The neuronal repellent SLIT2 is a target for repression by EZH2 in prostate cancer.

The neuronal repellent SLIT2 is repressed in a number of cancer types primarily through promoter hypermethylation. SLIT2, however, has not been studied in prostate cancer. Through genome-wide location analysis we identified SLIT2 as a target of polycomb group (PcG) protein EZH2. The EZH2-containing polycomb repressive complexes bound to the SLIT2 promoter inhibiting its expression. SLIT2 was downregulated in a majority of metastatic prostate tumors, showing a negative correlation with EZH2. This repressed expression could be restored by methylation inhibitors or EZH2-suppressing compounds. In addition, a low level of SLIT2 expression was associated with aggressive prostate, breast and lung cancers. Functional assays showed that SLIT2 inhibited prostate cancer cell proliferation and invasion. Thus, this study showed for the first time the epigenetic silencing of SLIT2 in prostate tumors, and supported SLIT2 as a potential biomarker for aggressive solid tumors. Importantly, PcG-mediated repression may serve as a precursor for the silencing of SLIT2 by DNA methylation in cancer.

APE/Ref-1 is controlled by both redox and cAMP-dependent mechanisms in rat thyroid cells.

APE/Ref-1 is a multifunctional protein possessing both redox and DNA repair functions. Through its redox activity, APE/Ref-1 controls the DNA-binding function of several transcriptional regulators (AP1, NF-kappaB, p53, Pax proteins). We have previously shown that APE/Ref-1 upregulates the transcriptional activity of the thyroid-specific transcription factor Pax8. In thyroid cells, APE/Ref-1 can be detected both in the nuclear and cytoplasmatic compartments. In this study regulatory mechanisms acting on APE/Ref-1 were revealed using the FRTL-5 cell line. TSH induces both cytoplasm-to-nucleus translocation and neosynthesis of APE/Ref-1 protein. Interestingly, only neosynthesis is dependent on cAMP signalling. In contrast, the cytoplasm-to-nucleus translocation is dependent on redox-mediated mechanisms. Based upon the data shown in this study and in others, a bimodal control of APE/Ref-1 by TSH can be delineated.

Missense mutations of human homeoboxes: A review.

The homeodomain (encoded by the homeobox) is the DNA-binding domain of a large variety of transcriptional regulators involved in controlling cell fate decisions and development. Mutations of homeobox-containing genes cause several diseases in humans. A variety of missense mutations giving rise to human diseases have been described. These mutations are an excellent model to better understand homeodomain molecular functions. To this end, homeobox missense mutations giving rise to human diseases are reviewed. Seventy-four independent homeobox mutations have been observed in 17 different genes. In the same genes, 30 missense mutations outside the homeobox have been observed, indicating that the homeodomain is more easily affected by single amino acids changes than the rest of the protein. Most missense mutations have dominant effects. Several data indicate that dominance is mostly due to haploinsufficiency. Among proteins having the homeodomain as the only DNA-binding domain, three "hot spot" regions can be delineated: 1) at codon encoding for Arg5; 2) at codon encoding for Arg31; and 3) at codons encoding for amino acids of recognition helix. In the latter, mutations at codons encoding for Arg residues at positions 52 and 53 are prevalent. In the recognition helix, Arg residues at positions 52 and 53 establish contacts with phosphates in the DNA backbone. Missense mutations of amino acids that contribute to sequence discrimination (such as those at positions 50 and 54) are present only in a minority of cases. Similar data have been obtained when missense mutations of proteins possessing an additional DNA-binding domain have been analyzed. The only exception is observed in the POU1F1 (PIT1) homeodomain, in which Arg58 is a "hot spot" for mutations, but is not involved in DNA recognition.

Nestin is a neuroepithelial target gene of thyroid transcription factor-1, a homeoprotein required for forebrain organogenesis.

