Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

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. Three samples were analyzed (total input and immunoprecipitated for each samples).

ORGANISM(S): Mus musculus

SUBMITTER: Martin Paquette

PROVIDER: E-GEOD-52417 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

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)

Project description:Synthetic selective thyroid hormone (TH) receptor (TR) modulators (STRMs) exhibit beneficial effects on dyslipidemias in animals and humans and reduce obesity, fatty liver and insulin resistance in preclinical animal models. STRMs differ from native THs in preferential binding to the TR? subtype versus TR?, increased uptake into liver and reduced uptake into other tissues. However, selective modulators of other nuclear receptors (NRs) exhibit important gene-selective actions which have been attributed to differential effects on receptor conformation and dynamics and these effects can have profound influences in animals and humans. While there are suggestions that STRMs could exhibit such gene-specific actions, the extent to which these effects are actually observed in vivo has not been explored. Here, we show that saturating concentrations of the main active form of TH, triiodothyronine (T3), and the prototype STRM GC-1 induce identical gene-sets in livers of euthyroid and hypothyroid mice and a human cultured hepatoma cell line that only expresses TR?, HepG2. We find one case in which GC-1 exhibits a modest gene-specific reduction in potency versus T3, at angiopoietin-like factor 4 (ANGPTL4) in HepG2. Investigation of the latter effect confirms that GC-1 acts through TR? to directly induce this gene. However, this gene-selective GC-1 activity is not related to unusual T3 response element (TRE) sequence, unlike previously documented promoter-selective STRM actions. Together, our data suggest that T3 and GC-1 exhibit almost identical gene regulation properties and that gene-selective actions of GC-1 and similar STRMs will be subtle and rare. We treated 9-week old euthyroid male C57/Bl6 mice with vehicle or ligand (T3 or GC-1) by a single oral gavage (n=5 per treatment) and also performed a similar study in which mice were first made hypothyroid by two week feeding on iodine deficient diet (n=3-4 per treatment), analyzing mRNA 24h post treatment [Illumina]. We also treated HepG2 with vehicle or 10nM ligand (T3 or GC1; n=3 / treatment), analyzing mRNA 24h post treatment [Affymetrix].

Project description:Although the effects of thyroid hormones (TH) on the brain development have been extensively studied perinatally, effects of TH of maternal origin on the fetal brain development have been largely unexplored. We applied a high throughput study on the mouse models with aberrant TH levels on gestation day (GD) 16, before the onset of fetal thyroid function. Although 3 day treatment with methimazole (MMI) and perchlorate significantly decreased TH levels in fetal cerebral cortex, few changes in the abundance of mRNA were revealed by the microarray analysis. Injection TH to dams 12 hours before sacrifice on GD 16 induced 161 genes significantly changed in fetal cortex. Nine out of 10 selected genes were confirmed with RT-PCR, including known TH responsive gene Klf9 and other novel TH responsive genes such as Appbp2, Ppap2b and Fgfr1op2. TH regulation of the expression of these genes was also confirmed with cultured N2a? cells. Thyroid responsive elements (TREs) in the promoters of these genes were identified using electrophoresis mobility shift assay. TH effect on microRNA (miRNA) expression in developing cortex on GD 16 and postnatal day (PND) 15 was investigated with microarray and RT-PCR. Some of miRNAs and precursors decreased in fetal cortex from the dams injected with TH on GD 16, including miR-16 and miR-106. Using 3’ untranslate region reporter vector, we identified Klf9 is one of the target genes of miR-106, while Ppap2b is the target of miR-16. These results indicated that TH regulation on gene expression could through TR-TRE interaction and through regulating target miRNA expression. This study is the first report to identify TH responsive genes and miRNAs genome wide in the early fetal brain; it provides evidence to further understand the mechanism of TH effect on brain development. Timed-pregnant C57BL/6 mice (n=20; Harlan, Indianapolis, IN) arrived on gestational day (GD) 11 and were housed individually in plastic cages under a 12:12 light cycle (0600 h–1800 h) with food available ad libitum. Mice were divided randomly into one of 4 groups (control, hypo, hypo+ and hyper; 5 per group) . Mice in the control and hyper groups were provided with fresh drinking water containing 2% sucrose from GD 13 to GD 16 until sacrifice. Mice in hypo and hypo+ were provided with fresh drinking water daily containing 1% Perchlorate (Per) and 0.025% Methimazole (MMI) supplemented with 2% sucrose (to mask bitterness) from GD 13 to sacrifice on GD 16. Twelve hours prior to sacrifice on GD 16 all mice received a single subcutaneous injection. Control and hypo mice received vehicle (0.9% saline in 100 µl volume); the hypo+ group received 25.0 µg/100g body weight thyroxine (T4) with 2.5 µg/100g body weight T3 to restore a physiological level of TH; the hyper group received 100.0 µg/100g body weight T4 with 10.0 µg/100g body weight T3 to model hyperthyroidism. Dams were killed by exposure to CO2. Fetuses were dissected from the uterine horn and embryonic membranes then frozen on pulverized dry ice. Tissue samples were taken from the fetuses to determine sex using PCR for SRY (Yang J and RT Zoeller, 2002). Fetal cerebral cortices were removed from each mouse and used for microarray and RT-PCR analysis. Total RNA was extracted from half cortices of individual female fetuses using RNeasy Lipid tissue Mini kits (Qiagen, Germantown, MD) according to the manufacturer's instructions. The quality of total RNA was evaluated by A260/A280 ratio (found to be at least 1.8 for each sample) and by electrophoresis on the Agilent Bioanalyzer. The Yale Center for the Neuroscience Microarray Consortium carried out the Affymetrix microarray analysis using The GeneChip Rat Genome 230 2.0 Array. Preparation of labeled cRNA for hybridization onto Affymetrix GeneChips followed the recommended Affymetrix protocol. Control versus each of the three treatment groups: hyper, hypo, and hypo+. 1 of 20 samples removed due to poor quality.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure