TR Binding Research Articles

Binding and Activity of Tetrabromobisphenol A Mono-Ether Structural Analogs to Thyroid Hormone Transport Proteins and Receptors

Background:Tetrabromobisphenol A (TBBPA) mono-ether structural analogs, identified as the by-products or transformation products of commercial TBBPA bis-ether derivatives, have been identified as emerging widespread pollutants. However, there is very little information regarding their toxicological effects.Objective:We aimed to explore the potential thyroid hormone (TH) system–disrupting effect of TBBPA mono-ether structural analogs.Methods:The binding potencies of chemicals toward human TH transport proteins [transthyretin (TTR) and thyroxine-binding globulin (TBG)] and receptors [ ligand-binding domain (LBD) and ] were determined by fluorescence competitive binding assays. Molecular docking was used to simulate the binding modes of the chemicals with the proteins. The cellular TR-disrupting potencies of chemicals were assessed by a GH3 cell proliferation assay. The intracellular concentrations of the chemicals were measured by high-performance liquid chromatography and mass spectrometry.Results:TBBPA mono-ether structural analogs bound to TTR with half maximal inhibitory concentrations ranging from to but did not bind to TBG. They also bound to both subtypes of TR-LBDs with 20% maximal inhibitory concentrations ranging from to . The docking results showed that the analogs fit into the ligand-binding pockets of TTR and TR-LBDs with binding modes similar to that of TBBPA. These compounds likely induced GH3 cell proliferation via TR [with the lowest effective concentrations (LOECs) ranging from to ] and further enhanced TH-induced GH3 cell proliferation (with LOECs ranging from to ). Compared with TBBPA, TBBPA-mono(2,3-dibromopropyl ether) showed a 4.18-fold higher GH3 cell proliferation effect and 105-fold higher cell membrane transportation ability.Conclusion:This study provided a possible mechanism underlying the difference in TTR or TR binding by novel TBBPA structural analogs. These compounds might exert TH system–disrupting effects by disrupting TH transport in circulation and TR activity in TH-responsive cells. https://doi.org/10.1289/EHP6498

SAT-446 Transcriptome Comparison of a Natural T3 Regulated Process and a Treatment with T3

Thyroid hormones (TH) act mainly on the expression of the genome. It is well accepted that differential gene expression following treatment with TH recapitulates the expression profiles taking place during natural processes induced by these hormones. Although both processes seem to correlate at the scale of a few target genes, this hasn’t been addressed in a systematic manner. Our objectives were first to compare transcriptome variations after TH treatment with transcriptome variations during a TH controlled natural process and second to evaluate the proportion of the direct TH-response. The measurement of gene expression at genome scale (transcriptome) is obtained by sequencing all the messenger RNAs (RNA-seq). Direct response was sorted out by linking the transcription start sites of target genes using RNA-PET analysis with TR binding sites mapping using chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). Indeed, ChIA-PET not only allow to map TR binding sites but also the physical interactions between them and transcription start sites of regulated genes. Our model is one of the striking developmental processes orchestrated by TH: amphibian metamorphosis. Tadpole transformation is marked by dramatic changes including de novo morphogenesis (limb), tissue remodelling (brain, intestine...) and organ resorption through apoptosis (tail). These changes involve cascades of gene regulation initiated by TH and their receptors. Because metamorphosis has close and interesting parallels with the perinatal period in mammals (including human), metamorphosis is thus an attractive model to analyze in a physiological context, the functions and mechanisms of action of TH. Here, we have focused on one Xenopus tropicalis organs, the tail fin skin which will disappear through cell death. We have compared natural metamorphosis with 24h of 10nM triiodothyronine (T3) treatment. We were able to observe several differences between T3 treatment and natural development. First, the genes regulated by T3 only correspond to a proportion of genes differentially expressed during metamorphosis. Second, T3-response genes start to be regulated well before tail regression (several days). Finally, T3-direct target genes represent a few percent of all the genes differentially expressed during tail regression. In conclusion, the comparison of transcriptomes of natural and induced metamorphosis allow us to reach a more precise understanding of TH action

Posttranslational Modification of Thyroid Hormone Nuclear Receptor by Phosphorylation.

TR phosphorylation promotes TR binding to DNA and heterodimerization with RXR. TRβ phosphorylation is induced by thyroid hormone on the cell membrane and phosphorylation by extracellular signal-regulated kinases (ERK), presumably at serine 142. TRα1 N-termini harbors two phosphorylation sites at serine 12 and serine 28/29. Serine 12 is phosphorylated by casein 2 and serine 28/29 by protein kinase A. Mutation analysis of TRα2 identified 2 serine sites, S472 and S473, as the substrates for casein kinase II. Phosphorylated TRα2 does not bind to DNA and dephosphorylated TRα binds to DNA and antagonizes TRα1 binding. Phosphorylation of TR is critical for TR function and T3 signaling and approaches to detection and analysis of phosphorylated TR are described.

