Thyroid Hormone Induces DNA Demethylation in Xenopus Tadpole Brain

Abstract Thyroid hormone (T3) plays pivotal roles in vertebrate development, acting via nuclear T3 receptors (TRs) that regulate gene transcription by promoting post-translational modifications to histones. Methylation of cytosine residues in deoxyribonucleic acid (DNA) also modulates gene transcription, and our recent finding of predominant DNA demethylation in the brain of Xenopus tadpoles at metamorphosis, a T3-dependent developmental process, caused us to hypothesize that T3 induces these changes in vivo. Treatment of premetamorphic tadpoles with T3 for 24 or 48 hours increased immunoreactivity in several brain regions for the DNA demethylation intermediates 5-hydroxymethylcytosine (5-hmC) and 5-carboxylcytosine, and the methylcytosine dioxygenase ten-eleven translocation 3 (TET3). Thyroid hormone treatment induced locus-specific DNA demethylation in proximity to known T3 response elements within the DNA methyltransferase 3a and Krüppel-like factor 9 genes, analyzed by 5-hmC immunoprecipitation and methylation sensitive restriction enzyme digest. Chromatin-immunoprecipitation (ChIP) assay showed that T3 induced TET3 recruitment to these loci. Furthermore, the messenger ribonucleic acid for several genes encoding DNA demethylation enzymes were induced by T3 in a time-dependent manner in tadpole brain. A TR ChIP-sequencing experiment identified putative TR binding sites at several of these genes, and we provide multiple lines of evidence to support that tet2 contains a bona fide T3 response element. Our findings show that T3 can promote DNA demethylation in developing tadpole brain, in part by promoting TET3 recruitment to discrete genomic regions, and by inducing genes that encode DNA demethylation enzymes.

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Microglial exosomes facilitate α-synuclein transmission in Parkinson’s disease

Brain ◽

10.1093/brain/awaa090 ◽

2020 ◽

Vol 143 (5) ◽

pp. 1476-1497 ◽

Cited By ~ 12

Author(s):

Min Guo ◽

Jian Wang ◽

Yanxin Zhao ◽

Yiwei Feng ◽

Sida Han ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Protein Aggregation ◽

Brain Regions ◽

Dependent Manner ◽

Nigrostriatal Pathway ◽

Stereotaxic Injection ◽

Promising Therapeutic Target ◽

The Brain

Abstract Accumulation of neuronal α-synuclein is a prominent feature in Parkinson’s disease. More recently, such abnormal protein aggregation has been reported to spread from cell to cell and exosomes are considered as important mediators. The focus of such research, however, has been primarily in neurons. Given the increasing recognition of the importance of non-cell autonomous-mediated neurotoxicity, it is critical to investigate the contribution of glia to α-synuclein aggregation and spread. Microglia are the primary phagocytes in the brain and have been well-documented as inducers of neuroinflammation. How and to what extent microglia and their exosomes impact α-synuclein pathology has not been well delineated. We report here that when treated with human α-synuclein preformed fibrils, exosomes containing α-synuclein released by microglia are fully capable of inducing protein aggregation in the recipient neurons. Additionally, when combined with microglial proinflammatory cytokines, these exosomes further increased protein aggregation in neurons. Inhibition of exosome synthesis in microglia reduced α-synuclein transmission. The in vivo significance of these exosomes was demonstrated by stereotaxic injection of exosomes isolated from α-synuclein preformed fibrils treated microglia into the mouse striatum. Phosphorylated α-synuclein was observed in multiple brain regions consistent with their neuronal connectivity. These animals also exhibited neurodegeneration in the nigrostriatal pathway in a time-dependent manner. Depleting microglia in vivo dramatically suppressed the transmission of α-synuclein after stereotaxic injection of preformed fibrils. Mechanistically, we report here that α-synuclein preformed fibrils impaired autophagy flux by upregulating PELI1, which in turn, resulted in degradation of LAMP2 in activated microglia. More importantly, by purifying microglia/macrophage derived exosomes in the CSF of Parkinson’s disease patients, we confirmed the presence of α-synuclein oligomer in CD11b+ exosomes, which were able to induce α-synuclein aggregation in neurons, further supporting the translational aspect of this study. Taken together, our study supports the view that microglial exosomes contribute to the progression of α-synuclein pathology and therefore, they may serve as a promising therapeutic target for Parkinson’s disease.

