Brenda J. Mengeling

Alternative mRNA splicing of corepressors generates variants that play opposing roles in adipocyte differentiation.

Background: The SMRT and NCoR corepressors play key biological roles in transcriptional repression. Results: Alternative mRNA splicing produces corepressor variants that can exert opposite effects on adipocyte differentiation. Conclusion: Corepressors are diversified by alternative mRNA splicing, allowing one locus to encode multiple proteins with distinct functions. Significance: Changes in alternative splicing may help drive the differentiation and customize the physiology of specific cell types. The SMRT and NCoR corepressors partner with, and help mediate repression by, a wide variety of nuclear receptors and non-receptor transcription factors. Both SMRT and NCoR are expressed by alternative mRNA splicing, resulting in the production of a series of interrelated corepressor variants that differ in their tissue distribution and in their biochemical properties. We report here that different corepressor splice variants can exert opposing transcriptional and biological effects during adipocyte differentiation. Most notably, the NCoRω splice variant inhibits, whereas the NCoRδ splice variant promotes, adipogenesis. Furthermore, the ratio of NCoRω to NCoRδ decreases during adipogenic differentiation. We propose that this alteration in corepressor splicing helps convert the cellular transcriptional program from one that maintains the pre-adipocyte in an undifferentiated state to a new transcriptional context that promotes differentiation and helps establish the proper physiology of the mature adipocyte.

Aberrant Corepressor Interactions Implicated in PML-RARα and PLZF-RARα Leukemogenesis Reflect an Altered Recruitment and Release of Specific NCoR and SMRT Splice Variants

Human acute promyelocytic leukemia is causally linked to chromosomal translocations that generate chimeric retinoic acid receptor-α proteins (x-RARα fusions). Wild-type RARα is a transcription factor that binds to the SMRT/NCoR family of corepressors in the absence of hormone but releases from corepressor and binds coactivators in response to retinoic acid. In contrast, the x-RARα fusions are impaired for corepressor release and operate in acute promyelocytic leukemia as dominant-negative inhibitors of wild-type RARα. We report that the two most common x-RARα fusions, PML-RARα and PLZF-RARα, have gained the ability to recognize specific splice variants of SMRT and NCoR that are poorly recognized by RARα. These differences in corepressor specificity between the normal and oncogenic receptors are further magnified in the presence of a retinoid X receptor heteromeric partner. The ability of retinoids to fully release corepressor from PML-RARα differs for the different splice variants, a phenomenon relevant to the requirement for supraphysiological levels of this hormone in differentiation therapy of leukemic cells. We propose that this shift in the specificity of the x-RARα fusions to a novel repertoire of corepressors contributes to the dominant-negative and oncogenic properties of these oncoproteins and helps explain previously paradoxical aspects of their behavior.

Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform.

To adapt the use of GH3.TRE-Luc reporter gene cell line for a quantitative high-throughput screening (qHTS) platform, we miniaturized the reporter gene assay to a 1536-well plate format. 1280 chemicals from the Library of Pharmacologically Active Compounds (LOPAC) and the National Toxicology Program (NTP) 1408 compound collection were analyzed to identify potential thyroid hormone receptor (TR) agonists and antagonists. Of the 2688 compounds tested, eight scored as potential TR agonists when the positive hit cut-off was defined at ≥10% efficacy, relative to maximal triiodothyronine (T3) induction, and with only one of those compounds reaching ≥20% efficacy. One common class of compounds positive in the agonist assays were retinoids such as all-trans retinoic acid, which are likely acting via the retinoid-X receptor, the heterodimer partner with the TR. Five potential TR antagonists were identified, including the antiallergy drug tranilast and the anxiolytic drug SB 205384 but also some cytotoxic compounds like 5-fluorouracil. None of the inactive compounds were structurally related to T3, nor had been reported elsewhere to be thyroid hormone disruptors, so false negatives were not detected. None of the low potency (>100µM) TR agonists resembled T3 or T4, thus these may not bind directly in the ligand-binding pocket of the receptor. For TR agonists, in the qHTS, a hit cut-off of ≥20% efficacy at 100 µM may avoid identification of positives with low or no physiological relevance. The miniaturized GH3.TRE-Luc assay offers a promising addition to the in vitro test battery for endocrine disruption, and given the low percentage of compounds testing positive, its high-throughput nature is an important advantage for future toxicological screening.

