Effects of 2-iodohexadecanal in the physiology of thyroid cells.

Iodide has direct effects on thyroid function. Several iodinated lipids are biosynthesized by the thyroid and they were postulated as intermediaries in the action of iodide. Among them, 2-iodohexadecanal (2-IHDA) has been identified and proposed to play a role in thyroid autoregulation. The aim of this study was to compare the effect of iodide and 2-IHDA on thyroid cell physiology. For this purpose, FRTL-5 thyroid cells were incubated with the two compounds during 24 or 48 h and several thyroid parameters were evaluated such as: iodide uptake, intracellular calcium and H2O2 levels. To further explore the molecular mechanism involved in 2-IHDA action, transcript and protein levels of genes involved in thyroid hormone biosynthesis, as well as the transcriptional expression of these genes were evaluated in the presence of iodide and 2-IHDA. The results obtained indicate that 2-IHDA reproduces the action of excess iodide on the "Wolff-Chaikoff" effect as well as on thyroid specific genes transcription supporting its role in thyroid autoregulation.

Functional inactivation of thyroid transcription factor-1 in PCCl3 thyroid cells.

Thyroid transcription factor-1 (TTF-1) is a key regulator of thyroid development and function. In order to identify the genes whose expression depends on TTF-1 transcriptional activity within the thyrocyte we analyzed the consequence of the functional inactivation of this factor in PCCl3 cells. The expression of a fusion protein composed of the DNA binding domain of TTF-1 and of the strong repressive domain of the engrailed protein resulted in a dramatic loss of epithelial cell morphology and in proliferation arrest. These changes were reversed when the inhibition of endogenous TTF-1 was relieved. No change was observed when a similar fusion protein containing point mutations abolishing DNA binding activity was produced in the cells. Besides the expected down-regulation of expression of the main genes linked to the differentiated thyroid function, we observed a decreased expression of the transcription factors Hhex, Pax 8 and TTF-2 and of E-cadherin. By contrast, both ThOX-1 and DUOXA-1 genes were up-regulated, as well as the ones encoding vimentin and several proteins involved in cell cycle arrest. Our data thus extend the known roles of TTF-1 in thyroid development and in the expression of differentiated function in the adult organ to the control of epithelial morphology and of cell division in mature thyrocytes.

Delimitation and functional characterization of the bidirectional THOX-DUOXA promoter regions in thyrocytes.

The THOX and DUOXA genes encode components of the oxidative machinery involved in thyroid hormone biosynthesis. Both of these genes are duplicated in mammalian genomes and are positioned in a head-to-head configuration, THOX1 facing DUOXA1 and THOX2 facing DUOXA2, respectively. The intergenic regions in both couples of genes exhibit dissimilar compositions, being highly GC-rich in the case of THOX1-DUOXA1 but not in the other case. In this study we localized precisely the transcription starts of all four genes using the RLM-RACE technique. It revealed that the distance between THOX1 and DUOXA1 transcription units is of about 70bp only, whereas THOX2 and DUOXA2 transcription starts are separated by 170bp. Analysis of these putative promoter regions revealed the presence of several potential binding sites for transcription factor Sp1 within the THOX1-DUOXA1 intergenic space, and of a TATA box and an Inr element in front of DUOXA2 and THOX2 genes, respectively. The putative promoter regions were inserted into a specifically designed vector harbouring two distinct reporter genes facing each other and their activity was investigated in transient transfection experiments in rat thyroid PCCl3 cells. Both regions exhibited bidirectional promoter activity in the assay. Gel shift experiments using extracts obtained from PCCl3 cells demonstrated the existence of at least one functional Sp1 binding site within the THOX1-DUOXA1 promoter. When Sp1 binding was abolished by mutation of the DNA sequence, a clear reduction in promoter activity in both THOX1 and DUOXA1 directions was observed in the functional assay. As these promoter sequences are well conserved in mammalian genomes, it appears very likely that the results we obtained here in the rat may be extended to the other species.

Identification of a short basic peptide motif able to drive copy-number dependent nuclear accumulation of a linked protein.

The repetitive [RTRG](6) peptide was fortuitously identified as a potent nuclear localization signal when linked to the green fluorescent reporter protein. Replacing the arginines by lysines, or the threonines by glycines, both resulted in a decreased nuclear targeting ability of the peptide within this context. By contrast, the sequence [RT](12) proved able to drive nuclear accumulation of the linked protein as efficiently as the starting peptide. Remarkably, [RTRG](n) peptides where n=2 to 6 showed a gradual, copy-number dependent, increase in their ability to target the green fluorescent protein to the cell nucleus. As a consequence, the nuclear to cytoplasmic concentration ratio of the linked protein within the cell could be adjusted to different values depending on the number of repeats used in the fusion. Our observation may open the way to the use of [RTRG](n) repeats of given lengths (n=2 to 6) for fixing the nuclear-cytoplasmic partition of shuttling protein domains in the course of their functional study.