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Is respiratory activity in the brain mitochondria responsive to thyroid hormone action?: a critical re-evaluation

Biochemical Journal ◽

10.1042/bj3020857 ◽

1994 ◽

Vol 302 (3) ◽

pp. 857-860 ◽

Cited By ~ 35

Author(s):

S S Katyare ◽

C S Bangur ◽

J L Howland

Keyword(s):

Thyroid Hormone ◽

Malate Dehydrogenase ◽

Respiratory Activity ◽

Hormone Treatment ◽

Brain Mitochondria ◽

Dependent Manner ◽

Glutamate Pyruvate ◽

Activity State ◽

Cytochrome Content

The effects of in vivo treatment with graded doses (0.5-1.5 micrograms/g body weight) of thyroid hormones, tri-iodothyronine (T3) and thyroxine (T4), for 4 consecutive days to euthyroid rats on the respiratory activity of isolated brain mitochondria were examined. T4 stimulated coupled State-3 respiration with glutamate, pyruvate + malate, ascorbate + tetramethyl-p-phenylenediamine and succinate, in a dose-dependent manner; T3 was effective only at the highest (1.5 micrograms) dose employed. T4 was more effective than T3 in stimulating respiratory activity. State-4 respiratory rates were in general not influenced except in the case of the ascorbate + tetramethyl-p-phenylenediamine system. Primary dehydrogenase activities, i.e. glutamate dehydrogenase, malate dehydrogenase and succinate dehydrogenase, were stimulated about 2-fold; interestingly mitochondrial but not cytosolic malate dehydrogenase activity was influenced under these conditions. The hormone treatments did not greatly influence the mitochondrial cytochrome content. The results therefore suggest that thyroid hormone treatment not only stimulates primary dehydrogenase activities but may also directly influence the process of mitochondrial electron transfer.

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Liganded Thyroid Hormone Receptor Induces Nucleosome Removal and Histone Modifications to Activate Transcription during Larval Intestinal Cell Death and Adult Stem Cell Development

Endocrinology ◽

10.1210/en.2011-1736 ◽

2012 ◽

Vol 153 (2) ◽

pp. 961-972 ◽

Cited By ~ 27

Author(s):

Kazuo Matsuura ◽

Kenta Fujimoto ◽

Liezhen Fu ◽

Yun-Bo Shi

Keyword(s):

Cell Death ◽

Gene Regulation ◽

Thyroid Hormone ◽

Chromatin Remodeling ◽

Histone Modifications ◽

Intestinal Cell ◽

Mrna Levels ◽

Dependent Manner ◽

Vertebrate Development

Thyroid hormone (T3) plays an important role in regulating multiple cellular and metabolic processes, including cell proliferation, cell death, and energy metabolism, in vertebrates. Dysregulation of T3 signaling results in developmental abnormalities, metabolic defects, and even cancer. We used T3-dependent Xenopus metamorphosis as a model to study how T3 regulates transcription during vertebrate development. T3 exerts its metamorphic effects through T3 receptors (TR). TR recruits, in a T3-dependent manner, cofactor complexes that can carry out chromatin remodeling/histone modifications. Whether and how histone modifications change upon gene regulation by TR during vertebrate development is largely unknown. Here we analyzed histone modifications at T3 target genes during intestinal metamorphosis, a process that involves essentially total apoptotic degeneration of the simple larval epithelium and de novo development of the adult epithelial stem cells, followed by their proliferation and differentiation into the complex adult epithelium. We demonstrated for the first time in vivo during vertebrate development that TR induces the removal of core histones at the promoter region and the recruitment of RNA polymerase. Furthermore, a number of histone activation and repression marks have been defined based on correlations with mRNA levels in cell cultures. Most but not all correlate with gene expression induced by liganded TR during development, suggesting that tissue and developmental context influences the roles of histone modifications in gene regulation. Our findings provide important mechanistic insights on how chromatin remodeling affects developmental gene regulation in vivo.

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Vitamin C Enhances PARPi Efficacy for the Treatment of AML

Blood ◽

10.1182/blood-2021-153884 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1168-1168

Author(s):

John P Brabson ◽

Tiffany Leesang ◽

Byron Fang ◽

Jingjing Wang ◽

Victoria Strippoli ◽

...

