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Relaxin is a peptide hormone that, in humans, is encoded by two genes referred to as H1 and H2, both located into chromosome 9p24.1. We have searched for polymorphisms in the 5'-flanking sequence of these genes. Both genes possess a CT repeat followed by a GT repeat. CT and GT repeats of the H2 gene are longer than those of the H1 gene. Moreover, CT and GT repeats of the H2 gene, but not those of the H1 gene, show length polymorphism. Protein-DNA interaction experiments suggest that difference between the H1 and H2 GT repeats may have arisen because the requirements of the transcriptional regulation of the two genes are different.

Pax proteins are transcriptional regulators that play important roles during embryogenesis. These proteins recognize specific DNA sequences via a conserved element: the paired domain (Prd domain). The low level of organized secondary structure, in the free state, is a general feature of Prd domains; however, these proteins undergo a dramatic gain in α-helical content upon interaction with DNA (‘induced fit’). Pax8 is expressed in the developing thyroid, kidney and several areas of the central nervous system. In humans, mutations of the Pax8 gene, which are mapped to the coding region of the Prd domain, give rise to congenital hypothyroidism. Here, we have investigated the molecular defects caused by a mutation in which leucine at position 62 is substituted for an arginine. Leu62 is conserved among Prd domains, and contributes towards the packing together of helices 1 and 3. The binding affinity of the Leu62Arg mutant for a specific DNA sequence (the C sequence of thyroglobulin promoter) is decreased 60-fold with respect to the wild-type Pax8 Prd domain. However, the affinities with which the wild-type and the mutant proteins bind to a non-specific DNA sequence are very similar. CD spectra demonstrate that, in the absence of DNA, both wild-type Pax8 and the Leu62Arg mutant possess a low α-helical content; however, in the Leu62Arg mutant, the gain in α-helical content upon interaction with DNA is greatly reduced with respect to the wild-type protein. Thus the molecular defect of the Leu62Arg mutant causes a reduced capability for induced fit upon DNA interaction.

Redox potential controls the DNA-binding activity of several transcription factors. In some cases, the regulation of DNA-binding activity by the redox state is mediated by the Ref-1 nuclear protein. In this study, we demonstrate that Ref-1 is able to induce "in vitro" the DNA-binding activity of the Pax-8 paired domain. In co-transfection experiments, Ref-1 increases the Pax-8 activating effect on thyroglobulin promoter. Moreover, immunoreactivity data suggest that, in nuclear extracts of thyroid cells, the levels of Ref-1 correlate with the amounts of reduced Pax-8. Therefore, the regulation of the Pax-8 DNA-binding activity by redox potential, that we have demonstrated occurring "in vitro", could represent a means to control "in vivo" the function of Pax proteins. Alignment of the Paired domains sequences present in the Protein Data Bank demonstrates a strong conservation of Cys residues, suggesting that the redox regulation of the Paired domain DNA-binding activity is widely conserved along phylogenesis.

The homeodomain (HD) is a 60 amino acid-long DNA-binding domain. A large fraction of HDs binds with high affinity sequences containing the 5'-TAAT-3' core motif. However, NK-2 class HDs recognizes sequences containing the 5'-CAAG-3' core motif. By using a cell transfection approach, here we show that modification of residues located in the N-terminal arm (at positions 6, 7 and 8) and in the recognition helix (at position 54) is enough to swap the "in vivo" binding specificity of TTF-1 HD (which is a member of the NK-2 class HD) from 5'-CAAG-3' to 5'-TAAT-3'-containing targets. The role of residue at position 54 is also supported by data obtained with the HD of the Drosophila engrailed protein. These data support the notion that DNA-binding specificity "in vivo" is dictated by few critical residues.

PAX8 is a transcription factor with a role in ontogenesis of the urinary tract. The aim of the present investigation was to investigate PAX8 expression in normal bladder and in non-invasive urothelial tumours. Nine cases of normal urothelial mucosa, 2 cases of papillary urothelial neoplasia of low malignant potential, 12 cases of low grade non-invasive papillary urothelial carcinoma and 16 cases of high grade non-invasive papillary urothelial carcinoma were investigated by immunohistochemistry. PAX8 mRNA expression was evaluated by RT-PCR in a different set of normal bladder mucosas and tumours. In addition, PAX8 expression was evaluated by RT-PCR in bladder from 2 human embryos and in several continuous cell lines derived from bladder tumours (5637, RT-112, TCC-SUP, HT 1376). In immunohistochemical studies, PAX8 was expressed in 28 out of 30 non-invasive urothelial tumours, but not in the normal adult bladders. In RT-PCR studies, PAX8 was expressed in 13 out of 13 bladder tumours but not in the 6 normal bladder mucosa. Contrary to that in adults, PAX8 was expressed in 2 cases of bladder mucosa from 16-week-old embryos. PAX8 was expressed in all the cell lines from bladder tumours. Both in the bladder tumours and cell lines PAX8 expression was highly heterogeneous in terms of the splicing isoforms. Treatment of cell lines with sodium butyrate (NaB) induced several changes of the splicing isoforms. Therefore, only subsets of molecular events that determine the PAX8 mRNA splicing heterogeneity in bladder tumours are sensitive to NaB treatment.