Coactivator recruitment is enhanced by thyroid hormone receptor trimers

Thyroid hormone receptors (TRs) are hormone-regulated transcription factors. TRs are generally thought to bind to their DNA target sites as homodimers or as TR/retinoid X receptor (RXR) heterodimers. However, we have shown that certain TR isoforms, such as TRbeta0, can bind as trimers to a subset of naturally occurring DNA elements. We report here that this trimeric mode of DNA recognition by TRbeta0 also results in an enhanced recruitment of coactivators in vitro and increased transcriptional activation in cells compared to TRbeta0 dimers. At least part of this enhanced coactivator recruitment reflects a selectively enhanced avidity of the TRbeta0 trimer for a specific LXXLL interaction motif within the p160 coactivators. TRbeta0 trimers also recruit certain coactivators at lower concentrations of T3 hormone and exhibit distinct coactivator stoichiometries than do TRbeta0 dimers. We conclude that trimer formation confers isoform-specific DNA recognition and transcriptional regulatory properties that are not observed for TR dimers.

Chasing the Elusive Benzofuran Impurity of the THR Antagonist NH-3: Synthesis, Isotope Labeling, and Biological Activity

We have synthesized and established the structure of a long-suspected, but hitherto unknown, benzofuran side product (EBI) formed during the synthesis of NH-3. Understanding the mechanism of its formation has enabled isotope (D) labeling. We further developed a highly efficient method for separating EBI from NH-3. Interestingly, EBI was found to be a very potent thyroid hormone receptor (THR) agonist, while NH-3 is an antagonist. In this process, we have also achieved a significantly improved synthesis of NH-3.

Pituitary specific retinoid-X receptor ligand interactions with thyroid hormone receptor signaling revealed by high throughput reporter and endogenous gene responses.

Disruption of thyroid hormone (TH) signaling can compromise vital processes both during development and in the adult. We previously reported on high-throughput screening experiments for man-made TH disruptors using a stably integrated line of rat pituitary cells, GH3.TRE-Luc, in which a thyroid hormone receptor (TR) response element drives luciferase (Luc) expression. In these experiments, several retinoid/rexinoid compounds activated the reporter. Here we show that all-trans and 13-cis retinoic acid appear to function through the heterodimer partners of TRs, retinoid-X receptors (RXRs), as RXR antagonists abrogated retinoid-induced activation. The retinoids also induced known endogenous TR target genes, showing good correlation with Luc activity. Synthetic RXR-specific agonists significantly activated all tested TR target genes, but interestingly, retinoid/rexinoid activation was more consistent between genes than the extent of T3-induced activation. In contrast, the retinoids neither activated the Luc reporter construct in transient transfection assays in the human hepatocarcinoma cell line HuH7, nor two of the same T3-induced genes examined in pituitary cells. These data demonstrate the suitability and sensitivity of GH3.TRE-Luc cells for screening chemical compound libraries for TH disruption and suggest that the extent of disruption can vary on a cell type and gene-specific bases, including an underappreciated contribution by RXRs.

A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling

The essential role of thyroid hormone (TH) signaling in mammalian development warrants the examination of man-made chemicals for its disruption. Among vertebrate species, the molecular components of TH signaling are highly conserved, including the thyroid hormone receptors (TRs), their heterodimer binding partners the retinoid-X receptors (RXRs), and their DNA recognition sequences (TREs). This molecular conservation allows examination of potential TH disruption in the tractable, in vivo model system of amphibian metamorphosis. Metamorphosis requires TH signaling for both instigation and progression, and it provides dramatic and well-characterized phenotypes involving different cell fates. Here we describe a quantitative, precocious-metamorphosis assay suite we developed using one-week post-fertilization (PF) Xenopus laevis tadpoles in order to assess disruption of TH signaling. Tadpoles at this developmental stage (Nieuwkoop-Faber (NF)-48) are competent to respond to TH hormone, although not yet producing TH, along many metamorphic pathways, and they are uniform in size. This allowed us to quantify changes in morphology associated with natural metamorphosis (e.g. gill and tail resorption, brain expansion, and craniofacial remodeling) after five days of treatment. Using the same tadpoles from morphological measurements, we quantified a 20-fold increase in TH-induced cellular proliferation in the rostral head region by whole-mount immunocytochemistry. At the molecular level, we used F3-generation tadpoles from a transgenic X. laevis line, which expresses luciferase under the control of a native TRE, to assess the ability of compounds to disrupt TR function. The luciferase reporter showed over 10-fold activation by physiologic concentrations of TH. We used the synthetic TR antagonist NH-3 to demonstrate the feasibility of our assay suite to measure inhibition of TH activity at the level of the receptor. Finally, we assessed the capabilities of suspected TH-disrupting chemicals tetrabrominated diphenyl ether 47 (BDE-47) and tetrabromobisphenol A (TBBPA). We found that BDE-47 displays general toxicity rather than TH disruption, as it did not increase brain width nor affect the TRE-luciferase reporter. However, TBBPA, a suspected TR antagonist, although not effective in antagonizing cell proliferation, significantly inhibited the TRE-luciferase reporter, suggesting that it bears closer scrutiny as a TH disruptor. Overall the assay suite has important advantages over the classical tadpole metamorphosis assays with respect to the uniformity of animal size, small test volume, reproducibility, and short test period. The assays are performed before endogenous TH production and free feeding start, which further reduces complexity and variability.