Keyword(s):

Vitamin C ◽

Cell Lines ◽

Combination Treatment ◽

Dna Demethylation ◽

Dependent Manner ◽

Publicly Traded ◽

Colony Forming Capacity ◽

Murine Leukemia

Abstract Poly-ADP-ribose polymerase inhibitors (PARPi) are currently in clinical trial to determine their therapeutic efficacy for the treatment of acute myeloid leukemia (AML). We have shown that vitamin C (VitC), an essential micronutrient and co-factor of Ten-Eleven translocation (TET) proteins, enhances AML sensitivity to PARPi, potentially due to an increased dependency on base-excision repair (BER) enzymes needed to remove TET-catalyzed oxidized methylcytosine bases via active DNA demethylation. TET2 is the most frequently mutated TET gene in patients with AML, and vitamin C treatment can mimic genetic restoration of TET2 function, leading to DNA demethylation, differentiation, and leukemia cell death. Whether vitamin C efficacy in combination with PARPi depends on the level of TET2 functional alleles is not yet known and may stratify whether TET2 wild-type or mutant patients should be targeted by vitamin C adjuvant therapy. We have generated primary murine AML-ETO9a+ and MLL-AF9+ leukemia models with Tet2 +/+, Tet2 +/- and Tet2 -/- alleles to determine the Tet2-dependent efficacy of PARPi treatment when combined with vitamin C. Furthermore, we have performed CRISPR gene knockout and drug library screening in human AML cell lines in combination with vitamin C treatment, and tested a panel of 10 AML cell lines with titrating concentrations of PARPi (Olaparib, Talazoparib, Veliparib and Rucaparib) alone or in combination with vitamin C (L-ascorbic acid) mimicking physiological to pharmacological in vivo doses. Primary murine AML cells and human cell lines were assayed for colony-forming capacity, differentiation, cell cycling, viability and effects on DNA methylation, levels of oxidized 5-mC and gene expression upon combination treatment in vitro and in vivo. TET2 mutant PDX and primary murine AMLs treated in vivo with L-ascorbate (4g/kg) and Olaparib (50mg/kg) by daily IP injection were also monitored for disease burden, cellular differentiation and survival. Vitamin C is known to drive the TET-catalyzed iterative oxidation of 5-methylcytosine (5-mC) leading to the formation of 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). We show that VitC-PARPi combination treatment causes an accumulation of oxidized 5-mC intermediates in the AML genome that correlates with increased yH2AX formation in mid-S phase and cell cycle stalling. Vitamin C reduces the IC 50 of Olaparib and Talazoparib by greater than 10-fold in human AML cells lines and primary murine leukemia cells, and treatment in combination promotes myeloid differentiation and blocks colony-forming capacity greater than either alone. In both our in vitro and in vivo studies, Tet2 +/- AML cells exhibit increased sensitivity to vitamin C treatment alone or in combination with PARPi compared to either Tet2 +/+ or Tet2 -/- cells, suggesting that patients with TET2 haploinsufficiency, which represents the majority of TET2 mutant cases, could benefit the most from combined treatment. Our findings confirm that vitamin C can act synergistically with PARPi to block AML cell viability, reduce colony-forming capacity, and decrease leukemia burden in PDX and primary murine leukemia models in a TET2 allelic dose-dependent manner. The combinatorial effect works at clinically relevant concentrations of PARPi, and low-pharmacological doses of vitamin C. These studies suggest that vitamin C can be used as a non-toxic therapeutic adjuvant to PARPi therapy for the treatment of AML. Disclosures Neel: Northern Biologics, LTD: Current equity holder in publicly-traded company, Other: Co- Founder; SAB: Other: Co-Founder; Navire Pharma: Consultancy, Current equity holder in publicly-traded company; Jengu Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Co-Founder; Arvinas, Inc: Consultancy, Current equity holder in publicly-traded company; Recursion Pharma: Current equity holder in publicly-traded company.

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High levels of thyroid hormone impair regulatory T cells function via reduced PD-1 expression

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgab191 ◽

2021 ◽

Author(s):

Yi Zhong ◽

Ting-Ting Lu ◽

Xiao-Mei Liu ◽

Bing-Li Liu ◽

Yun Hu ◽

...

