Anti-diabetic rosiglitazone remodels the adipocyte transcriptome by redistributing transcription to PPARγ-driven enhancers.

Rosiglitazone (rosi) is a powerful insulin sensitizer, but serious toxicities have curtailed its widespread clinical use. Rosi functions as a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), the adipocyte-predominant nuclear receptor (NR). The classic model, involving binding of ligand to the NR on DNA, explains positive regulation of gene expression, but ligand-dependent repression is not well understood. We addressed this issue by studying the direct effects of rosi on gene transcription using global run-on sequencing (GRO-seq). Rosi-induced changes in gene body transcription were pronounced after 10 min and correlated with steady-state mRNA levels as well as with transcription at nearby enhancers (enhancer RNAs [eRNAs]). Up-regulated eRNAs occurred almost exclusively at PPARγ-binding sites, to which rosi treatment recruited coactivators, including MED1, p300, and CBP. In contrast, transcriptional repression by rosi involved a loss of coactivators from eRNA sites devoid of PPARγ and enriched for other transcription factors, including AP-1 factors and C/EBPs. Thus, rosi activates and represses transcription by fundamentally different mechanisms that could inform the future development of anti-diabetic drugs.

Deacetylase-independent function of HDAC3 in transcription and metabolism requires nuclear receptor corepressor.

Histone deacetylases (HDACs) are believed to regulate gene transcription by catalyzing deacetylation reactions. HDAC3 depletion in mouse liver upregulates lipogenic genes and results in severe hepatosteatosis. Here we show that pharmacologic HDAC inhibition in primary hepatocytes causes histone hyperacetylation but does not upregulate expression of HDAC3 target genes. Meanwhile, deacetylase-dead HDAC3 mutants can rescue hepatosteatosis and repress lipogenic genes expression in HDAC3-depleted mouse liver, demonstrating that histone acetylation is insufficient to activate gene transcription. Mutations abolishing interactions with the nuclear receptor corepressor (NCOR or SMRT) render HDAC3 nonfunctional in vivo. Additionally, liver-specific knockout of NCOR, but not SMRT, causes metabolic and transcriptomal alterations resembling those of mice without hepatic HDAC3, demonstrating that interaction with NCOR is essential for deacetylase-independent function of HDAC3. These findings highlight nonenzymatic roles of a major HDAC in transcriptional regulation in vivo and warrant reconsideration of the mechanism of action of HDAC inhibitors.

Transient Maternal Hypothyroxinemia Potentiates the Transcriptional Response to Exogenous Thyroid Hormone in the Fetal Cerebral Cortex Before the Onset of Fetal Thyroid Function: A Messenger and MicroRNA Profiling Study.

Thyroid hormone (TH) is essential for brain development both before and after birth. We have used gene expression microarrays to identify TH-regulated genes in the fetal cerebral cortex prior to the onset of fetal thyroid function to better understand the role of TH in early cortical development. TH levels were transiently manipulated in pregnant mice by treatment with goitrogens from gestational day (GD) 13-16 and/or by injection of TH 12 h before sacrifice on GD 16. The transcriptional response to exogenous TH in the GD 16 fetal cortex was potentiated by transient goitrogen treatment, suggesting that the hypothyroxinemic brain is a different substrate upon which TH can act, or that robust compensatory mechanisms are induced by transient hypothyroxinemia. Several known TH-responsive genes were identified including Klf9, and several novel TH-responsive genes such as Appbp2, Ppap2b, and Fgfr1op2 were identified in which TH response elements were confirmed. We also identified specific microRNAs whose expression in the fetal cortex was affected by TH treatment, and determined that Ppap2b and Klf9 are the target genes of miR-16 and miR-106, respectively. Thus, a complex redundant functional network appears to coordinate TH-mediated gene expression in the developing brain.

FoxO1 deacetylation regulates thyroid hormone-induced transcription of key hepatic gluconeogenic genes.