Thyroid transcription factor-1 (TTF-1, also known as NKX2.1 and T/EBP), a transcription factor belonging to the NKX-2 family of homeodomain-containing genes, plays an essential role in the organogenesis of the thyroid gland, lung, and ventral forebrain. Nestin is an intermediate filament protein strongly expressed in multipotential neuroepithelial stem cells and rapidly down-regulated during postnatal life. Here we show that stable fibroblastic clones expressing TTF-1 acquire a phenotype reminiscent of neuroepithelial cells in culture and up-regulate the endogenous nestin gene. TTF-1 transactivates in HeLa and NIH3T3 cells a reporter gene driven by a central nervous system-specific enhancer element from the second intron of the rat nestin gene, where it recognizes a DNA-binding site (NestBS) whose sequence resembles a nuclear hormone/cAMP-responsive element very different from canonical TTF-1 binding sites. Nuclear extracts from the head of mouse embryos form a retarded complex with NestBS of the same mobility of the extracts obtained from TTF1-expressing clones, which is either abolished or supershifted in the presence of two different antibodies recognizing the TTF-1 protein. Thus, the neuroepithelial marker nestin is a direct central nervous system-specific target gene of TTF-1, leading to the hypothesis that it might be the effector through which TTF-1 plays its role in the organogenesis of the forebrain.

Correlations between individual clinical manifestations and CTG repeat amplification in myotonic dystrophy.

Myotonic dystrophy (DM) is a multisystemic disease caused by the expansion of a CTG repeat, located in the 3'-untranslated region of the DMPK gene. The number of CTG repeats broadly correlates with the overall severity of the disease. However, correlations between CTG repeat number and presence/absence or severity of individual clinical manifestations in the same patients are yet scarce. In this study the number of CTG repeats detected in blood cells of 24 DM subjects was correlated with the severity of single clinical manifestations. The presence/absence of muscular atrophy, respiratory insufficiency, cardiac abnormalities, diabetes, cataract, sleep disorders, sterility or hypogonadism is not related to the number of CTG repeats. Muscular atrophy and respiratory insufficiency are present with the highest frequency, occurring in 96 and 92% of the cases, respectively. A significant correlation was found with age of onset (r = -0.57, p<0.01), muscular disability (r = 0.46, p<0.05), intellective quotient (r = -0.58, p<0.01) and short-term memory (r= -0.59, p<0.01). Therefore, the CTG repeat number has a predictive value only in the case of some clinical manifestations, this suggesting that pathogenetic mechanisms of DM may differ depending on the tissue.

Thyroid-specific gene expression is differentially influenced by intracellular glutathione level in FRTL-5 cells.

Alteration of the redox potential has been proposed as a mechanism influencing gene expression. Reduced glutathione (GSH) is one of the cellular scavengers involved in the regulation of the redox potential. To test the role that GSH may play in thyroid cells, we cultured a differentiated rat thyroid cell strain (FRTL-5) in the presence of L-buthionine-(S,R)-sulfoximine (BSO). BSO affects GSH synthesis by irreversibly inhibiting gamma-glutamylcysteine synthetase (EC 6.3.2.2), a specific enzyme involved in GSH synthesis. BSO-treated FRTL-5 cells show a great decrease in the GSH level, whereas malondialdehyde increases in the cell culture medium as a sign of lipid peroxidation. In these conditions the activity of two thyroid-specific promoters, thyroglobulin (Tg) and thyroperoxidase (TPO), is strongly reduced in transient transfection experiments. As both Tg and TPO promoters depend upon the thyroid-specific transcription factors, thyroid-specific transcription factor-1 (TTF-1) and Pax-8 for full transcriptional activity, we tested whether reduction of GSH concentration impairs the activity of these transcription factors. After BSO treatment of FRTL-5 cells, both transcription factors fail to trans-activate the respective chimerical targets, C5 and B-cell specific activating protein promoters, containing, respectively, multimerized TTF-1- or Pax-8-binding sites only as well as the Tg and TPO natural promoters. Northern analysis revealed that endogenous Tg messenger RNA (mRNA) expression is also reduced by BSO treatment, whereas endogenous TPO expression is not modified. Furthermore, the Pax-8 mRNA steady state concentration does not change in BSO-treated cells, whereas TTF-1 mRNA slightly decreases. Immunoblotting analysis of FRTL-5 nuclear extracts does not show significant modification of the Pax-8 concentration in BSO-treated cells, whereas a decrease of 25% in TTF-1 protein is revealed. Furthermore, BSO treatment decreases the DNA-binding activity to the respective consensus sequence of both transcription factors. Finally, different mechanisms seem to act on TTF-1 and Pax-8 functional impairment in BSO-treated cells. Indeed, with a lowered GSH concentration, the overexpressed Pax-8 still activates transcription efficiently, whereas, on the contrary, the overexpressed TTF-1 does not recover its transactivation capability when the respective chimerical target sequences are used (C5 and BSAP). When the natural Tg and TPO promoter sequences are used, overexpression of Pax-8 parallels the effect on both promoters observed using the chimeric target sequences, whereas overexpression of TTF-1 increases TPO promoter transcriptional activity only.