Pituitary NR4A1 is negatively regulated by thyroid hormone without direct binding of thyroid hormone receptors on the gene

We previously reported that TRH stimulated pituitary TSHβ gene expression via an immediate increase in NR4A1 in thyrotrophs. We demonstrated that NR4A1 mRNA levels are regulated by thyroid hormone. Pituitary NR4A1 mRNA levels were decreased in mice injected with L-T4. NR4A1 promoter activity was increased by the overexpression of TRβs, and these increases were decreased by T3, and the −27∼+152 bp region was responsible for these changes in vitro. An EMSA showed the lack of TRβs-isoforms binding, and a ChIP assay demonstrated the recruitment of TRβs and NCoR in the −147∼+148 bp region in the absence of T3, whereas T3 induced their release. Experiments on the overexpression and knockdown of NCoR, and using the mutant TRs supported the involvement of NCoR in the TR-induced stimulation. These results demonstrate that thyroid hormone down-regulated basal NR4A1 mRNA levels in the pituitary, and the direct binding of TR was not required.

The impact of viral RNA on the association free energies of capsid protein assembly: bacteriophage MS2 as a case study.

A large number of single-stranded RNA viruses assemble their capsid and their genomic material simultaneously. The RNA viral genome plays multiple roles in this process that are currently only partly understood. In this work, we investigated the thermodynamic basis of the role of viral RNA on the assembly of capsid proteins. The viral capsid of bacteriophage MS2 was considered as a case study. The MS2 virus capsid is composed of 60 AB and 30 CC protein dimers. We investigated the effect of RNA stem loop (the translational repressor TR) binding to the capsid dimers on the dimer-dimer relative association free energies. We found that TR binding results in destabilization of AB self-association compared with AB and CC association. This indicates that the association of the AB and CC dimers is the most likely assembly pathway for the MS2 virus, which explains the experimental observation of alternating patterns of AB and CC dimers in dominant assembly intermediates of the MS2 virus. The presence of viral RNA, therefore, dramatically channels virus assembly to a limited number of pathways, thereby enhancing the efficiency of virus self-assembly process. Interestingly, Thr59Ser and Thr45Ala mutations of the dimers, in the absence of RNA stem loops, lead to stabilization of AB self-association compared with the AB and CC associations, thereby channelling virus assembly towards a fivefold (AB)5 pentamer intermediate, providing a testable hypothesis of our thermodynamic arguments.

Reversing thyroid-hormone-mediated repression of a HSV-1 promoter via computationally guided mutagenesis.

Thyroid hormones (THs) and their DNA-binding nuclear receptors (TRs) direct transcriptional regulation in diverse ways depending on the host cell environment and specific promoter characteristics of TH-sensitive genes. This study sought to elucidate the impact on transcriptional repression of nucleotide sequence or orientation within TR binding sites - the TH response elements (TREs) of TH-sensitive promoters - to better understand ligand-dependent transcriptional repression of wild-type promoters. Computational analysis of the HSV-1 thymidine kinase (TK) gene TRE bound by TR and retinoid X receptor (RXR) revealed a single TRE point mutation sufficient to reverse the TRE orientation. In vitro experiments showed that the TRE point mutation had distinct impacts on promoter activity, sufficient to reverse the TH-dependent negative regulation in neuroendocrine differentiated cells. This point mutation altered the promoter's regulatory mechanism by discrete changes in transcription factor TR occupancy and altered enrichment of the repressive chromatin modification of histone-3-lysine-9-trimethyl (H3K9Me3). Insights relating to this negative TRE (nTRE) mechanism aids our understanding of other nTREs and TRE mutations associated with TH and herpes diseases.

The allosteric switching mechanism in bacteriophage MS2.

We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.

The flow of genetic information – the role of thyroid hormones in the regulation of gene expression