Trialkyltin Rexinoid-X Receptor Agonists Selectively Potentiate Thyroid Hormone Induced Programs of Xenopus laevis Metamorphosis

The trialkyltins tributyltin (TBT) and triphenyltin (TPT) can function as rexinoid-X receptor (RXR) agonists. We recently showed that RXR agonists can alter thyroid hormone (TH) signaling in a mammalian pituitary TH-responsive reporter cell line, GH3.TRE-Luc. The prevalence of TBT and TPT in the environment prompted us to test whether they could also affect TH signaling. Both trialkyltins induced the integrated luciferase reporter alone and potentiated TH activation at low doses. Trimethyltin, which is not an RXR agonist, did not. We turned to a simple, robust, and specific in vivo model system of TH action: metamorphosis of Xenopus laevis, the African clawed frog. Using a precocious metamorphosis assay, we found that 1nM TBT and TPT, but not trimethyltin, greatly potentiated the effect of TH treatment on resorption phenotypes of the tail, which is lost at metamorphosis, and in the head, which undergoes extensive remodeling including gill loss. Consistent with these responses, TH-induced caspase-3 activation in the tail was enhanced by cotreatment with TBT. Induction of a transgenic reporter gene and endogenous collagenase 3 (mmp13) and fibroblast-activating protein-α (fap) genes were not induced by TBT alone, but TH induction was significantly potentiated by TBT. However, induction of other TH receptor target genes such as TRβ and deiodinase 3 by TH were not affected by TBT cotreatment. These data indicate that trialkyltins that can function as RXR agonists can selectively potentiate gene expression and resultant morphological programs directed by TH signaling in vivo.

RXR Ligands Modulate Thyroid Hormone Signaling Competence in Young Xenopus laevis Tadpoles

Appropriate thyroid hormone (TH) signaling through thyroid hormone receptors (TRs) is essential for vertebrate development. Amphibian metamorphosis is initiated and sustained through the action of TH on TRs, which are conserved across vertebrates. TRs heterodimerize with retinoid X receptors (RXRs) on thyroid hormone response elements (TREs) in the genome; however, in most cell line and adult animal studies, RXR ligands do not affect expression of TR target genes. We used a quantitative, precocious metamorphosis assay to interrogate the effects of the RXR agonist bexarotene (Bex) and the RXR antagonist UVI 3003 (UVI) on T3-induced resorption phenotypes in Xenopus laevis tadpoles 1 week postfertilization. Bex potentiated gill and tail resorption, and UVI abrogated T3 action. These results held in transgenic tadpoles bearing a TRE-driven luciferase reporter. Therefore, we used poly-A-primed RNA sequencing transcriptomic analysis to determine their effects on T3-induced gene expression. We also assayed the environmental pollutant tributyltin (TBT), which is an RXR agonist. We found that the proteases that carry out resorption were potentiated by Bex and TBT but were not significantly inhibited by UVI. However, several transcription factors from multiple families (sox4, fosl2, mxd1, mafb, nfib) were all inhibited by UVI and potentiated by Bex and TBT. All required T3 for induction. Time course analysis of gene expression showed that although the agonists could potentiate within 12 hours, the antagonist response lagged. These data indicate that the agonists and antagonist are not necessarily functioning through the same mechanism and suggest that RXR liganding may modulate TH competence in metamorphic signaling.