In the TTF-1 homeodomain the contribution of several amino acids to DNA recognition depends on the bound sequence.

The thyroid transcription factor-1 homeodomain (TTF-1HD) shows a peculiar DNA binding specificity, preferentially recognizing sequences containing the 5'-CAAG-3' core motif. Most other homeodomains instead recognize sites containing the 5'-TAAT-3' core motif. Here, we show that TTF-1HD efficiently recognizes another sequence, called D1, devoid of the 5'-CAAG-3' core motif. Different experimental approaches indicate that TTF-1HD contacts the D1 sequence in a manner which is different to that used to interact with sequences containing the 5'-CAAG-3' core motif. The binding activities that mutants of TTF-1HD display with the D1 sequence or with the sequence containing the 5'-CAAG-3' core motif indicate that the role of several DNA-contacting amino acids is different. In particular, during recognition of the D1 sequence, backbone-interacting amino acids not relevant in binding to sequences containing the 5'-CAAG-3' core motif play an important role. In the TTF-1HD, therefore, the contribution of several amino acids to DNA recognition depends on the bound sequence. These data indicate that although a common bonding network exists in all of the HD/DNA complexes, peculiarities important for DNA recognition may occur in single cases.

A network of specific minor-groove contacts is a common characteristic of paired-domain-DNA interactions.

Pax proteins are a family of transcription factors conserved during evolution and able to bind specific DNA sequences through a domain called a "paired domain'. The DNA-binding specificity of the Pax-8 paired domain was investigated. Site-selection experiments indicate that Pax-8 binds to a consensus sequence similar to those bound by Pax-2 and Pax-5. When consensus sequences of various paired domains are observed in light of recent structural studies describing paired-domain-DNA interaction [Xu, Rould, Jun, Desplan and Pabo (1995) Cell 80, 639-650], it appears that base-pairs contacted in the minor groove are conserved, while most of the base-pairs contacted in the major groove are not. Therefore a network of specific minor groove contacts is a common characteristic of paired-domain-DNA interactions. The functional importance of such a network was successfully tested by analysing the effect of consensus-based mutations on the Pax-8 binding site of the thyroglobulin promoter.

TTF-1 gene expression in human lung tumours.

Tissue-specific transcription factors control cell determination and differentiation. TTF-1 is a tissue-specific transcription factor expressed in the thyroid and lung. We investigated the expression of TTF-1 in normal human lung, and in various histopathological types of lung cancers by immunohistochemistry. In normal lung, TTF-1 expression was restricted to bronchial and alveolar epithelial cells. TTF-1 expression was found in 7 of the 29 cases of non-small cell lung carcinomas. In these tumours, the expression of TTF-1 did not correlate with the histological degree of differentiation. Results obtained using RNase protection assay confirmed that TTF-1 was expressed only in a subset of non-small cell carcinomas. TTF-1, as expected, was not expressed in neoplasms having a neuroendocrine cell origin, such as carcinoids. Interestingly, TTF-1 was always expressed in small cell lung carcinomas. These findings indicate that: (i) small cell lung carcinomas could originate from the endothermal cell lineage and (ii) dedifferentiation processes that operate in these neoplasms do not affect molecular mechanisms necessary for TTF-1 gene expression.

