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.

Effects of tissue factor induced by oxygen free radicals on coronary flow during reperfusion.

Tissue factor is a transmembrane protein that activates the extrinsic coagulation pathway by binding factor VII. Endothelial cells, being in contact with circulating blood, do not normally express tissue factor. Here we provide evidence that oxygen free radicals induce tissue factor messenger RNA transcription and expression of tissue factor procoagulant activity in endothelial cells in culture. Isolated, perfused rabbit hearts exposed to exogenous oxygen free radicals also showed a marked increase in tissue factor activity within the coronary circulation. Furthermore, in ex vivo and in vivo hearts subjected to ischemia and reperfusion, a condition associated with a production of oxygen free radicals in large amounts, a marked increase in tissue factor activity occurred. This phenomenon could be abolished by oxygen radical scavengers. This increase in tissue factor activity during postischemic reperfusion was accompanied by a significant decrease in coronary flow, suggesting that increase in tissue factor activity with the consequent activation of the coagulation cascade might impair coronary flow during reperfusion and possibly contribute to the occurrence of reperfusion injury.

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 rho = -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.

Dissociation between transformed and differentiated phenotype in rat thyroid epithelial cells after transformation with a temperature-sensitive mutant of the Kirsten murine sarcoma virus.

Differentiated rat thyroid epithelial cells, infected in vitro with a temperature-sensitive mutant of the Kirsten murine sarcoma virus, expressed at the permissive temperature (33 degrees C) some phenotypic properties typical of transformed cells, including morphological features, colony formation in agar, and induction of tumors in newborn animals. Specific functional markers of these differentiated cells, i.e., synthesis/secretion of thyroglobulin, synthesis of thyroglobulin mRNA and iodide uptake, were blocked during growth at 33 degrees C. Normal morphology, failure to grow in agar, and the requirement of hormones for optimal growth were all restored after shifting to the temperature nonpermissive for transformation (39 degrees C), though the typical differentiated functions remained blocked. Infection with a leukemia helper virus clone (Moloney or Kirsten murine leukemia virus) did not lead to the loss of the differentiated phenotype of rat epithelial thyroid cells, thus demonstrating that the loss of the differentiated phenotype is caused by the sarcoma virus component. These results indicate that the expression of some of the phenotypic properties of transformed differentiated rat thyroid epithelial cells is under the direct control of the p21 thermosensitive activity, whereas the block in the expression of two typical differentiation markers of thyroid epithelial cells is irreversible and probably controlled by different mechanisms.

p53 genes mutated in the DNA binding site or at a specific COOH-terminal site exert divergent effects on thyroid cell growth and differentiation.

Expression of mutated versions of the p53 gene deranged the differentiation program of thyroid cells and resulted in deregulated growth. Specifically, p53 mutants in several residues of the DNA-binding region induced thyrotropin (TSH) -independent growth and inhibition of the expression of thyroid-specific genes. The loss of the differentiated phenotype invariably correlated with the blockage of the expression of the genes coding for the thyroid transcriptional factors PAX-8 and TTF2. Conversely, thyroid cells transfected with a p53 gene mutated at codon 392, located outside the DNA-binding region, stimulated the expression of differentiation genes in the absence of the TSH, and induced TSH-independent growth. cAMP intracellular levels were higher in thyroid cells transfected with the p53 gene mutated at the 392 site than in the untransfected thyroid cells, but lower in the cells transfected with the other mutated p53 genes. Fra-1 and c-jun were induced by p53, resulting in increased AP-1 levels. The results of this study suggest that p53 exerts effects on cAMP transduction pathway in thyroid cells, which are exquisitely sensitive to cAMP.

The v-erbA oncogene selectively inhibits iodide uptake in rat thyroid cells.

v-erbA is the oncogenic form of the c-erbA proto-oncogene, which encodes the receptor for thyroid hormones. The expression of the v-erbA oncogene in thyroid differentiated cells, PC Cl 3, inhibits iodide uptake and thyrotropin-dependent growth, whereas it has no effect on the expression of the other thyroid specific markers, i.e. thyroglobulin, thyroperoxidase and thyrotropin receptor. The activity of transcription factor AP-1, evaluated by a specific DNA binding assay and by transcription of AP-induced promoter (TRE) is enhanced in PC v-erbA cells. v-erbA mutants in the DNA binding domain do not affect the iodide uptake of thyroid cells nor AP-1 activity. We suggest that this transcriptional activation mediates the selective effects of v-erbA on the expression of thyroid specific markers.

Novel mutations of thyrotropin receptor gene in thyroid hyperfunctioning adenomas. Rapid identification by fine needle aspiration biopsy.