Hepatic gluconeogenesis is a concerted process that integrates transcriptional regulation with hormonal signals. A major regulator is thyroid hormone (TH), which acts through its nuclear receptor (TR) to induce the expression of the hepatic gluconeogenic genes, phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC). Forkhead transcription factor FoxO1 also is an important regulator of these genes; however, its functional interactions with TR are not known. Here, we report that TR-mediated transcriptional activation of PCK1 and G6PC in human hepatic cells and mouse liver was FoxO1-dependent and furthermore required FoxO1 deacetylation by the NAD(+)-dependent deacetylase, SirT1. siRNA knockdown of FoxO1 decreased, whereas overexpression of FoxO1 increased, TH-dependent transcriptional activation of PCK1 and G6PC in cultured hepatic cells. FoxO1 siRNA knockdown also decreased TH-mediated transcription in vivo. Additionally, TH was unable to induce FoxO1 deacetylation or hepatic PCK1 gene expression in TH receptor ß-null (TRß(-/-)) mice. Moreover, TH stimulated FoxO1 recruitment to the PCK1 and G6PC gene promoters in a SirT1-dependent manner. In summary, our results show that TH-dependent deacetylation of a second metabolically regulated transcription factor represents a novel mechanism for transcriptional integration of nuclear hormone action with cellular energy status.

4-Hydroxy-PCB106 acts as a direct thyroid hormone receptor agonist in rat GH3 cells.

Polychlorinated biphenyls (PCBs) may interfere with thyroid hormone (TH) action by interacting directly with the TH receptor (TR). We found that the hydroxylated PCB metabolite, 4-OH-CB106, bound to the human TRbeta1 and significantly elevated endogenous growth hormone (GH) expression in GH3 cells in a manner similar to that of T(3) itself. This effect was also observed using a consensus TH response element (TRE) in a luciferase expression system, and was blocked by a single base-pair substitution in this TRE. In addition, we found that 4-OH-CB106 did not alter the ability of TRbeta1 to physically interact with the TRE in the GH promoter, or with SRC1 or NCoR. These effects were directly parallel to effects of T(3), indicating that 4-OH-CB106 exerts a direct agonistic effect on the TRbeta1.

Maternal thyroid hormone increases HES expression in the fetal rat brain: an effect mimicked by exposure to a mixture of polychlorinated biphenyls (PCBs).

Thyroid hormone is known to be essential for normal brain development both before and after birth, but much less is known about the role of thyroid hormone development before birth. In rodents, thyroid hormone of maternal origin can selectively regulate gene expression in the fetal cortex; HES1 was identified as a putative thyroid hormone responsive gene in the fetal cortex. Using in situ hybridization, we now confirm that thyroid hormone administration to pregnant rats can increase the abundance of HES1 mRNA in the fetal cortex on gestational day 16 (G16). In separate experiments, we found that maternal exposure to polychlorinated biphenyls (PCBs) increases HES expression similarly. Western analysis of proteins extracted from fetal cortex did not confirm that Notch-1 or Notch-3 activation was associated with treatment effects on HES expression. However, considering the role of HES proteins in fate specification of cortical neurons, these findings suggest that thyroid hormone, and PCB exposure, may influence fate specification of cortical neurons.

Nuclear receptor co-repressors are required for the histone-deacetylase activity of HDAC3 in vivo.

Histone deacetylase 3 (HDAC3) is an epigenome-modifying enzyme that is required for normal mouse development and tissue-specific functions. In vitro, HDAC3 protein itself has minimal enzyme activity but gains its histone-deacetylation function from stable association with the conserved deacetylase-activating domain (DAD) contained in nuclear receptor co-repressors NCOR1 and SMRT. Here we show that HDAC3 enzyme activity is undetectable in mice bearing point mutations in the DAD of both NCOR1 and SMRT (NS-DADm), despite having normal levels of HDAC3 protein. Local histone acetylation is increased, and genomic HDAC3 recruitment is reduced though not abrogated. Notably, NS-DADm mice are born and live to adulthood, whereas genetic deletion of HDAC3 is embryonic lethal. These findings demonstrate that nuclear receptor co-repressors are required for HDAC3 enzyme activity in vivo and suggest that a deacetylase-independent function of HDAC3 may be required for life.

Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy.

For more than a century, thyroid hormones (THs) have been known to exert powerful catabolic effects, leading to weight loss. Although much has been learned about the molecular mechanisms used by TH receptors (TRs) to regulate gene expression, little is known about the mechanisms by which THs increase oxidative metabolism. Here, we report that TH stimulation of fatty acid ß-oxidation is coupled with induction of hepatic autophagy to deliver fatty acids to mitochondria in cell culture and in vivo. Furthermore, blockade of autophagy by autophagy-related 5 (ATG5) siRNA markedly decreased TH-mediated fatty acid ß-oxidation in cell culture and in vivo. Consistent with this model, autophagy was altered in livers of mice expressing a mutant TR that causes resistance to the actions of TH as well as in mice with mutant nuclear receptor corepressor (NCoR). These results demonstrate that THs can regulate lipid homeostasis via autophagy and help to explain how THs increase oxidative metabolism.

The interaction between nuclear receptor corepressor and histone deacetylase 3 regulates both positive and negative thyroid hormone action in vivo.

Thyroid hormone (TH) plays a critical role in development, growth, and metabolism by binding to nuclear TH receptors to modulate gene expression. In the absence of TH, TH receptors repress genes that are TH-activated by recruiting the nuclear receptor corepressor (NCoR), which exists in a tight complex with histone deacetylase 3 (HDAC3). Here we explored the actions of TH in the deacetylase activating domain mutant (DADm) mouse, whose NCoR-HDAC3 interaction is genetically disrupted. Several TH-activated genes were derepressed in the liver of euthyroid and hypothyroid DADm mice, consistent with the corepressor paradigm and a critical role of the NCoR-HDAC3 interaction in basal repression. The role of corepressors in genes that are down-regulated by TH is less well understood. Remarkably, circulating TSH levels were increased in euthyroid DADm mice, and the pituitary expression of TSHalpha, a classic TH-down-regulated gene, was modestly but significantly elevated regardless of TH status. Thus, the NCoR interaction with HDAC3 modulates expression of both positively- and negatively-regulated genes by TH in vivo.

Identification of thyroid hormone receptor binding sites and target genes using ChIP-on-chip in developing mouse cerebellum.

Thyroid hormone (TH) is critical to normal brain development, but the mechanisms operating in this process are poorly understood. We used chromatin immunoprecipitation to enrich regions of DNA bound to thyroid receptor beta (TRbeta) of mouse cerebellum sampled on post natal day 15. Enriched target was hybridized to promoter microarrays (ChIP-on-chip) spanning -8 kb to +2 kb of the transcription start site (TSS) of 5000 genes. We identified 91 genes with TR binding sites. Roughly half of the sites were located in introns, while 30% were located within 1 kb upstream (5') of the TSS. Of these genes, 83 with known function included genes involved in apoptosis, neurodevelopment, metabolism and signal transduction. Two genes, MBP and CD44, are known to contain TREs, providing validation of the system. This is the first report of TR binding for 81 of these genes. ChIP-on-chip results were confirmed for 10 of the 13 binding fragments using ChIP-PCR. The expression of 4 novel TH target genes was found to be correlated with TH levels in hyper/hypothyroid animals providing further support for TR binding. A TRbeta binding site upstream of the coding region of myelin associated glycoprotein was demonstrated to be TH-responsive using a luciferase expression system. Motif searches did not identify any classic binding elements, indicating that not all TR binding sites conform to variations of the classic form. These findings provide mechanistic insight into impaired neurodevelopment resulting from TH deficiency and a rich bioinformatics resource for developing a better understanding of TR binding.