Human Thyroid Oxidases genes promoter activity in thyrocytes does not appear to be functionally dependent on Thyroid Transcription Factor-1 or Pax8.

Thyroid Oxidases (ThOX/DUOX) genes encode proteins that are thought to play a crucial role in the biosynthesis of thyroid hormone by providing the oxidizing agent required to allow the organification of iodine. The expression of these genes is not restricted to the thyroid, but the corresponding mRNAs are found in the thyrocyte more abundantly than in several other cell types. It raises the question whether the same transcription factors, namely Thyroid Transcription Factor-1 (TTF-1) and Pax8, that control the expression of other genes involved in the differentiated thyroid function, also regulate ThOX/DUOX gene transcription in the thyrocyte. We set up a functional co-transfection assay in which fusion proteins composed of the DNA-binding domain of either TTF-1 or Pax8 fused to the repressive domain of the drosophila engrailed protein were used to competitively counteract the activity of endogenous TTF-1 or Pax8 factor in the differentiated thyroid cell line PCCl3. Contrary to the Thyroglobulin or Thyroid Peroxidase promoter, the known regulatory elements of the human ThOX/DUOX genes displayed no reduction in transcriptional activity when either TTF-1 or Pax8 competitor was produced in the cell, indicating that the presently characterized control elements of human ThOX/DUOX genes are not responsive to these thyroid-specific transcription factors.

Sequences downstream of the bHLH domain of the Xenopus hairy-related transcription factor-1 act as an extended dimerization domain that contributes to the selection of the partners.

XHRT1 is a member of the HRT/Hey protein subfamily that are known as Notch effectors. XHRT1 is expressed in the developing floor plate and encodes a basic helix-loop-helix (bHLH) transcription repressor. Here, we show that XHRT1 misexpression in the neural plate inhibits differentiation of neural precursor cells and thus may be important for floor plate cells to prevent them from adopting a neuronal fate. Deletion analysis indicated that inhibition of differentiation by XHRT1 requires the DNA-binding bHLH motif and either the Orange domain or the C-terminal region. XHRT1 could efficiently homodimerize and heterodimerize with hairy proteins. Among those hairy genes, Xhairy2b shows extensive overlap of expression with XHRT1 in floor plate precursors and may be a biologically relevant XHRT1 partner. Dimerization is mediated through both the bHLH and downstream sequences, the Orange domain being particularly important for the efficiency of the interaction. Using chimeric constructs between XHRT1 and the ESR9 bHLH-O protein that does not interact with Xhairy1 and Xhairy2b, we found that both the bHLH domain and downstream sequences of XHRT1 were required for heterodimerization with Xhairy2b, while only the XHRT1 sequences downstream of the Orange domain are required for the interaction with Xhairy1. Together, these results suggest that XHRT1 plays a role in floor plate cell development and highlight the importance of the Orange and downstream sequences in dimerization and in the selection of the bHLH partners.

Transcriptional repression by the bHLH-Orange factor XHRT1 does not involve the C-terminal YRPW motif.

Hairy-related transcription factors (HRTs) constitute a recently identified subfamily of basic-helix-loop-helix transcription factors containing an Orange domain (bHLH-O factors). As compared to the related HES proteins, HRTs exhibit distinct DNA-binding activities in vitro and the molecular mechanisms underlying their transcriptional activity remain poorly understood. We have identified here the sequence "ggCACGTGcc" as predominant binding site for Xenopus HRT1 (XHRT1). In transiently transfected 3T3 cells, XHRT1 represses the expression of a luciferase reporter gene under the control of multimerized XHRT1 binding sites. Deletion analysis indicated that repression by XHRT1 requires the presence of the DNA-binding bHLH motif and the Orange domain. However, the presence of the sequence motif YRPWGTEIGAF located at the very C-terminus of XHRT1 is dispensable. Accordingly, the groucho co-repressor, which is known to mediate transcriptional repression by HES factors through binding their C-terminal WRPW sequence, does not recognize the related YRPW motif present in the C-terminal part of XHRT1 significantly in vitro. As the C-terminus of HRTs is well conserved, our observation indicates that this part of HRTs, unlike the corresponding part of HES proteins, does not recruit the groucho co-repressor efficiently.

The control of thyroid-specific gene expression: what exactly have we learned as yet?

The identification of transcription factors TTF 1 and Pax 8 and the demonstration of their pivotal role in thyroid development and in the control of thyroid-specific gene expression, although representing remarkable openings in our understanding of cell-specific transcription in the thyroid, still leave a lot of open questions. Recent work investigating the development of thyroid-specific gene expression in transgenic mouse models, now reveal that some basic assumptions have to be reconsidered also. Altogether, currently available data indicate that the regulatory machinery undergoes significant changes during thyroid organogenesis and confirm the existence of still unknown factors whose roles appear at least as critical as the ones played by TTF 1 and Pax 8 in the control of specific gene expression.