Keyword(s):

T Cells ◽

Thyroid Hormone ◽

Regulatory T Cells ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Dependent Manner ◽

Peripheral Blood Mononuclear

Abstract Context Regulatory T cells (Tregs) dysfunction plays an important role in the development and progression of Graves’ disease (GD). Programmed cell death 1 (PD-1) prompts FoxP3 in Tregs expression and enhances the suppressive activity of Tregs. Whether abnormal expression of PD-1 contributes to the breakdown of Tregs and the role of thyroid hormone in the PD-1 expression of Tregs in GD remain substantially undefined. Objective To evaluate the role of PD-1 in Tregs function and triiodothyronine (T3) in PD-1 expression in patients with GD and mice treated with T3. Methods We recruited 30 patients with GD and 30 healthy donors. PD-1 expression in Tregs and Tregs function were determined. To evaluate the effects of thyroid hormone on PD-1 expression in Tregs, we used T3 for the treatment of human peripheral blood mononuclear cells (PBMCs). We then treated mice with T3 to confirm the effect of thyroid hormone on PD-1 expression in Tregs and Tregs function in vivo. Results PD-1 expression in Tregs and the suppressive function of Tregs significantly decreased in patients with GD. T3 reduced PD-1 expression in human Tregs in a concentration- and time-dependent manner in vitro. High levels of circulating T3 reduced PD-1 expression in Tregs, impaired Tregs function, and disrupted T-helper cell (Th1 and Th2) balance in mice treated with T3. Conclusions Tregs dysfunction in GD patients might be due to down-regulation of PD-1 expression in Tregs induced by high levels of serum T3.

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Regulation of thyroid hormone activation via the liver X-receptor/retinoid X-receptor pathway

Journal of Endocrinology ◽

10.1677/joe-09-0448 ◽

2010 ◽

Vol 205 (2) ◽

pp. 179-186 ◽

Cited By ~ 20

Author(s):

Marcelo A Christoffolete ◽

Márton Doleschall ◽

Péter Egri ◽

Zsolt Liposits ◽

Ann Marie Zavacki ◽

...

Keyword(s):

Thyroid Hormone ◽

Thyroid Hormone Receptor ◽

Liver X Receptor ◽

Retinoid X Receptor ◽

Luciferase Reporter ◽

Dependent Manner ◽

Diet Induced Obesity ◽

Ectopic Liver ◽

Dose Dependent

Thyroid hormone receptor (TR) and liver X-receptor (LXR) are the master regulators of lipid metabolism. Remarkably, a mouse with a targeted deletion of both LXRα and LXRβ is resistant to western diet-induced obesity, and exhibits ectopic liver expression of the thyroid hormone activating type 2 deiodinase (D2). We hypothesized that LXR/retinoid X-receptor (RXR) signaling inhibits hepatic D2 expression, and studied this using a luciferase reporter containing the human DIO2 (hDIO2) promoter in HepG2 cells. Given that, in contrast to mammals, the chicken liver normally expresses D2, the chicken DIO2 (cDIO2) promoter was also studied. 22(R)-OH-cholesterol negatively regulated hDIO2 in a dose-dependent manner (100 μM, approximately twofold), while it failed to affect the cDIO2 promoter. Truncations in the hDIO2 promoter identified the region −901 to −584 bp as critical for negative regulation. We also investigated if 9-cis retinoic acid (9-cis RA), the ligand for the heterodimeric partner of TR and LXR, RXR, could regulate the hDIO2 promoter. Notably, 9-cis RA repressed the hDIO2 luciferase reporter (1 μM, approximately fourfold) in a dose-dependent manner, while coexpression of an inactive mutant RXR abolished this effect. However, it is unlikely that RXR homodimers mediate the repression of hDIO2 since mutagenesis of a DR-1 at −506 bp did not interfere with 9-cis RA-mediated repression. Our data indicate that hDIO2 transcription is negatively regulated by both 22(R)-OH-cholesterol and 9-cis RA, which is consistent with LXR/RXR involvement. In vivo, the inhibition of D2-mediated tri-iodothyronine (T3) production by cholesterol/9-cis RA could function as a feedback loop, given that T3 decreases hepatic cholesterol levels.