Expression of thyroid transcription factor 1 gene can be regulated at the transcriptional and posttranscriptional levels.

The complete structure of the gene for thyroid transcription factor 1 (TTF-1), both in rats and humans, has been determined. The rat TTF-1 gene shows three transcriptional start sites and contains two introns, one of which is alternatively spliced. Nuclear run-on and transient transfection experiments indicate that TTF-1 gene expression can be controlled at different levels. Using thyroid and nonthyroid cell lines, it can be shown that transcriptional mechanisms are involved in controlling thyroid-specific expression of the TTF-1 gene. In contrast, in thyroid cells expressing an activated Ki-ras oncogene, the steady-state level of TTF-1 mRNA is greatly reduced, while transcription of the TTF-1 gene is only moderately affected, suggesting that the accumulation of TTF-1 mRNA can be regulated by a posttranscriptional, Ras-sensitive mechanism.

Definition of the DNA-binding specificity of TTF-1 homeodomain by chromatographic selection of binding sequences.

The homeodomain of the thyroid transcription factor-1 (TTF-1HD) shows a peculiar DNA-binding specificity, preferentially recognizing sequences having the 5'-CAAG-3' core motif. In order to detail the DNA-binding specificity of this protein, a TTF-1HD-Sepharose column chromatography was used. A sequential selection and amplification of sequences was performed. TTF-1HD binding activity for selected and unselected sequences was measured. The presence of the 5'-CAAG-3' core motif was necessary, but not sufficient, to obtain the maximal binding activity for TTF-1HD. However, several of the selected sequences do not contain the 5'-CAAG-3' core motif and are bound by TTF-1HD only 2-fold less with respect to sequences bound with the highest affinity. Therefore, these data indicate that TTF-1HD specifically recognizes a spectrum of sequences wider than previously determined.

The thyroid transcription factor-1 gene is a candidate target for regulation by Hox proteins.

Vertebrate Hox homeobox genes are transcription factors which regulate antero-posterior axial identity in embryogenesis, presumably through activation and/or repression of downstream target genes. Some of these targets were reported to code for molecules involved in cell-cell interactions, whereas no relationship has yet been demonstrated between Hox genes and other transcription factors involved in determining and/or maintaining tissue specificity. The thyroid transcription factor-1 (TTF-1) is a homeodomain-containing protein required for expression of thyroid-specific genes. A 862 bp 5' genomic fragment of the rat TTF-1 gene, conferring thyroid-specific expression to a reporter gene, was sufficient to mediate transactivation by the human HOXB3 gene in co-transfection assay in both NIH3T3 or HeLa cells. HOXB3 is expressed in early mammalian embryogenesis in the anterior neuroectoderm, branchial arches and their derivatives, including the area of the thyroid primordia and thyroid gland. Transcription of the TTF-1 promoter is induced only by HOXB3, while its paralogous gene HOXD3 or other Hox genes expressed more posteriorly (HOXA4, HOXD4, HOXC5, HOXC6, HOXC8 and Hoxd-8) have no effect. Transactivation by HOXB3 is mediated by two binding sites containing an ATTA core located at -100 and +30 from the transcription start site. DNase I footprinting experiments show that the two sites bind HOXB3 protein synthesized in both Escherichia coli and eukaryotic cells, as well as nuclear factor(s) present in protein extracts obtained from mouse embryonic tissues which express group 3 Hox genes and TTF-1. Some of the DNA-protein complexes formed by the embryonic extracts are indistinguishable from those generated by HOXB3.(ABSTRACT TRUNCATED AT 250 WORDS)

The tissue-specific expression of the thyroglobulin gene requires interaction between thyroid-specific and ubiquitous factors.