Background The homeobox gene HEX is expressed in several cell types during different phases of animal development. It encodes for a protein localized in both the nucleus and the cytoplasm. During early mouse development, HEX is expressed in the primitive endoderm of blastocyst. Later, HEX is expressed in developing thyroid, liver, lung, as well as in haematopoietic progenitors and endothelial cells. Absence of nuclear expression has been observed during neoplastic transformation of the thyroid follicular cells. Aim of the present study was to evaluate the localization and the function of the protein HEX in normal and tumoral breast tissues and in breast cancer cell lines. Methods HEX expression and nuclear localization were investigated by immunohistochemistry in normal and cancerous breast tissue, as well as in breast cancer cell lines. HEX mRNA levels were evaluated by real-time PCR. Effects of HEX expression on Sodium Iodide Symporter (NIS) gene promoter activity was investigated by HeLa cell transfection. Results In normal breast HEX was detected both in the nucleus and in the cytoplasm. In both ductal and lobular breast carcinomas, a great reduction of nuclear HEX was observed. In several cells from normal breast tissue as well as in MCF-7 and T47D cell line, HEX was observed in the nucleolus. MCF-7 treatment with all-trans retinoic acid enhanced HEX expression and induced a diffuse nuclear localization. Enhanced HEX expression and diffuse nuclear localization were also obtained when MCF-7 cells were treated with inhibitors of histone deacetylases such as sodium butyrate and trichostatin A. With respect to normal non-lactating breast, the amount of nuclear HEX was greatly increased in lactating tissue. Transfection experiments demonstrated that HEX is able to up-regulate the activity of NIS promoter. Conclusion Our data indicate that localization of HEX is regulated in epithelial breast cells. Since modification of localization occurs during lactation and tumorigenesis, we suggest that HEX may play a role in differentiation of the epithelial breast cell.

Pax6 controls eye, pancreas and brain morphogenesis. In humans, heterozygous PAX6 mutations cause aniridia and various other congenital eye abnormalities. Most frequent PAX6 missense mutations are located in the paired domain (PD), while very few missense mutations have been identified in the homeodomain (HD). In the present report, we describe a molecular analysis of the human PAX6 R242T missense mutation, which is located in the second helix of the HD. It was identified in a male child with partial aniridia in the left eye, presenting as a pseudo-coloboma. Gel-retardation assays revealed that the mutant HD binds DNA as well as the wild-type HD. In addition, the mutation does not modify the DNA-binding properties of the PD. Cell transfection assays indicated that the steady-state levels of the full length mutant protein are higher than those of the wild-type one. In cotransfection assays a PAX6 responsive promoter is activated to a higher extent by the mutant protein than by the wild-type protein. In vitro limited proteolysis assays indicated that the presence of the mutation reduces the sensitivity to trypsin digestion. Thus, we suggest that the R242T human phenotype could be due to abnormal increase of PAX6 protein, in keeping with the reported sensitivity of the eye phenotype to increased PAX6 dosage.

Inhibitors of histone deacetylases (HDACs) activate the sodium iodide symporter (NIS) expression in thyroid tumor cells. In this study, mechanisms accounting for these effects were investigated. Various human thyroid tumor cell lines (ARO, BCPAP, FRO, TPC-1) were treated with the HDAC inhibitors Na butyrate (NaB) and tricostatin A (TSA), and the effects on the expression of NIS and several thyroid-specific transcription factors together with the activity of NIS promoter were evaluated. TSA and NaB increased NIS mRNA levels in all cell lines. Among thyroid-specific transcription factors, only expression of PAX8 in ARO cells was increased. Down-regulation of thyroid-specific transcription factor-1 expression was observed in BCPAP and TPC-1 cell lines. Thyroid-specific transcription factor-2 mRNA was reduced in FRO, BCPAP, and TPC-1 cells. Histone acetylation had no significant effects on HEX expression. Altogether, these data indicatethattheincreaseofNISexpressionisnotmediatedby modification of expression of thyroid-specific transcription factors. Accordingly, in transfection experiments performed in the HeLa cell line (which does not express thyroid-specific transcriptionfactors),treatmentwithTSAandNaBincreased NIS promoter activity. Stimulation of NIS promoter activity was also obtained by overexpressing histone acetylating proteins pCAF and p300 in HeLa cells. Conversely, overexpression of the HDAC 1 enzyme inhibited basal activity of the NIS promoter. Effects of TSA and NaB on NIS expression were also evaluated in nonthyroid cell lines MCF-7, Hep-G2, and SAOS-2. In all cell lines TSA and NaB greatly increased NIS mRNA levels. We concluded that control of NIS expression by inhibitionofHDACappearsnottobemediatedbycell-specific mechanisms, suggesting it as a potential strategy to induce radioiodine sensitivity in different human tumors. (Endocrinology 146: 3967–3974, 2005)