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Topical Thyroid Hormone Accelerates Wound Healing in Mice

Endocrinology ◽

10.1210/en.2005-0192 ◽

2005 ◽

Vol 146 (10) ◽

pp. 4425-4430 ◽

Cited By ~ 56

Author(s):

Joshua D. Safer ◽

Tara M. Crawford ◽

Michael F. Holick

Keyword(s):

Wound Healing ◽

Thyroid Hormone ◽

Topical Application ◽

Dependent Manner ◽

Accelerate Wound Healing ◽

Previous Hypothesis ◽

Dose Dependent ◽

Physiologic Role

Although the physiologic role of thyroid hormone in skin is not well understood, mounting evidence suggests that T3 plays an important role in epidermal proliferation. The goal of this project was to evaluate whether the topical application of supraphysiologic doses of T3 could accelerate wound healing. We evaluated mice treated with topical T3vs. the same mice receiving vehicle alone (Novasome A). Ten-millimeter diameter (79 mm2) dorsal skin wounds were established in all animals, and wounds were remeasured 4 d after injury. All animals were evaluated twice: once with the T3 treatment and once with the vehicle alone. Daily topical application of 150 ng T3 resulted in 58% greater wound closure relative to wounds on the same animals receiving vehicle alone (P < 0.001). Furthermore, we determined that wound healing-associated keratin 6 protein expression in hair follicle keratinocytes increased in a dose-dependent manner in vivo during topical T3 treatment. The data support our previous hypothesis that T3 is necessary for optimal wound healing. Now, we further suggest that topical thyroid hormone may be an inexpensive agent to hasten healing of certain wounds.

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Effect of Chromatin-Remodeling Agents in Hepatic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem CellsIn VitroandIn Vivo

Stem Cells International ◽

10.1155/2016/3038764 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 7

Author(s):

Danna Ye ◽

Tong Li ◽

Philip Heraud ◽

Rangsun Parnpai

Keyword(s):

Bone Marrow ◽

Liver Damage ◽

Dna Methyltransferase ◽

Trichostatin A ◽

Specific Gene ◽

Dependent Manner ◽

Hepatic Differentiation ◽

Rat Bone

Epigenetic events, including covalent histone modifications and DNA methylation, play fundamental roles in the determination of lineage-specific gene expression and cell fates. The aim of this study was to determine whether the DNA methyltransferase inhibitor (DNMTi) 5-aza-2′-deoxycytidine (5-aza-dC) and the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) promote the hepatic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) and their therapeutic effect on liver damage. 1 μM TSA and 20 μM 5-aza-dC were added to standard hepatogenic medium especially at differentiation and maturation steps and their potential function on hepatic differentiationin vitroandin vivowas determined. Exposure of rBM-MSCs to 1 μM TSA at both the differentiation and maturation steps considerably improved hepatic differentiation. TSA enhanced the development of the hepatocyte shape, promoted the chronological expression of hepatocyte-specific markers, and improved hepatic functions. In contrast, treatment of rBM-MSCs with 20 μM 5-aza-dC alone or in combination with TSA was ineffective in improving hepatic differentiationin vitro. TSA and/or 5-aza-dC derived hepatocytes-like cells failed to improve the therapeutic potential in liver damage. We conclude that HDACis enhance hepatic differentiation in a time-dependent manner, while DNMTis do not induce the hepatic differentiation of rBM-MSCsin vitro. Theirin vivofunction needs further investigation.

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Differential regulation of DNA methylation versus histone acetylation in cardiomyocytes during HHcy in vitro and in vivo: an epigenetic mechanism

Physiological Genomics ◽

10.1152/physiolgenomics.00168.2013 ◽

2014 ◽

Vol 46 (7) ◽

pp. 245-255 ◽

Cited By ~ 35

Author(s):

Pankaj Chaturvedi ◽

Anuradha Kalani ◽

Srikanth Givvimani ◽

Pradip Kumar Kamat ◽

Anastasia Familtseva ◽

...

