No abstract

Pax genes encode for transcription factors essential for tissue development in many species. Pax8, the only member of the family expressed in the thyroid tissue, is involved in the morphogenesis of the gland and in the transcriptional regulation of thyroid-specific genes. TTF-1, a homeodomain-containing factor, is also expressed in the thyroid tissue and has been demonstrated to play a role in thyroid-specific gene expression. Despite the presence of Pax8 and TTF-1 also in a few other tissues, the simultaneous expression of the two transcription factors occurs only in the thyroid, supporting the idea that Pax8 and TTF-1 might cooperate to influence thyroid-specific gene expression. In this report, we describe a physical and functional interaction between these two factors. The fusion protein GST-Pax8 is able to bind TTF-1 present in thyroid or in non-thyroid cell extracts, and by using bacterial purified TTF-1 we demonstrate that the interaction is direct. By co-immunoprecipitation, we also show that the interaction between the two proteins occurs in vivo in thyroid cells. Tissue-specific transcriptional regulation is often mediated by a complex of cis-acting elements. The vast majority of the promoters of genes expressed in a cell type-specific fashion contains a variety of recognition sequences for tissue-specific and ubiquitous transcription factors. It is well known that tissue-specific transcriptional regulation is mediated by a set of transcription factors whose combination is unique to the cell type. Thyroid follicular cells, the most abundant cell population of the thyroid gland, represent a useful model system to elucidate the mechanism operating in the establishment and maintenance of cell type-specific expression. Thyrocytes are responsible for thyroid hormone synthesis and are characterized by the expression of a specific set of genes such as thyroglobulin (Tg) 1 and thyroperoxidase (TPO), which are exclusively expressed in this cell type (1, 2), and by the expression of genes expressed only in a few tissues other than the thyroid, such as the thyrotropin-stimulating hormone receptor and the sodium/ iodide symporter.The Tg and TPO promoters have been extensively studied, and multiple factors have been shown to be required for their expression (3, 4). To date, three transcription factors that specifically bind to and regulate these promoters have been cloned (2). The three transcription factors are as follows: thyroid transcription factor-1 (TTF-1), thyroid transcription factor-2 (TTF-2), and Pax8. TTF-1 (also named NKx 2.1 and T/EBP) is a homeodomain-containing protein expressed in embryonic diencephalon, thyroid, and lung (5). TTF-2 is a forkhead domaincontaining protein expressed in pituitary and thyroid (6), and Pax8 is a member of the murine Pax family of paired domaincontaining genes that is expressed in kidney, in the developing excretory system, and in the thyroid (7). We have focused our studies on the molecular mechanisms of action of TTF-1 and Pax8. These two transcription factors are pre...

p63, a p53 family member, is essential for the development of various stratified epithelia and is one of the earliest markers of many ectodermal structures, including the epidermis, oral mucosa, apical ectodermal ridge, and mammary gland. Genetic regulatory mechanisms controlling p63 spatial expression during development have not yet been defined. Using a genomic approach, we identified an evolutionarily conserved cis-regulatory element, located 160 kb downstream of the first p63 exon, which functions as a keratinocyte-specific enhancer and is sufficient to recapitulate expression of the endogenous gene during mouse embryogenesis. Dissection of the p63 enhancer activity revealed a positive autoregulatory loop in which the p63 proteins directly bind to and are essential regulators of the enhancer. Accordingly, transactivating p63 isoforms induce endogenous p63 expression in cells that do not normally express this gene, whereas dominant negative isoforms suppress p63 expression in keratinocytes. In addition the transcription factor AP-2 also binds to the enhancer and cooperates with p63 to induce its activity. These results demonstrate that a long-range autoregulatory loop is involved in the regulation of p63 expression during embryonic development and in adult cells.

The PAX8 gene, mapped on 2q12-q14, encodes for a transcription factor involved in thyroid cell proliferation and differentiation. Five mutations in PAX8 have been so far described in both sporadic and rare familial forms of thyroid dysgenesis with proposed autosomal dominant inheritance, all associated with thyroid hypoplasia and/or dysfunction. Fifty-four subjects with congenital hypothyroidism detected during neonatal screening and associated with an ultrasound or scintiscan picture of thyroid dysgenesis were investigated for PAX8 mutations. The entire PAX8 coding region with exon-intron boundaries was amplified from genomic DNA, and a mutational screening was performed by denaturing HPLC followed by direct sequencing when denaturing HPLC elution abnormalities appeared. A new heterozygous deletion (c.989_992delACCC) in exon 7 causing a frameshift with premature stop codon after codon 277 was identified in a subject with thyroid hypoplasia. This mutation is the only one so far identified that lies outside the paired domain. The predicted mutant protein completely lacks the C-terminal region but contains the paired box, octapeptide, and homeodomain. It retains the ability to bind a paired-domain sequence in vitro but is transcriptionally inactive. These results provide evidence that the C-terminal region is essential for transcriptional activity. The new mutation has been inherited from the completely euthyroid mother. It was also present in a brother with slightly elevated TSH only. Thus, it is associated with thyroid dysgenesis in the proband and both euthyroidism and compensated hypothyroidism in her family. This suggests that other factors/genes may modulate phenotypic expression.