Mutations of the RUNX2 gene result in dominantly inherited cleidocranial dysplasia (CCD). RUNX2 encodes for an osteoblast-specific transcription factor, which recognizes specific DNA sequences by the runt domain. DNA binding is stabilized by the interaction with the protein CBFbeta, which induces structural modifications of the runt domain. A novel 574GA RUNX2 missense mutation has been found in members of a family clinically diagnosed with CCD. This mutation causes the glycine at position 192 to change to arginine (G192R), in loop 9 of the runt domain. Unlike other residues of loop 9, G192 does not establish DNA contacts. Accordingly, the G192R mutant showed a 50% reduction in binding activity compared to the wild-type runt domain. However, the mutation completely abolished the activating properties of the protein on osteocalcin promoter. Moreover, the G192R mutant exerts a dominant-negative effect when overexpressed. Computer modeling indicated that the G192R mutation perturbs not only loop 9, but also other parts of the runt domain, suggesting impairment of the interaction with CBFbeta.

The homeobox-containing gene Hex is expressed in several cell types, including thyroid follicular cells, in which it regulates the transcription of tissue- specific genes. In this study the regulation of Hex promoter activity was investigated. Using co- transfection experiments, we demonstrated that the transcriptional activity of the Hex gene promoter in rat thyroid FRTL-5 cells is approximately 10-fold greater than that observed in HeLa and NIH 3T3 cell lines (which do not normally express the Hex gene). To identify the molecular mechanisms underlying these differences, we evaluated the effect of the thyroid- specific transcription factor TTF-1 on the Hex promoter activity. TTF-1 produced 3-4-fold increases in the Hex promoter activity. Gel- retardation assays and mutagenesis experiments revealed the presence of functionally relevant TTF-1 binding sites in the Hex promoter region. These in vitro data may also have functional relevance in vivo, since a positive correlation between TTF-1 and Hex mRNAs was demonstrated in human thyroid tissues by means of RT-PCR analysis. The TTF-1 effect, however, is not sufficient to explain the difference in Hex promoter activity between FRTL-5 and cells that do not express the Hex gene. For this reason, we tested whether Hex protein is able to activate the Hex promoter. Indeed, co-transfection experiments indicate that Hex protein is able to increase the activity of its own promoter in HeLa cells approximately 4-fold. TTF-1 and Hex effects are additive: when transfected together in HeLa cells, the Hex promoter activity is increased 6-7-fold. Thus, the contemporary presence of both TTF-1 and Hex could be sufficient to explain the higher transcriptional activity of the Hex promoter in thyroid cells with respect to cell lines that do not express the Hex gene. These findings demonstrate the existence of direct cross-regulation between thyroid-specific transcription factors.

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. Hum Mutat 18:361–374, 2001. © 2001 Wiley-Liss, Inc.

BACKGROUND Extragonadal yolk sac tumors of the gastrointestinal tract are extremely rare neoplasms. Their greater rarity compared with other extragonadal yolk sac tumors suggests that different pathogenetic mechanisms could be involved according to the site of origin. This report describes a case of a combined yolk sac tumor and adenocarcinoma that arose in a gastric stump in a man age 61 years 43 years after he underwent distal gastric resection and gastrojejunostomy (Billroth II operation) for a benign duodenal ulcer. The coexistence of an adenocarcinomatous component with the yolk sac component suggests that the two histologic patterns may represent distinct phenotypes arising from a common mucosal epithelial cell. METHODS Immunohistochemical and molecular techniques were used to define the mutation pattern of p53 in both components of the tumor. RESULTS Single-strand conformation polymorphism and sequencing analyses demonstrated the same pattern of p53 mutation in the adenocarcinomatous and yolk sac tumor components. CONCLUSIONS This finding suggests that the two tumors could have been derived from the same cellular clone and supports the hypothesis that the two components represented a heterogeneous differentiation of the same tumor. Cancer 1999;85:1910–6. © 1999 American Cancer Society.

Pax proteins are transcription factors that control differentiation of several cell types. In adult organisms Pax-8 is expressed in the follicular thyroid cell where it interacts with sequences of thyroglobulin and thyroperoxidase promoters. In this study, we provide evidence indicating that Pax-8 protein levels regulate thyroglobulin gene transcription. The most critical approach consisted in increasing Pax-8 protein levels by transfecting thyroid cells with a Pax-8 expression vector. In this situation the thyroglobulin promoter transcriptional activity was significantly increased with respect to untransfected cells. In contrast, the transfection of thyroid transcription factor-1 (TTF-1) expression vector causes a modest decrease of thyroglobulin promoter activity, rather than an increase. Northern blots of human papillary cancers reveal a significant correlation between Pax-8 and thyroglobulin mRNAs. Gel-retardation assays suggest that the mechanism by which the Pax-8 protein levels modulate thyroglobulin promoter activity may occur through competition with TTF-1 for a common binding site. Since we also demonstrate that Pax-8 expression is subjected to TSH control, our data strongly suggest that Pax-8 protein levels could represent an important determinant for the regulation of thyroid cells.

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of α-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into α-helices or β-strands can be induced. Therefore our data indicate that the inducibility of α-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development.

In this study, the effect of the reducing sugar glyceraldehyde 3-phosphate on protein/DNA interaction has been investigated. Treatment with glyceraldehyde 3-phosphate of oligonucleotides recognized by various transcription factors severely inhibits protein binding. The inhibitory effect is time and dose-dependent. Treatment with glyceraldehyde 3-phosphate of the homeodomain protein TTF-1 HD has also an inhibitory effect on the interaction with DNA, again in a time and dose-dependent manner. These "in vitro" effects could have "in vivo" counterparts and therefore contribute to molecular alterations observed either when intracellular protein are exposed to high doses of reducing sugars (i.e. in diabetes) or after a long time exposure (i.e. in Gzero-arrested cells during aging).

Ref-1 (called also APE) is a bifunctional protein playing a role in a large variety of cell functions. It is a major member of the DNA base excision repair system. Moreover, through reduction of cysteine residues, Ref-1 controls the activity of several transcription factors. It has been previously demonstrated that TSH up-regulates Ref-1 gene expression in thyroid cells. By using the rat FRTL-5 cell line, we demonstrate that TSH controls Ref-1 intracellular localization. Western blot experiments indicate that addition of TSH to the culture medium increases the Ref-1 cytoplasm-to-nucleus translocation. This phenomenon occurs at early times of TSH stimulation and is not dependent on protein neosynthesis. The Ref-1 cellular compartmentalization was also investigated in human thyroid tumors. A Ref-1 nuclear/cytoplasmic ratio difference between normal and cancerous thyroid tissues was observed. These results suggest that Ref-1 localization may have a critical role in the control of thyroid cell functions.

The Ref-1 (also called APE or HAP1) protein is a bifunctional enzyme impacting on a wide variety of important cellular functions. It acts as a major member of the DNA base excision repair pathway. Moreover, Ref-1 stimulates the DNA-binding activity of several transcription factors (TFs) through the reduction of highly reactive cysteine residues. Therefore, it represents a mechanism that regulates eukaryotic gene expression in a fast way. However, it has been demonstrated that external stimuli directly act on Ref-1 by increasing its expression levels, a time-consuming mechanism representing a paradox in terms of rapidity of TF regulation. In this paper we demonstrate that this is only an apparent paradox. Exposure of B lymphocytes to H(2)O(2)induced a rapid and sustained increase in Ref-1 protein levels in the nucleus as evaluated by both western blot analysis and by pulse-chase experiments. A time course, two color in situ immunocytochemistry indicated that the up-regulation of Ref-1 in the nucleus at30 min was primarily the consequence of translocation of its cytoplasmic form. This early nuclear accumulation is effective in modulating the DNA-binding activity of the B cell-specific activator protein BSAP/Pax-5. In fact, EMSA experiments demonstrate that a transient interaction with Ref-1 up-regulates the DNA-binding activity of BSAP/Pax-5. Moreover, in a co-transfection experiment, Ref-1 increased the BSAP/Pax-5 activating effect on an oligomerized BSAP/Pax-5 binding site of the CD19 promoter by 5- to 8-fold. Thus, Ref-1 mediates its effect by up-regulating the DNA-binding activity of BSAP/Pax-5, accounting for a new and fast outside/inside pathway of signaling in B cells.