Hyperfunctioning thyroid adenomas are clonal neoplasms with the intrinsic capacity of growing and differentiate independently of thyroid-stimulating hormone (TSH). We analysed the mRNA encoding thyrotropin receptor of 11 adenomas obtained by fine needle aspiration biopsy (FNAB) and found 7 mutants all located in three aminoacids clustered in the sixth transmembrane domain of the receptor. These mutations were somatic and specifically present in the tumour tissue. DNA sequence revealed that 80 to 90% of the mutations can be rapidly screened and identified by restriction enzyme analysis of the amplified cDNA obtained from the FNABs. The mutation Thr->Ile was introduced in the wild type receptor and expressed in mouse fibroblasts. These cells constitutively activate the transcription of a reporter gene under the control of cyclic AMP responsive element.

Effects of glucocorticoids on activation of c-jun N-terminal, extracellular signal-regulated, and p38 MAP kinases in human pulmonary endothelial cells.

Mitogen-activated protein kinases (MAPK) play a central role in signal transduction by regulating many nuclear transcription factors involved in inflammatory, immune, and proliferative responses. The aim of this study was to investigate, in human pulmonary endothelial cells, the effects of synthetic glucocorticosteroids on activation of c-jun N-terminal kinases, extracellular signal-regulated kinases, and p38 subgroups of the MAPK family. Human microvascular endothelial cells from lung were stimulated for 2 h with either H(2)O(2) (2 mM), IL-1beta (10 ng/mL), or tumour necrosis factor-alpha (10 ng/mL). Under these conditions, a remarkable increase in the phosphorylation pattern of c-jun N-terminal kinases, extracellular signal-regulated kinases 1/2, and p38 was detected. Pretreatment for 12 h with dexamethasone (100 nM) was able to prevent phosphorylation-dependent MAPK activation in stimulated cells, without substantially affecting the expression levels of these enzymes. Our results suggest that inhibition of MAPK signaling pathways in human pulmonary endothelial cells may significantly contribute, by interfering with activation of several different transcription factors, to the antiinflammatory and immunosuppressive effects of glucocorticosteroids.

Apolipoprotein A-I reverse transcriptase-polymerase chain reaction analysis for detection of hematogenous colon cancer dissemination.

Detection of systemic tumor dissemination in colon carcinoma patients might be important for selection of appropriate treatment modalities. It has been previously shown that Apolipoprotein A-I (Apo A-I) is expressed in human intestinal epithelial cells, and in some human colon carcinoma cell lines. We examined the expression of Apo A-I mRNA in 14 human primary colon carcinomas by Northern blot and/or reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. An Apo A-I specific transcript was found in up to 70% of the colon carcinomas. We developed an RT-PCR assay for Apo A-I transcripts, to identify circulating carcinoma cells in the peripheral blood of colon cancer patients. The Apo A-I RT-PCR assay was optimized using limiting dilution of an Apo A-I positive cancer cell line mixed with peripheral blood from healthy donor. In this system, up to 10 colon carcinoma cells were detected in 5 ml of peripheral blood. We examined Apo A-I mRNA expression in peripheral blood samples from 4 healthy donors, 20 colon carcinoma patients, and 11 individuals with tumor disease other than colon cancer. No Apo A-I mRNA was detected in the healthy donors and in the patients without colon cancer. Two out of 10 patients with metastatic colon carcinoma were positive by this assay, whereas Apo A-I mRNA was not found in any of the blood samples from the 10 radically resected colon carcinoma patients. These data suggest that Apo A-I RT-PCR assay is a highly specific and sensitive assay, although a low number of advanced colon carcinoma patients was found to be positive.

Screening of thyrotropin receptor mutations by fine-needle aspiration biopsy in autonomous functioning thyroid nodules in multinodular goiters.

Multinodular goiter (MNG) is characterized by nodules of different size and function. Areas of increased function may emerge, appearing as single, or more frequently, multiple autonomously functioning thyroid nodules (AFTN). The molecular mechanism for the autonomous growth and function of these nodules has been related to mutations in the thyrotropin receptor (TSHR) that constitutively activate the adenylyl cyclase. We searched for mutations in a limited area of the TSHR gene, covering the major mutational hotspot, in 38 AFTNs found in 37 patients with MNGs. We used reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction enzyme analysis of fine-needle aspiration biopsy (FNAB) samples to rapidly identify 4 of the more frequently occurring TSHR mutations: D619G, F631C, T632I and D633E. Mutations were identified in 5 nodules (1 D619G mutation and 4 T632I mutations). Subsequently, the entire transmembrane portion of the TSHR gene was sequenced in a random sample of 12 AFTN samples that were free of mutations by RT-PCR and restriction enzyme analysis. By direct sequencing we identified a new mutation, F666L, in the seventh transmembrane domain in a sample from 1 nodule. Analysis of FMA samples of AFTN is an effective approach to identify TSHR gene mutations because individual mutations may be associated with different growth and function in vitro, our approach might, allow correlation of a given mutation with the clinical behavior in vivo.

