Dataset Information

Pendrin is a novel in vivo downstream target gene of the TTF-1/Nkx-2.1 homeodomain transcription factor in differentiated thyroid cells.

ABSTRACT: Thyroid transcription factor gene 1 (TTF-1) is a homeobox-containing gene involved in thyroid organogenesis. During early thyroid development, the homeobox gene Nkx-2.5 is expressed in thyroid precursor cells coincident with the appearance of TTF-1. The aim of this study was to investigate the molecular mechanisms underlying thyroid-specific gene expression. We show that the Nkx-2.5 C terminus interacts with the TTF-1 homeodomain and, moreover, that the expression of a dominant-negative Nkx-2.5 isoform (N188K) in thyroid cells reduces TTF-1-driven transcription by titrating TTF-1 away from its target DNA. This process reduced the expression of several thyroid-specific genes, including pendrin and thyroglobulin. Similarly, down-regulation of TTF-1 by RNA interference reduced the expression of both genes, whose promoters are sensitive to and directly associate with TTF-1 in the chromatin context. In conclusion, we demonstrate that pendrin and thyroglobulin are downstream targets in vivo of TTF-1, whose action is a prime factor in controlling thyroid differentiation in vivo.

SUBMITTER: Dentice M

PROVIDER: S-EPMC1280265 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Pendrin is a novel in vivo downstream target gene of the TTF-1/Nkx-2.1 homeodomain transcription factor in differentiated thyroid cells.

Dentice Monica M Luongo Cristina C Elefante Antonia A Ambrosio Raffaele R Salzano Salvatore S Zannini Mariastella M Nitsch Roberto R Di Lauro Roberto R Rossi Guido G Fenzi Gianfranco G Salvatore Domenico D

Molecular and cellular biology 20051101 22

Thyroid transcription factor gene 1 (TTF-1) is a homeobox-containing gene involved in thyroid organogenesis. During early thyroid development, the homeobox gene Nkx-2.5 is expressed in thyroid precursor cells coincident with the appearance of TTF-1. The aim of this study was to investigate the molecular mechanisms underlying thyroid-specific gene expression. We show that the Nkx-2.5 C terminus interacts with the TTF-1 homeodomain and, moreover, that the expression of a dominant-negative Nkx-2.5 ...[more]

PMID: 16260629

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Project description:BACKGROUND: The preoperative characterization of thyroid nodules is a challenge for the clinicians. Fine-needle aspiration (FNA) is the commonly used pre-operative technique for diagnosis of malignant thyroid tumor. However, many benign lesions, with indeterminate diagnosis following FNA, are referred to surgery. There is an urgent need to identify biomarkers that could be used with the FNA to distinguish benign thyroid nodules from malignant tumors. The purpose of the study is to examine the level of expression of the helicase-like transcription factor (HLTF) in relation to neoplastic progression of thyroid carcinomas. METHODS: The presence of HLTF was investigated using quantitative and semi-quantitative immunohistochemistry in a series of 149 thyroid lesion specimens. Our first clinical series was composed of 80 patients, including 20 patients presenting thyroid adenoma, 40 patients presenting thyroid papillary carcinoma, 12 patients presenting thyroid follicular carcinoma and 8 patients presenting anaplastic carcinoma. These specimens were assessed quantitatively using computer assisted microscopy. Our initial results were validated on a second clinical series composed of 40 benign thyroid lesions and 29 malignant thyroid lesions using a semi-quantitative approach. Finally, the HLTF protein expression was investigated by Western blotting in four thyroid cancer cell lines. RESULTS: The decrease of HLTF staining was statistically significant during thyroid tumor progression in terms of both the percentage of mean optical density (MOD), which corresponds to the mean staining intensity (Kruskall-Wallis: p < 0.0005), and the labelling index (LI), which corresponds to the percentage of immunopositive cells (Kruskall-Wallis: p < 10-6). Adenomas presented very pronounced nuclear HLTF immunostaining, whereas papillary carcinomas exhibited HLTF only in the cytoplasm. The number of HLTF positive nuclei was clearly higher in the adenomas group (30%) than in the papillary carcinomas group (5%).The 115-kDa full size HLTF protein was immunodetected in four studied thyroid cancer cell lines. Moreover, three truncated HLTF forms (95-kDa, 80-kDa and 70-kDa) were also found in these tumor cells. CONCLUSIONS: This study reveals an association between HLTF expression level and thyroid neoplastic progression. Nuclear HLTF immunostaining could be used with FNA in an attempt to better distinguish benign thyroid nodules from malignant tumors.

Project description:Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, a comprehensive study on the microRNAs and their targets has not been undertaken. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, we identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. We knocked down each of these 15 microRNAs by the piggyback method and found knockdown of three microRNAs, mir-7148, let-7b , and mir-223 in adult zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. We also found enhanced thrombosis using arterial laser thrombosis assay in a group of zebrafish larvae after mir-7148, let-7b , and mir-223 knockdowns. These results suggested mir-7148, let-7b , and mir-223 are repressors for thrombocyte production. We then explored miRWalk database for let-7b downstream targets and then selected those that are expressed in thrombocytes, and from this list based on their role in differentiation selected 14 genes, rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8 , and lbx1b that encode transcriptional regulators. The qRT-PCR analysis of expression levels of the above genes following let-7b knockdown showed changes in the expression of 13 targets. We then studied the effect of the 13 targets on thrombocyte production and identified 5 genes, irf5, tgif1, irf8, cebpa , and rorca that showed thrombocytosis and one gene, ikzf1 that showed thrombocytopenia. Furthermore, we tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223 . We also identified that tgif1, cebpa, ikzf1, irf5 , irf8 , and ikzf1 play a role in thrombopoiesis. Since the ikzf1 gene showed a differential expression profile in let-7b and mir-223 knockdowns but resulted in thrombocytopenia in ikzf1 knockdown in both adults and larvae we also studied an ikzf1 mutant and showed the mutant had thrombocytopenia. Taken together, these studies showed that thrombopoiesis is controlled by a network of transcription regulators that are regulated by multiple microRNAs in both positive and negative manner resulting in overall inhibition of thrombopoiesis.

Dataset Information

Pendrin is a novel in vivo downstream target gene of the TTF-1/Nkx-2.1 homeodomain transcription factor in differentiated thyroid cells.

Publications

Pendrin is a novel in vivo downstream target gene of the TTF-1/Nkx-2.1 homeodomain transcription factor in differentiated thyroid cells.

Similar Datasets

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