Keyword(s):

Dna Methylation ◽

Histone Acetylation ◽

Dna Methyltransferase ◽

Epigenetic Mechanism ◽

Dependent Manner ◽

Histone Deacetylase 1 ◽

H3k9 Acetylation ◽

Dose Dependent

The mechanisms of homocysteine-mediated cardiac threats are poorly understood. Homocysteine, being the precursor to S-adenosyl methionine (a methyl donor) through methionine, is indirectly involved in methylation phenomena for DNA, RNA, and protein. We reported previously that cardiac-specific deletion of N-methyl-d-aspartate receptor-1 (NMDAR1) ameliorates homocysteine-posed cardiac threats, and in this study, we aim to explore the role of NMDAR1 in epigenetic mechanisms of heart failure, using cardiomyocytes during hyperhomocysteinemia (HHcy). High homocysteine levels activate NMDAR1, which consequently leads to abnormal DNA methylation vs. histone acetylation through modulation of DNA methyltransferase 1 (DNMT1), HDAC1, miRNAs, and MMP9 in cardiomyocytes. HL-1 cardiomyocytes cultured in Claycomb media were treated with 100 μM homocysteine in a dose-dependent manner. NMDAR1 antagonist (MK801) was added in the absence and presence of homocysteine at 10 μM in a dose-dependent manner. The expression of DNMT1, histone deacetylase 1 (HDAC1), NMDAR1, microRNA (miR)-133a, and miR-499 was assessed by real-time PCR as well as Western blotting. Methylation and acetylation levels were determined by checking 5′-methylcytosine DNA methylation and chromatin immunoprecipitation. Hyperhomocysteinemic mouse models (CBS+/−) were used to confirm the results in vivo. In HHcy, the expression of NMDAR1, DNMT1, and matrix metalloproteinase 9 increased with increase in H3K9 acetylation, while HDAC1, miR-133a, and miR-499 decreased in cardiomyocytes. Similar results were obtained in heart tissue of CBS+/− mouse. High homocysteine levels instigate cardiovascular remodeling through NMDAR1, miR-133a, miR-499, and DNMT1. A decrease in HDAC1 and an increase in H3K9 acetylation and DNA methylation are suggestive of chromatin remodeling in HHcy.

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A Mechanism to Enhance Cellular Responsivity to Hormone Action: Krüppel-Like Factor 9 Promotes Thyroid Hormone Receptor-β Autoinduction During Postembryonic Brain Development

Endocrinology ◽

10.1210/en.2015-1980 ◽

2016 ◽

Vol 157 (4) ◽

pp. 1683-1693 ◽

Cited By ~ 11

Author(s):

Fang Hu ◽

Joseph R. Knoedler ◽

Robert J. Denver

Keyword(s):

Thyroid Hormone ◽

Gene Transfer ◽

Target Genes ◽

Thyroid Hormone Receptor ◽

Upstream Region ◽

Mrna Levels ◽

Dependent Manner ◽

Functional Dna ◽

Mutant Forms

Abstract Thyroid hormone (TH) receptor (TR)-β (trb) is induced by TH (autoinduced) in Xenopus tadpoles during metamorphosis. We previously showed that Krüppel-like factor 9 (Klf9) is rapidly induced by TH in the tadpole brain, associates in chromatin with the trb upstream region in a developmental stage and TH-dependent manner, and forced expression of Klf9 in the Xenopus laevis cell line XTC-2 accelerates and enhances trb autoinduction. Here we investigated whether Klf9 can promote trb autoinduction in tadpole brain in vivo. Using electroporation-mediated gene transfer, we transfected plasmids into premetamorphic tadpole brain to express wild-type or mutant forms of Klf9. Forced expression of Klf9 increased baseline trb mRNA levels in thyroid-intact but not in goitrogen-treated tadpoles, supporting that Klf9 enhances liganded TR action. As in XTC-2 cells, forced expression of Klf9 enhanced trb autoinduction in tadpole brain in vivo and also increased TH-dependent induction of the TR target genes klf9 and thbzip. Consistent with our previous mutagenesis experiments conducted in XTC-2 cells, the actions of Klf9 in vivo required an intact N-terminal region but not a functional DNA binding domain. Forced expression of TRβ in tadpole brain by electroporation-mediated gene transfer increased baseline and TH-induced TR target gene transcription, supporting a role for trb autoinduction during metamorphosis. Our findings support that Klf9 acts as an accessory transcription factor for TR at the trb locus during tadpole metamorphosis, enhancing trb autoinduction and transcription of other TR target genes, which increases cellular responsivity to further TH action on developmental gene regulation programs.

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