Thyroid Hormone Receptor alpha-1 Directly Controls Transcription of the beta-Catenin Gene in Intestinal Epithelial Cells

Thyroid hormones, T3 and T4, are known regulators of intestine development. The best characterized example is the remodeling of the gastrointestinal tract during amphibian metamorphosis. Thyroid hormones act via nuclear receptors, the TRs, which are T3-dependent transcription factors. We previously showed that intestinal epithelial cell proliferation is controlled by thyroid hormones and the TRalpha gene. To analyze the mechanisms responsible, we studied the expression of genes belonging to and/or activated by the Wnt/beta-catenin pathway, a major actor in the control of physiological and pathological epithelial proliferation in the intestine. We show that T3-TR1 controls the transcription of the beta-catenin gene in an epithelial cell-autonomous way. This is parallel to positive regulation of proliferation-controlling genes such as type D cyclins and c-myc, known targets of the Wnt/-beta-catenin. In addition, we show that the regulation of the beta-catenin gene is direct, as TR binds in vitro and in chromatin in vivo to a specific thyroid hormone-responsive element present in intron 1 of this gene. This is the first report concerning in vivo transcriptional control of the beta-catenin gene. As Wnt/beta-catenin plays a crucial role in intestinal tumorigenesis, our observations open a new perspective on the study of TRs as potential tumor inducers

Thyroid Hormone Receptor alpha-1 Directly Controls Transcription of the beta-Catenin Gene in Intestinal Epithelial Cells

Thyroid hormones, T3 and T4, are known regulators of intestine development. The best characterized example is the remodeling of the gastrointestinal tract during amphibian metamorphosis. Thyroid hormones act via nuclear receptors, the TRs, which are T3-dependent transcription factors. We previously showed that intestinal epithelial cell proliferation is controlled by thyroid hormones and the TRalpha gene. To analyze the mechanisms responsible, we studied the expression of genes belonging to and/or activated by the Wnt/beta-catenin pathway, a major actor in the control of physiological and pathological epithelial proliferation in the intestine. We show that T3-TR1 controls the transcription of the beta-catenin gene in an epithelial cell-autonomous way. This is parallel to positive regulation of proliferation-controlling genes such as type D cyclins and c-myc, known targets of the Wnt/-beta-catenin. In addition, we show that the regulation of the beta-catenin gene is direct, as TR binds in vitro and in chromatin in vivo to a specific thyroid hormone-responsive element present in intron 1 of this gene. This is the first report concerning in vivo transcriptional control of the beta-catenin gene. As Wnt/beta-catenin plays a crucial role in intestinal tumorigenesis, our observations open a new perspective on the study of TRs as potential tumor inducers

A point mutation in the AF-2 domain of thyroid hormone receptor alpha1 expressed after CRE mediated recombination partially recapitulates hypothyroidism.

Thyroid hormones act directly on transcription by binding to TRα1, TRβ1, TRβ2 nuclear receptors, regulating many aspects of post-natal development and homeostasis. To precisely analyze the implication of the widely expressed TRα1 isoform in this pleiotropic action, we have generated transgenic mice with a point mutation in the TRα1 coding sequence, which is expressed only after CRE/loxP mediated DNA recombination. The amino-acid change prevents interaction between TRα1 and histone acetyltransferase coactivators and the release of corepressors. Early expression of this dominant-negative receptor deeply affects post-natal development and adult homeostasis, recapitulating many aspects of congenital and adult hypothyroidism, except in tissues and cells where TRβ1 and TRβ2 are predominantly expressed. Both respective abundance and intrinsic properties of TRα1 and TRβ1/2 seems to govern specificity of action

Thyroid hormone receptor {beta} (TR{beta}) and liver X receptor (LXR) regulate carbohydrate response element binding protein (ChREBP) expression in a tissue selective manner.

Thyroid hormone- (TR) and Liver X- (LXR)receptors are transcription factors involved in lipogenesis. Both receptors recognize the same consensus DNA response element in vitro. It was previously shown that their signalling pathways interact in the control of cholesterol elimination in the liver. In the present study ChREBP, a major transcription factor controlling the activation of glucose-induced lipogenesis in liver, is characterized as a direct target of thyroid hormones(TH) in liver and white adipose tissue(WAT), the two main lipogenic tissues in mice. Using genetic and molecular approaches ChREBP is shown to be specifically regulated by TRbeta, but not by TRalpha in vivo even in WAT where both TR isoforms are expressed. However this isotype specificity is not found in vitro. This TRbeta specific regulation correlates with the loss of TH-induced lipogenesis in TRbeta-/- mice. Fasting/refeeding experiments show that TRbeta is not required for the activation of ChREBP expression particularly marked in WAT following refeeding. However TH can stimulate ChREBP expression in WAT even under fasting conditions suggesting completely independent pathways. Since ChREBP has been described as an LXR target, the interaction of LXR and TRbeta in ChREBP regulation was assayed both in vitro and in vivo. Each receptor recognizes a different response element on the ChREBP promoter, located only eight base pairs apart.There is a crosstalk between LXR and TRbeta signalling on the ChREBP promoter in liver but not in WAT where LXR does not regulate ChREBP expression. The molecular basis for this crosstalk has been determined in in vitro systems