Menin stimulates homology-directed DNA repair.

Menin, the nuclear protein encoded by the Multiple Endocrine Neoplasia type 1 (MEN1) gene, acts as a tumor suppressor. It interacts with a large number of proteins involved in chromatin modification, transcription, cell cycle checkpoint and DNA repair, though its exact function is not clear. We report that in human cells menin stimulates homology-directed (HD) DNA repair induced by the rare endonuclease I-SceI and it accumulates with Chk1 at the site of the double strand break. In addition, menin and Chk1 interact in vivo. Deletion of the first 228 amino acids of menin impairs the interaction with Chk1 and the ability to stimulate HD repair, suggesting that the complex menin-Chk1 on the damaged chromatin facilitates homologous recombination.

[In vitro expression of the thyroid gland genome. Difference between chromatin and DNA]. / Expression in vitro du génome thyroïden. Différence entre chromatine et ADN.

In vitro, transcription by thyroid chromatin has been compared to that carried out by deproteinized DNA prepared from the same tissue. The chromatin-primed transcripts are represented by 4S and 10S RNAs, whereas the DNA-primed transcript is heterogenous and polydisperse. The sequences expressed in vitro have been analyzed by hydridization of 4S and 10S RNA to the total native thyroid DNA. Most of the sequences transcribed are represented by intermediate repetitive-associated an unique sequence. Furthermore, they are expressed only by chromatin and not by DNA. Thus, thyroid chromatin very likely holds in vitro the same fonctional restriction exhibited in vivo, whereas the transcriptional behaviour of thyroid DNA is totally aspecific.

Sequence organization of porcine DNA.

The sequence organization of porcine DNA isolated from thyroid has been analyzed by hydroxylapatite (HAP) chromatography. The reassociation of 0.4 kilobase (Kb) DNA fragments shows, besides the presence of 5% inverted repeat sequences (foldback DNA), that 45% of the genome is represented by high (10%) and intermediate (35%) repetitive components, whereas the remaining 50% is unique sequences. 30% of the unique sequences consists of 1,000 nucleotide fragments interspersed with repetitive elements 400 nucleotides in length. The remaining 20% is longer unique sequences (10,000 nucleotides) apparently not linked to repetitive elements.

Repetitive and non-repetitive sequences in the transcript in vitro of porcine thyroid chromatin.

Purified pig thyroid chromatin has been transcribed in vitro with Escherichia coli RNA polymerase. The transcript, analyzed by DNA-RNA hybridization, shows two major kinetic components: 40% of the transcript is copied by repetitive sequences present 100 times per haploid genome; another 25% anneals to DNA with a rate constant Kh 10-4 M - S-1, typical of single-copy sequences. The transcript annealed at cot = 40 M - S to fractions of 2000-nucleotide DNA, when banded in neutral CsCl gradient only hybridizes to the heavy side of the main band. At cot = 3000 M - S, another hybridizing fraction appears on the light side of the main band of the gradient. The reassociation properties of these fractions show that the heavy DNA fraction is reiterated about 100 times per haploid genome, whereas the light DNA appears as a unique sequence, associated to small repetitive elements. The transcript, analyzed by formamide/sucrose gradient, shows two peaks with sedimentation coefficients of 10 S and 4 S, respectively. The 10-S RNA, hybridized to native 2000-nucleotide-length DNA, has a Kh of 10-4 M - S-1 and a cot1/2 of 10(3) M - S, typical of single-copy sequences.

Ki-ras oncogene interferes with the expression of cyclic AMP-dependent promoters.

The expression of thyroglobulin and other thyroid-specific markers depends upon the activation of protein kinase A (PKA) by cyclic AMP. A rat thyroid cell line dedifferentiates when transformed with Ki-ras oncogene. The decrease in thyroglobulin gene expression parallels a reduction in the level of PKA nuclear catalytic subunit. We find that the activity of cAMP-responsive elements and thyroglobulin promoters is down-regulated in Ras-transformed cells. Transcription of a third cAMP-regulated gene, H-ferritin, is similarly reduced. cAMP-responsive element and H-ferritin expression were stimulated when intracellular cAMP levels were increased. Reactivation of the thyroglobulin promoter required depletion of PKC in addition to increased cAMP. We also find that v-Ras activation leads to a significant increase in membrane-bound PKC. These data support the idea that v-Ras via PKC inhibits the transmission of cAMP-PKA signals to the nucleus. We suggest that the thyroglobulin promoter is more sensitive than other cAMP-dependent promoters to reduced nuclear levels of PKA catalytic subunit.