Thyroid-specific expression of the rat thyroglobulin gene is mediated by transcriptional control. Sufficient DNA sequence information to confer thyroid-specific expression to a heterologous gene is contained between positions -168 and +39. DNA-binding studies have demonstrated that this region interacts with two thyroid-specific factors (TTF-1 and TTF-2), and a ubiquitous factor (UFA). Here we have characterized three elements within the promoter, A, K, and C, which are important for promoter activity in thyroid cells. We have shown by mutational analysis that the interaction of TTF-1 with the A and C regions. UFA with the A region, and TTF-2 with the K region are required for full promoter activity. The complex interactions in the A region can be replaced by the substitution of the UFA/TTF-1-binding site with a high-affinity TTF-1 binding site. There is a correlation between the presence of TTF-1 and TTF-2 DNA-binding activities and the expression of thyroglobulin, which implies that the mechanism restricting thyroglobulin expression to thyroid cells is mediated through the control of the expression, or the activity, of TTF-1 and TTF-2.

A thyroid-specific nuclear protein essential for tissue-specific expression of the thyroglobulin promoter.

A rat thyroglobulin promoter fragment, capable of directing thyroid-specific transcription, binds at least three different factors, TTF-1, TTF-2 and UFA, which are all present in nuclear extracts of the differentiated rat thyroid cell line FRTL-5. TTF-1 and TTF-2 are FRTL-5 specific, as demonstrated by their absence in nuclear extracts prepared from cell lines that do not express any thyroid-differentiated function, while UFA is present in all cell lines tested. TTF-1 has been extensively purified. It binds to the rat thyroglobulin promoter at three different sites which share sequence homology. Mutations in two of the three sites decrease both binding of TTF-1 in vitro and promoter function in vivo. This suggests that the tissue-specific expression of the thyroglobulin genes is mediated, at least in part, by the presence of a transcription factor exclusively in thyroid cells.

Distribution of tissue-specific tightly bound non-histone proteins in the first level of repeating chromatin structures.

Non-histone proteins, tightly bound to DNA, have been extracted from whole chromatin and core particles prepared from pig liver or kidney. We have investigated by bidimensional slab gel electrophoresis the distribution of this protein class in the first level of repeating structure of chromatin. Our results reveal that non-histone proteins tightly bound to DNA are a heterogeneous protein class. Some of them, particularly in the core particles, appear to be essentially the same in both tissues, though having differences in their isoelectric point, which may be attributed to postsynthetic modifications. We have calculated that this protein class is associated to only 10% of nucleosomes, these nucleosomes having, on the average, one protein molecule for each core DNA. The tissue-specific proteins have high molecular mass (ranging from 135 kDa to 70 kDa in liver, over 135 kDa in kidney) and, in kidney, a more basic isoelectric point. These proteins are mainly located outside the core particles; they could be situated in the spacer regions and/or be involved in determining higher levels of chromatin organization.

Distribution of tightly bound non-histone proteins in chromatin fractions obtained by DNAase II digestion.

Digestion of pig liver chromatin with DNAse II afforded three different fractions which were characterized in terms of their DNA, RNA and tightly bound non-histone protein content, their DNA fragment size and their template activity. Two of these fractions are soluble after digestion with DNAase II and have been separated on the basis of their different solubility in MgCl2. A third fraction is not solubilized even after extensive digestion, although the size of its DNA is comparable to that of the enzyme solubilized fractions. The three fractions show qualitatively and quantitatively different distribution of tightly bound non-histone proteins, with specific protein components in each fraction; furthermore the non-solubilized fraction is greatly enriched in proteins tightly bound to DNA. From all the data obtained it can be suggested that the tightly bound proteins of the insoluble fraction may play, directly or indirectly, a role in maintaining an organized chromatin structure.

Governing equations of motion for the otolith organs and their response to a step change in velocity of the skull.

The otolith organs are the linear motion sensors of the inner ear. They comprise an overdamped second-order system and respond to gravity and skull acceleration. The governing equations of motion which describe the relative displacement of the mass with respect to the skull are developed. When these equations are non-dimensionalized they indicate that the elastic term is almost negligible with respect to the viscous and inertial terms. For a step change in skull velocity an analytic solution is given for the elastic term equal to zero and numeric solutions are given for small values of the elastic term.