction endonuclease (NmeBII) that cleaves 5'-GmeATC-3'. drg is also present in 50% of Neisseria lactamica strains, but in most of them it is inactive because of the absence of an SRE-providing promoter. This is associated with the presence of GATmeC, suggesting an alternative restriction-modification system (RM) specific for 5'-GATC-3', similar to Sau3AI-RM of Staphylococcus aureus 3A, Lactococcus lactis KR2 and Listeria monocytogenes.

Rab-interacting lysosomal protein (RILP): the Rab7 effector required for transport to lysosomes.

Rab7 is a small GTPase that controls transport to endocytic degradative compartments. Here we report the identification of a novel 45 kDa protein that specifically binds Rab7GTP at its C-terminus. This protein contains a domain comprising two coiled-coil regions typical of myosin-like proteins and is found mainly in the cytosol. We named it RILP (Rab-interacting lysosomal protein) since it can be recruited efficiently on late endosomal and lysosomal membranes by Rab7GTP. RILP-C33 (a truncated form of the protein lacking the N-terminal half) strongly inhibits epidermal growth factor and low-density lipoprotein degradation, and causes dispersion of lysosomes similarly to Rab7 dominant-negative mutants. More importantly, expression of RILP reverses/prevents the effects of Rab7 dominant-negative mutants. All these data are consistent with a model in which RILP represents a downstream effector for Rab7 and both proteins act together in the regulation of late endocytic traffic.

A new vector for insertion of any DNA fragment into the chromosome of transformable Neisseriae.

A useful method for inserting any DNA fragment into the chromosome of Neisseriae has been developed. The method relies on recombination-proficient vector plasmid pNLE1, a pUC19 derivative containing (1) genes conferring resistance to ampicillin and erythromycin, as selectable markers; (2) a chromosomal region necessary for its integration into the Neisseria chromosome; (3) a specific uptake sequence which is required for natural transformation; (4) a promoter capable of functioning in Neisseria; and (5) several unique restriction sites useful for cloning. pNLE1 integrates into the leuS region of the neisserial chromosome at high frequencies by transformation-mediated recombination. The usefulness of this vector has been demonstrated by cloning the tetracycline-resistance gene (tet) and subsequently inserting the tet gene into the meningococcal chromosome.

Galectin-1 gene expression and methylation state in human T leukemia cell lines.

Galectin-1 has been demonstrated to be a mediator of T-cell apoptosis acting on activated T-cells and, in a selective manner, on different T leukemia cell lines. Here we show that the sensitivity to galectin-1 is associated with repression of the endogenous galectin-1 gene whereas non-sensitive cells express high levels of galectin-1. Repression of galectin-1 gene in sensitive cells is associated with hyper-methylation of the promoter region. Transient treatment of non-expressing cells with the demethylating agent 5-azacytidine led to irreversible demethylation and subsequent reactivation of galectin-1 gene.

Relaxation of insulin-like growth factor 2 imprinting and discordant methylation at KvDMR1 in two first cousins affected by Beckwith-Wiedemann and Klippel-Trenaunay-Weber syndromes.

Beckwith-Wiedeman syndrome (BWS) and Klippel-Trenaunay-Weber syndrome (KTWS) are different human disorders characterized, among other features, by tissue overgrowth. Deregulation of one or more imprinted genes located at chromosome 11p15.5, of which insulin-like growth factor 2 (IGF2) is the most likely candidate, is believed to cause BWS, whereas the etiology of KTWS is completely obscure. We report a case of BWS and a case of KTWS in a single family. The probands, sons of two sisters, showed relaxation of the maternal IGF2 imprinting, although they inherited different 11p15.5 alleles from their mothers and did not show any chromosome rearrangement. The patient with BWS also displayed hypomethylation at KvDMR1, a maternally methylated CpG island within an intron of the KvLQT1 gene. The unaffected brother of the BWS proband shared the same maternal and paternal 11p15.5 haplotype with his brother, but the KvDMR1 locus was normally methylated. Methylation of the H19 gene was normal in both the BWS and KTWS probands. Linkage between the insulin-like growth factor 2 receptor (IGF2R) gene and the tissue overgrowth was also excluded. These results raise the possibility that a defective modifier or regulatory gene unlinked to 11p15.5 caused a spectrum of epigenetic alterations in the germ line or early development of both cousins, ranging from the relaxation of IGF2 imprinting in the KTWS proband to disruption of both the imprinted expression of IGF2 and the imprinted methylation of KvDMR1 in the BWS proband. Analysis of these data also indicates that loss of IGF2 imprinting is not necessarily linked to alteration of methylation at the KvDMR1 or H19 loci and supports the notion that IGF2 overexpression is involved in the etiology of the tissue hypertrophy observed in different overgrowth disorders, including KTWS.

A novel member of the BTB/POZ family, PATZ, associates with the RNF4 RING finger protein and acts as a transcriptional repressor.