Neoplastic transformation inactivates specific trans-acting factor(s) required for the expression of the thyroglobulin gene.

The expression of rat thyroglobulin gene is repressed following the transformation of rat thyroid cells with Kirsten murine sarcoma virus. The expression of a reporter gene fused to the thyroglobulin promoter is down-regulated in transformed thyroid cells in transient or in stable transfection assays. DNase and exonuclease III cleavage-protection analysis reveals that a promoter binding activity located at -60 base pairs from the transcription start site is substantially reduced in transformed thyroid cells. The repression in the transformed cells of the reporter gene joined to the thyroglobulin promoter can be reversed by fusion with normal differentiated thyroid cells. Fusion of transformed thyroid cells to liver cells does not reactivate the reporter under control of the thyroglobulin promoter.

Structural organization of the 3' half of the rat thyroglobulin gene.

We report the structural organization of an 80 Kb segment of rat DNA, which encodes for about 40% of Thyroglobulin mRNA at the 3' end. The codogenic information included in this segment is splitted in 17 exons of homogeneous size (about 200 bp). The seven exons at the extreme 3' end have been precisely defined by DNA sequence analysis. No clear sequence homology is found among the exons, even though their coding capacity is quite similar, from 55 to 63 aminoacids residues. We located 2 hormonogenic (T4 forming) sites on the extreme 3' end of the gene in different exons. The DNA sequence coding for these functional sites shows a 70% homology in a 50 nucleotides segment. In addition we found a remnant of this sequence in other exons of the gene. Two large introns have been found on the 3' end of the gene: one is 17 Kb and the other one is more than 30 Kb long. On the basis of these findings and of preliminary studies on the remaining 5' end of the gene, we can predict that the minimum length of the rat TGB gene will be 150 Kb, which makes this gene the largest so far identified eukaryotic gene. We propose in addition that the 3' end exons arose by duplication of a common ancestor.

The level of thyroglobulin mRNA is regulated by TSH both in vitro and in vivo.

We have studied the effect of thyrotropin on the intracellular concentration of thyroglobulin and of its mRNA both in a differentiated rat thyroid cell line and in the rat thyroid in vivo. Our data demonstrate that the absence of thyrotropin causes a two fold decrease of both thyroglobulin and of its mRNA suggesting that thyroglobulin synthesis in thyroid cells is 50% constitutive.

Differential expression of thyroglobulin gene in normal and transformed thyroid cells.

We have measured the synthesis of thyroglobulin in two differentiated cell lines, FRTL-5 and FRTL-424, and two transformed thyroid cell lines, FRA and 1-5G. The untransformed cells actively synthesized and exported thyroglobulin in the medium: however, the FRTL-5 cell line synthesized seven times less thyroglobulin than the FRTL-424 cell line, even though both cell types contained equal amounts of functional thyroglobulin mRNA. In contrast the transformed cells expressed extremely low levels of thyroglobulin mRNA, even though there was no detectable change in gene structure or copy number as determined by Southern blot analysis. On the basis of these data we conclude that (a) the different levels of thyroglobulin synthesis in the two untransformed cell lines are due to stable post-transcriptional alterations in the biosynthesis of thyroglobulin and (b) the transformation of thyroid cells results in a substantial reduction in thyroglobulin gene expression.

Thyroid transcription factor 1 phosphorylation is not required for protein kinase A-dependent transcription of the thyroglobulin promoter.

Thyroid transcription factor 1 (TTF1) is a nuclear homeodomain protein that binds to and activates the promoters of several thyroid-specific genes, including that of the thyroglobulin gene (pTg). These genes are also positively regulated by thyroid-stimulating hormone/cyclic AMP (cAMP)/protein kinase A (PKA) signaling. We asked whether PKA directly activates TTF1. We show that cAMP/PKA activates pTg and a synthetic target promoter carrying TTF1 binding site repeats in several cell types. Activation depends on TTF1. Phosphopeptide mapping indicates that TTF1 is constitutively phosphorylated at multiple sites, and that cAMP stimulated phosphorylation of one site, serine 337, in vivo. However, alanine substitution at this residue or at all sites of phosphorylation did not reduce PKA activation of pTg. Thus, PKA stimulates TTF1 transcriptional activity in an indirect manner, perhaps by recruiting to or removing from the target promoter another regulatory factor(s).