TM4SF10 gene sequencing in XLMR patients identifies common polymorphisms but no disease-associated mutation.

BACKGROUND: The TM4SF10 gene encodes a putative four-transmembrane domains protein of unknown function termed Brain Cell Membrane Protein 1 (BCMP1), and is abundantly expressed in the brain. This gene is located on the short arm of human chromosome X at p21.1. The hypothesis that mutations in the TM4SF10 gene are associated with impaired brain function was investigated by sequencing the gene in individuals with hereditary X-linked mental retardation (XLMR). METHODS: The coding region (543 bp) of TM4SF10, including intronic junctions, and the long 3' untranslated region (3 233 bp), that has been conserved during evolution, were sequenced in 16 male XLMR patients from 14 unrelated families with definite, or suggestive, linkage to the TM4SF10 gene locus, and in 5 normal males. RESULTS: Five sequence changes were identified but none was found to be associated with the disease. Two of these changes correspond to previously known SNPs, while three other were novel SNPs in the TM4SF10 gene. CONCLUSION: We have investigated the majority of the known MRX families linked to the TM4SF10 gene region. In the absence of mutations detected, our study indicates that alterations of TM4SF10 are not a frequent cause of XLMR.

Characterization of a novel loss of function mutation of PAX8 in a familial case of congenital hypothyroidism with in-place, normal-sized thyroid.

Thyroid dysgenesis is the most common cause of congenital hypothyroidism, a relatively frequent disease affecting 1 in 3000-4000 newborns. Whereas most cases are sporadic, mutations in transcription factors implicated in thyroid development have been shown to cause a minority of cases transmitted as monogenic Mendelian diseases. PAX8 is one of these transcription factors, and so far, five mutations have been identified in its paired domain in patients with thyroid dysgenesis. We have identified a novel mutation of PAX8, in the heterozygous state, in a father and his two children both presenting with congenital hypothyroidism associated with an in-place thyroid of normal size at birth. In addition, one of the affected siblings displayed unilateral kidney agenesis. The mutation substitutes a highly conserved serine in position 54 of the DNA-binding domain of the protein (S54G mutation) by a glycine. Functional analyses of the mutant protein (PAX8-S54G) demonstrated that it is unable to bind a specific cis-element of the thyroperoxidase gene promoter in EMSAs and that it has almost completely lost the ability to act in synergy with Titf1 to transactivate transcription from the thyroglobulin promoter/enhancer. These results indicate that loss of function mutations of the PAX8 gene may cause congenital hypothyroidism in the absence of thyroid hypoplasia.

Identification of BOIP, a novel cDNA highly expressed during spermatogenesis that encodes a protein interacting with the orange domain of the hairy-related transcription factor HRT1/Hey1 in Xenopus and mouse.

Hairy-related transcription factor (HRT/Hey) genes encode a novel subfamily of basic helix-loop-helix (bHLH) transcription factors related to the Drosophila hairy and Enhancer-of-split (E(spl)) and the mammalian HES proteins that function as downstream mediators of Notch signaling. Using the yeast two-hybrid approach, a previously uncharacterized protein was identified in Xenopus that interacts with XHRT1 (originally referred to as bc8), one member of the HRT/Hey subclass. This protein is evolutionarily conserved in chordates. It binds to sequences adjacent to the bHLH domain of XHRT1 known as the Orange domain and has been named bc8 Orange interacting protein (BOIP). BOIP shows a rather uniform subcellular localization and is recruited to the nucleus upon binding to XHRT1. In Xenopus, XBOIP mRNA is detected by RNase protection analysis throughout embryogenesis. In the adult, the strongest expression is detected in testis. In the mouse, high levels of BOIP mRNA are also found in adult testis. No expression is detected in the embryo and in any of the other adult organs tested. In situ hybridization revealed that BOIP transcripts were detected almost exclusively in round spermatids and that this expression overlaps with that of Hey1 (HRT1), which is expressed throughout spermatogenesis. In view of the importance of the Orange domain for HRT/Hey function, the newly identified BOIP proteins may serve as regulators specifically of HRT1/Hey1 activity.

XHRT-1, a hairy and Enhancer of split related gene with expression in floor plate and hypochord during early Xenopus embryogenesis.

We have isolated a Xenopus homologue of the mammalian hairy and Enhancer of split related gene HRT1. XHRT1 expression in late gastrula and early neurula embryos is restricted to two stripes of cells in the medial neural plate and in dorsal endodermal cells. At later stages, XHRT1 is expressed in the floor plate, in hypochord cells and in the somitogenic and anterior presomitic mesoderm. By tailbud stage, XHRT1 is also highly expressed in the dorsal hindbrain, telencephalon and eye vesicles, olfactory placodes, pronephros, branchial arches and tail fin. We also show that XHRT1 expression in medial neural cells is induced by Notch signaling and that there are differences in the way XHRT1 and other H/E(spl) genes are regulated.