We have identified a novel human gene encoding a 59-kDa POZ-AT hook-zinc finger protein (PATZ) that interacts with RNF4, a mediator of androgen receptor activity, and acts as a transcriptional repressor. PATZ cDNA was isolated through a two-hybrid interaction screening using the RING finger protein RNF4 as a bait. In vitro and in vivo interaction between RNF4 and PATZ was demonstrated by protein-protein affinity chromatography and coimmunoprecipitation experiments. Such interaction occurred through a small region of PATZ containing an AT-hook DNA binding domain. Immunofluorescence staining and confocal microscopy showed that PATZ localizes in distinct punctate nuclear regions and colocalizes with RNF4. Functional analysis was performed by cotransfection assays: PATZ acted as a transcriptional repressor, whereas its partner RNF4 behaved as a transcriptional activator. When both proteins were overexpressed a strong repression of the basal transcription was observed, indicating that the association of PATZ with RNF4 switches activation to repression. In addition, RNF4 was also found to associate with HMGI(Y), a chromatin-modeling factor containing AT-hook domains.

The H19 endodermal enhancer is required for Igf2 activation and tumor formation in experimental liver carcinogenesis.

The expression of the linked but reciprocally imprinted Igf2 and H19 genes is activated in adult liver in the course of tumor development. By in situ hybridization analysis we have shown that both the Igf2 and H19 RNAs are expressed in the majority of the neoplastic nodules, and that hepatocellular carcinomas are developed in an experimental model of liver carcinogenesis. H19 is also highly activated in smaller and less distinct hyperplastic regions. The few neoplastic areas showing Igf2 but no H19 RNA display loss of the maternally inherited allele at the Igf2/H19 locus. These data are compatible with the existence of a common activation mechanism of these two genes during liver carcinogenesis and with a stronger H19 induction in the pre-neoplastic lesions. By using mice carrying a deletion of the H19 endodermal enhancer, we show that this regulatory element is necessary for the activation of the Igf2 and H19 genes upon induction of liver carcinogenesis. Furthermore, multiple sites of the H19 endodermal enhancer region become hypersensitive to DNase I when the carcinogenesis process is induced. Lastly, liver tumors developed in mice paternally inheriting the H19 enhancer deletion are found to have marked growth delays, increased frequency of apoptotic nuclei, and lack of Igf2 mRNA expression, thus indicating that this regulatory element plays a major role in the progression of liver carcinogenesis, since it is required for the activation of the anti-apoptotic Igf2 gene.

Role of histone acetylation and DNA methylation in the maintenance of the imprinted expression of the H19 and Igf2 genes.

H19 and Igf2 are linked and reciprocally imprinted genes. We demonstrate that the histones associated with the paternally inherited and unexpressed H19 allele are less acetylated than those associated with the maternal expressed allele. Cell growth in the presence of inhibitors of either histone deacetylase or DNA methylation activated the silent Igf2 allele, whereas derepression of the silent H19 allele required combined inhibition of DNA methylation and histone deacetylation. Our results indicate that histone acetylation as well as DNA methylation contribute to the somatic maintenance of H19 and Igf2 imprinting and that silencing of the imprinted alleles of these two genes is maintained via distinct mechanisms.

Control of galectin gene expression.

In this review we summarize the available information on the expression of mammalian galectins in normal and transformed cells. From all these studies it is apparent that each cell might express most of galectins; yet, during development or in various differentiation stages or under different physiological or pathological conditions, one or more galectins are preferentially expressed in each cell type. This implies a fine control of gene expression and suggests that such control should be coordinated. Nevertheless, to date very few studies have been performed on the mechanisms responsible for the regulation of galectin genes. We review the current knowledge on galectin promoter function. We believe that this area of galectin research will expand rapidly in the near future.

The small GTPases Rab5a, Rab5b and Rab5c are differentially phosphorylated in vitro.

Rab GTPases play a fundamental role in the regulation of membrane traffic. Three different Rab5 isoforms have been reported but no differences in their function in endocytosis have been discovered. As the Rab5 isoforms show a conserved consensus site for Ser/Thr phosphorylation, we investigated whether this site was phosphorylated. Here, we report that the three Rab5 proteins are differentially recognized by different kinases. Rab5a is efficiently phosphorylated by extracellular-regulated kinase 1 but not by extracellular-regulated kinase 2, while cdc2 kinase preferentially phosphorylates Ser-123 of Rab5b. These findings strongly suggest that phosphorylation could be important to differentially regulate the function of the Rab5 isoforms.

Intracistronic transcription termination in polysialyltransferase gene (siaD ) affects phase variation in Neisseria meningitidis.