The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells

International audienceEmbryonic stem cells ( ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self- renew has been shown to be governed by the transcription factors Oct4 ( Pou5f1) and Nanog. Oct4 appears to control cell- fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In nonmammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 ( spg; pou5f1) and Xenopus Pou91 ( XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC ( cESC), which display similar properties of pluripotency and long- term self- renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV ( cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self- renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self- renewal are not exclusive to mammal

The transforming acidic coiled coil (TACC1) protein modulates the transcriptional activity of the nuclear receptors TR and RAR

Background: The transcriptional activity of Nuclear hormone Receptors (NRs) is regulated by interaction with coactivator or corepressor proteins. Many of these cofactors have been shown to have a misregulated expression or to show a subcellular mislocalization in cancer cell lines or primary tumors. Therefore they can be factors involved in the process of oncogenesis. Results: We describe a novel NR coregulator, TACC1, which belongs to the Transforming Acidic Coiled Coil (TACC) family. The interaction of TACC1 with Thyroid Hormone Receptors (TR) and several other NRs has been shown in a yeast two-hybrid screen and confirmed by GST pulldown, colocalization and co-immunoprecipitation experiments. TACC1 interacts preferentially with unliganded NRs. In F9 cells, endogenous TACC1 localized in the chromatin-enriched fraction of the nucleus and interacted with Retinoid Acid Receptors (RAR alpha) in the nucleus. TACC1 depletion in the cell led to decreased RAR alpha and TR alpha ligand-dependent transcriptional activity and to delocalization of TR from the nucleus to the cytoplasm. Conclusions: From these experimental studies we propose that TACC1 might be a scaffold protein building up a transcriptional complex around the NRs we studied. This function of TACC1 might account for its involvement in several forms of tumour development

Single and Synergistic Effects of Cannabidiol and Δ-9-Tetrahydrocannabinol on Zebrafish Models of Neuro-Hyperactivity

In this study, we aimed to investigate the effect of the two main active cannabinoids extracted from cannabis: Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on two distinct behavioral models of induced neuro-hyperactivity. We have taken advantage of two previously developed zebrafish models of neuro-hyperactivity: a chemically induced pentylenetetrazole model and a genetic model caused by loss-of-function mutations in the GABA receptor subunit alpha 1 (GABRA1−/−). Both CBD and THC have a significant effect on the behavioral changes induced by both models. Importantly, we have also shown that when applied together at different ratios of THC to CBD (1:1, 1:5, and 1:10), there was a synergistic effect at a ratio of 1:1. This was particularly important for the genetically induced neuro-hyperactivity as it brought the concentrations of THC and CBD required to oppose the induced behavioral changes to levels that had much less of an effect on baseline larval behavior. The results of this study help to validate the ability of THC and CBD to oppose neuro-hyperactivity linked to seizure modalities. Additionally, it appears that individually, each cannabinoid may be more effective against the chemically induced model than against the GABRA1−/− transgenic model. However, when applied together, the concentration of each compound required to oppose the GABRA1−/− light-induced activity was lowered. This is of particular interest since the use of cannabinoids as therapeutics can be dampened by their side-effect profile. Reducing the level of each cannabinoid required may help to prevent off target effects that lead to side effects. Additionally, this study provides a validation of the complimentary nature of the two zebrafish models and sets a platform for future work with cannabinoids, particularly in the context of neuro-hyperactivity disorders such as epilepsy

A simplified method for identifying early CRISPR-induced indels in zebrafish embryos using High Resolution Melting analysis

BACKGROUND: The CRISPR/Cas9 system has become a regularly used tool for editing the genome of many model organisms at specific sites. However, two limiting steps arise in the process of validating guide RNA target sites in larvae and adults: the time required to identify indels and the cost associated with identifying potential mutant animals. RESULTS: Here we have combined and optimized the HotSHOT genomic DNA extraction technique with a two-steps Evagreen PCR, followed by a high-resolution melting (HRM) assay, which facilitates rapid identification of CRISPR-induced indels. With this technique, we were able to genotype adult zebrafish using genomic DNA extracted from fin-clips in less than 2 h. We were also able to obtain a reliable and early read-out of the effectiveness of guide RNAs only 4 h after the embryos were injected with the constructs for the CRISPR/Cas9 mutagenic system. Furthermore, through mutagenesis kinetic assay, we identified that the 2-cell stage is the earliest time point at which indels can be observed. CONCLUSIONS: By combining an inexpensive and rapid genomic DNA extraction method with an HRM-based assay, our approach allows for high-throughput genotyping of adult zebrafish and embryos, and is more sensitive than standard PCR approaches, permitting early identification of CRISPR-induced indels and with applications for other model organisms as well