Using the recognition code to swap homeodomain target specificity in cell culture.

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.

A deletion 3' to the PAX6 gene in familial aniridia cases.

PURPOSE: PAX6 mutations cause aniridia as well as other various congenital eye abnormalities. Aniridia can be due to both point mutations and chromosomal deletions/rearrangements. Therefore, a complete search for PAX6 gene alterations in aniridia subjects requires a technically complex approach involving the comprehension of fluorescence in situ hybridization (FISH) analysis. In the present study, an Italian casistic of aniridia patients has been investigated and a quantitative polymerase chain reaction (PCR) assay to detect PAX6 gene deletions was set up. METHODS: Twenty-one aniridia patients were screened for point mutations (missense, nonsense, splicing-affecting, and short insertion/deletion) by using single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (dHPLC). To reveal deletions not detectable by SSCP or dHPLC, a quantitative PCR approach was set up for the PAX6 structural gene and for regions 5' and 3' to it at the level of WT1 and ELP4, respectively. RESULTS: Point mutations were found in 7 out of 21 patients. Three out of twenty-one patients showed deletions at the level of the PAX6 structural gene. In addition, two familial cases showed an undamaged PAX6 gene but a deletion in the region 3' to it at level of the ELP4 gene. In one of the families, the presence of the deletion has been confirmed by linkage analysis of polymorphic markers. CONCLUSIONS: In our casistic, a significant fraction of familial aniridia patients appears to be caused by a 3' deletion to PAX6, suggesting that evaluation of this alteration should be included in routine procedures of aniridia patients analysis. The quantitative PCR assay described here represents a simple approach to accomplish this task.

Molecular analysis of a human PAX6 homeobox mutant.

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.

HEX expression and localization in normal mammary gland and breast carcinoma.

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.

PAX8 expression in human bladder cancer.

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.

Thyroid-specific transcription factors control Hex promoter activity.

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.

Effects of histone acetylation on sodium iodide symporter promoter and expression of thyroid-specific transcription factors.

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 indicate that the increase of NIS expression is not mediated by modification of expression of thyroid-specific transcription factors. Accordingly, in transfection experiments performed in the HeLa cell line (which does not express thyroid-specific transcription factors), treatment with TSA and NaB increased 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 inhibition of HDAC appears not to be mediated by cell-specific mechanisms, suggesting it as a potential strategy to induce radioiodine sensitivity in different human tumors.

Functional analysis of a novel RUNX2 missense mutation found in a family with cleidocranial dysplasia.

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 574G > A 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.