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Global Analysis of Thyroid Receptor Binding Sites Reveals Interaction with the Ddx54 and Thrsp Promoter in Juvenile Mouse Liver

ABSTRACT: Thyroid hormones (THs) play a critical role in development and throughout adulthood. THs act through the thyroid receptor (TR), which binds to the TH response element (TRE) to regulate the expression of target genes. Although TH action has been studied for decades, surprisingly few TREs have been well validated and characterized. In this study we used chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) to identify TR-binding sites in juvenile (postnatal day 15) mice liver. Microarray analysis revealed twelve TR-binding sites consistent between all analyzed samples. In silico analysis was carried out to search for moderately conserved classic TRE sequences within these novel binding regions, which led to the identification of six candidate TREs within three binding regions. Luciferase reporter assays confirmed the presence of a TRE in the promoter region of DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 (Ddx54) and thyroid hormone responsive SPOT14 (Thrsp). The TR/retinoid X receptor (RXR) heterodimer and RXR homodiner were shown to bind the promoter region of Ddx54 and drive gene expression in the presence of 9-cis-retinoic acid (9cRA). The promoter region of Thrsp was shown to allow binding of the TR/RXR heterodimer, and both T3 and 9cRA were able to significantly increase luciferase activity. The RXR homodimer was also able to bind the response element in the promoter region of Thrsp and increase luciferase activity. Overall, ChIP-chip analysis revealed a relatively limited number of TR-binding sites in juvenile mouse liver despite previous studies showing that numerous genes can be affected by TH disruption at that developmental stage, suggesting that TH action may also be mediated through other intermediates. Collectively the results provide an important step towards characterizing TR-binding sites and identifying the underlying drivers of TR-gene regulation.

ORGANISM(S): Mus musculus

PROVIDER: GSE52417 | GEO | 2013/11/16

SECONDARY ACCESSION(S): PRJNA228238

REPOSITORIES: GEO

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Project description:Thyroid hormones (THs) play a critical role in development and throughout adulthood. THs act through the thyroid receptor (TR), which binds to the TH response element (TRE) to regulate the expression of target genes. Although TH action has been studied for decades, surprisingly few TREs have been well validated and characterized. In this study we used chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) to identify TR-binding sites in juvenile (postnatal day 15) mice liver. Microarray analysis revealed twelve TR-binding sites consistent between all analyzed samples. In silico analysis was carried out to search for moderately conserved classic TRE sequences within these novel binding regions, which led to the identification of six candidate TREs within three binding regions. Luciferase reporter assays confirmed the presence of a TRE in the promoter region of DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 (Ddx54) and thyroid hormone responsive SPOT14 (Thrsp). The TR/retinoid X receptor (RXR) heterodimer and RXR homodiner were shown to bind the promoter region of Ddx54 and drive gene expression in the presence of 9-cis-retinoic acid (9cRA). The promoter region of Thrsp was shown to allow binding of the TR/RXR heterodimer, and both T3 and 9cRA were able to significantly increase luciferase activity. The RXR homodimer was also able to bind the response element in the promoter region of Thrsp and increase luciferase activity. Overall, ChIP-chip analysis revealed a relatively limited number of TR-binding sites in juvenile mouse liver despite previous studies showing that numerous genes can be affected by TH disruption at that developmental stage, suggesting that TH action may also be mediated through other intermediates. Collectively the results provide an important step towards characterizing TR-binding sites and identifying the underlying drivers of TR-gene regulation. Three samples were analyzed (total input and immunoprecipitated for each samples).

Project description:Thyroid hormone receptors (TRs) represent important regulators of development, homeostasis, cell proliferation and differentiation. Here we investigated the impact of thyroid hormone (T3) on growth and differentiation of human red blood cells. T3 was found to effectively accelerate differentiation of SCF/Epo dependent red cell progenitors in vitro concomitantly with inducing growth arrest. This T3 activity was further enhanced by 9-cis retinoic acid (9cRA) that activates retinoid X receptor (RXR), the obligate heterodimeric partner of TR. To identify molecular targets for T3 activity in red cells, we employed a genome wide approach with DNA microarrays. We demonstrate that T3 and 9cRA induces specific gene expression patterns of up-or down-regulated genes, including ALAD and GATA-2, respectively. This study also revealed gene clusters, indicating accelerated differentiation in response to treatment. Mining for T3 induced genes identified BTEB1 (KLF9) and GAR22 as TR target genes. BTEB1/ KLF9 (basic transcription element binding protein 1/ KrÃ¼ppel-like factor 9) is a known TR target gene. GAR22 (growth arrest specific 2 (GAS2)-related gene on chromosome 22), initially found as a putative tumor suppressor gene, was induced by T3 also in the presence of cycloheximide, identifying it as a direct target of TR. Thus, our study uncovers novel targets of TR action with a potential function in T3 induced differentiation and growth arrest of red cell progenitors. Experiment Overall Design: 13 hybridizations in two individual experiments. Experiment Overall Design: Experiment 1: Experiment Overall Design: 1_SCF/Epo, 1_Epo/insulin(8h), 1_Epo/insulin(24h), 1_Epo/insulin(48h) Experiment Overall Design: Experiment 2: Experiment Overall Design: 2_SCF/Epo, 2_Epo/insulin(8h), 2_Epo/insulin(24h), 2_Epo/insulin(48h) Experiment Overall Design: 2_Epo/insulin+T3(8h), 2_Epo/insulin+9cRA(8h) Experiment Overall Design: 2_Epo/insulin+T3+9cRA(8h), , 2_Epo/insulin+T3+9cRA(24h), 2_Epo/insulin+T3+9cRA(48h)

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Global Analysis of Thyroid Receptor Binding Sites Reveals Interaction with the Ddx54 and Thrsp Promoter in Juvenile Mouse Liver

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