Expression of serogroup B meningococcal capsular polysaccharide is subject to frequent phase variation. A reversible +1/-1 frameshift mutation within a poly(dC) repeat altering the reading frame of the polysialyltransferase gene (siaD ), thereby causing premature arrest of translation, is responsible for loss of capsule expression. After analysis of transcription of the siaD gene from an encapsulated strain and from two unencapsulated derivatives, we have found that the siaD mRNA in the unencapsulated strains is reduced in size as a result of premature transcription termination at a cryptic Rho-dependent site within the proximal region of the siaD cistron. Termination is sensitive to bicyclomycin, a natural inhibitor of Rho activity. Bicyclomycin decreased the rates of capsule re-expression (off-on) without affecting the rates of loss of capsule expression (on-off). This finding suggested the existence of a novel mechanism linking transcription elongation termination and mutation frequency. A genetic system was therefore developed to measure phase variation of siaD-ermC' gene fusions in wild type and Rho-defective Escherichia coli strains. These studies demonstrated that in the Rho-defective E. coli strain readthrough transcription of the mutated siaD gene caused a fourfold lower off-on phase variation rate than in the congenic Rho+ strain. Analysis of phase variation of siaD-ermC' gene fusions in a DNA mismatch-defective E. coli strain suggests that the effect of transcription on mutation rates required a functional mismatch repair system.

Hypermutation in pathogenic bacteria: frequent phase variation in meningococci is a phenotypic trait of a specialized mutator biotype.

Expression of serogroup B meningococcal capsular polysaccharide undergoes frequent phase variation involving reversible frameshift mutations within a homopolymeric repeat in the siaD gene. A high rate of phase variation is the consequence of a biochemical defect in methyl-directed mismatch repair. The mutator phenotype is associated to the absence of DNA adenine methyltransferase (Dam) activity in all pathogenic isolates and in 50% of commensal strains. Analysis of the meningococcal dam gene region revealed that in all Dam- strains a gene encoding a putative restriction endonuclease (drg) that cleaves only the methylated DNA sequence 5'-GmeATC-3' replaced the dam gene. Insertional inactivation of the dam and/or drg genes indicated that high rates of phase variation and hypermutator phenotype are caused by absence of a functional dam gene.

Interaction cloning and characterization of the cDNA encoding the human prenylated rab acceptor (PRA1).

Rab proteins are small GTPases involved in the regulation of intracellular membrane traffic in mammalian cells. In order to find Rab-interacting proteins we performed a two-hybrid screening using a human brain cDNA library. Here we report the isolation of a full-length human cDNA clone coding for a protein of 185 amino acids. This protein interacts strongly with the Rab4b, Rab5a, and Rab5c proteins and weakly with Rab4a, Rab6, Rab7, Rab17, and Rab22 in the two-hybrid assay. Comparison with the Data Bank revealed that this clone represents the human homolog of the previously isolated rat Prenylated Rab Acceptor (rPRA1). Analysis of mRNA expression shows a single abundant mRNA of about 0.8 kb ubiquitously expressed. Western blot analysis of the overexpressed protein shows a band of the expected size equally distributed between cytosol and membranes.

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.

Helicobacter pylori up-regulates cyclooxygenase-2 mRNA expression and prostaglandin E2 synthesis in MKN 28 gastric mucosal cells in vitro.

Helicobacter pylori has been suggested to play a role in the development of gastric carcinoma in humans. Also, mounting evidence indicates that cyclooxygenase-2 overexpression is associated with gastrointestinal carcinogenesis. We studied the effect of H. pylori on the expression and activity of cyclooxygenase-1 and cyclooxygenase-2 in MKN 28 gastric mucosal cells. H. pylori did not affect cyclooxygenase-1 expression, whereas cyclooxygenase-2 mRNA levels increased by 5-fold at 24 h after incubation of MKN 28 cells with broth culture filtrates or bacterial suspensions from wild-type H. pylori strain. Also, H. pylori caused a 3-fold increase in the release of prostaglandin E2, the main product of cyclooxygenase activity. This effect was specifically related to H. pylori because it was not observed with Escherichia coli and was independent of VacA, CagA, or ammonia. H. pylori isogenic mutants specifically lacking picA or picB, which are responsible for cytokine production by gastric cells, were less effective in the up-regulation of cyclooxygenase-2 mRNA expression and in the stimulation of prostaglandin E2 release compared with the parental wild-type strain. This study suggests that development of gastric carcinoma associated with H. pylori infection may depend on the activation of cyclooxygenase-2-related events.

Control of Rab5 and Rab7 expression by the isoprenoid pathway.

Rab proteins are small molecular mass GTP-ases involved in the regulation of vescicular transport. The ability of rab proteins to carry out their role in intracellular membrane traffic requires the post-translational attachment to their C-terminus of a geranylgeranyl group, an isoprenoid lipid moiety derived from mevalonate. Here we report that depletion of intracellular mevalonate by lovastatin in FRTL-5 thyroid cells specifically resulted in a four-fold increase of Rab5 and Rab7 protein levels. This increase was reversed within 4 h upon addition of mevalonate. Similarly lovastatin also induced, at same extent, mRNA levels. Lovastatin effect was not common to other prenylated proteins. Moreover incubation with cycloheximide abolished the observed increase in lovastatin treated cells, suggesting that the effect is mediated by newly synthesized protein. These findings demonstrate that Rab5 and Rab7 expression are regulated by the isoprenoid pathway.