Human genome encodes about 25 000 protein coding genes, among which some are constitutively expressed in all cells of the organism while the others are activated in response to specific extra- and intracellular signaling. Gene expression is regulated on multiple levels in a spatiotemporal manner. These levels include: genome, which contains all cellular DNA sequences (nuclear and extranuclear -mitochondrial), transcriptome, which contains all sequences of RNA synthesized in the cell (including long: pre-mRNA, mRNA, ncRNA, and short: snRNA, miRNA, and piRNA), proteome, which contains all proteins synthesized in the cell, metabolome, which includes all metabolic pathways in the organism, localizome, which includes all possible subcellular localizations of macromolecules in the organism, phenome, which is defined as a set of all phenotypes of the organism, which enables establishing of the real effects of particular genes and external environment on the phenotypic variability in humans. Thyroid hormones, 3,5,3'5'-tetraiodothyronine (thyroxine) and 3,5,3'-triiodothyronine (T3) participate in the regulation of key cellular processes, including proliferation, differentiation, apoptosis and metabolism. Together with other factors they initiate variable cellular processes, starting from activation of the flow and amplification of genetic information (via DNA-RNA-protein), and ending with elicitation of specific phenotypic traits of the cell. The action of thyroid hormone, is mediated mainly by interactions with nuclear receptors (THR), which belong to the superfamily of ligand dependent nuclear transcription factors. THR bind to specific sequences of response to thyroid hormone (TRE) in promoters of target genes and regulate their expression. Apart from classic, genomic mechanism of action, THRs can also act via non-genomic mechanism in which regulation of target genes is mediated by interactions with other receptors – located in plasma membrane or cytoplasm. THR are encoded by two genes, THRA (ERBA) and THRB. These genes, located in chromosome regions 17q21 (THRA) and 3p21-3p25 (THRB), encode for three functional receptors: TRα1, TRβ1, and TRβ2, and the receptor TRα2 which does not bind T3 and acts as an antagonist of the other receptors. TRα1 and TRβ1 are expressed in all tissue types and their expression and ratios at which they are expressed depend on tissue type and developmental stage. In most tissues and organs only one of the receptors is responsible for T3 mediated regulation of gene expression. For instance, TRα1 is the main receptor acting in bones, TRβ1 acts in liver and kidney, while TRβ2 acts in pituitary and hypothalamus where it regulates HPT axis. In the genomic mechanism THR regulate expression of target genes via binding to the sequences of response to thyroid hormone (TRE) located in their promoters. Typical TRE consists of two repeats of AGGTC(C/A)A sequence. The presence of so called positive TRE results in induction of gene expression after binding of TR and T3, while binding of THR without T3 results in inhibition of gene expression. Thus, deficiency of T3 in the cell will result in inhibition of expression of a given gene, for instance GH or a gene coding for iodothyronine deiodinase type 1. The genes with negative TRE are regulated in an opposite manner: binding of T3 to the receptor results in inhibition of gene expression. The negative regulation of gene expression via T3 enables, among others, the mechanism of feedback regulation of expression of THR and TSH via T3. In consequence, all disturbances of thyroid hormone signaling pathways can directly result in deregulation of homeostasis of the organism.

The Rhesus Rotavirus Gene Encoding VP4 Is a Major Determinant in the Pathogenesis of Biliary Atresia in Newborn Mice

Biliary atresia (BA) is a devastating disease of childhood for which increasing evidence supports a viral component in pathogenesis. The murine model of BA is induced by perinatal infection with rhesus rotavirus (RRV) but not with other strains of rotavirus, such as TUCH. To determine which RRV gene segment(s) is responsible for pathogenesis, we used the RRV and TUCH strains to generate a complete set of single-gene reassortants. Eleven single-gene "loss-of-function" reassortants in which a TUCH gene replaced its RRV equivalent and 11 single-gene "gain-of-function" reassortants in which an RRV gene replaced its TUCH equivalent were generated. Newborn BALB/c mice were inoculated with the reassortants and were monitored for biliary obstruction and mortality. In vitro, the ability to bind to and replicate within cholangiocytes was analyzed. Infection of mice with the "loss-of-function" reassortant R(T(VP4)), where gene 4 from TUCH was placed on an RRV background, eliminated the ability of RRV to cause murine BA. In a reciprocal fashion, the "gain-of-function" reassortant T(R(VP4)) resulted in murine BA with 88% mortality. Compared with those for RRV, R(T(VP4)) binding and titers in cholangiocytes were significantly attenuated, while T(R(VP4)) binding and titers were significantly increased over those for TUCH. Reassortants R(T(VP3)) and T(R(VP3)) induced an intermediate phenotype. RRV gene segment 4 plays a significant role in governing tropism for the cholangiocyte and the ability to induce murine BA. Gene segment 3 did not affect RRV infectivity in vitro but altered its in vivo effect.

Specific Histone Lysine 4 Methylation Patterns Define TR-Binding Capacity and Differentiate Direct T3 Responses

The diversity of thyroid hormone T(3) effects in vivo makes their molecular analysis particularly challenging. Indeed, the current model of the action of T(3) and its receptors on transcription does not reflect this diversity. Here, T(3)-dependent amphibian metamorphosis was exploited to investigate, in an in vivo developmental context, how T(3) directly regulates gene expression. Two, direct positively regulated T(3)-response genes encoding transcription factors were analyzed: thyroid hormone receptor β (TRβ) and TH/bZIP. Reverse transcription-real-time quantitative PCR analysis on Xenopus tropicalis tadpole brain and tail fin showed differences in expression levels in premetamorphic tadpoles (lower for TH/bZIP than for TRβ) and differences in induction after T(3) treatment (lower for TRβ than for TH/bZIP). To dissect the mechanisms underlying these differences, chromatin immunoprecipitation was used. T(3) differentially induced RNA polymerase II and histone tail acetylation as a function of transcriptional level. Gene-specific patterns of TR binding were found on the different T(3) -responsive elements (higher for TRβ than for TH/bZIP), correlated with gene-specific modifications of H3K4 methylation (higher for TRβ than for TH/bZIP). Moreover, tissue-specific modifications of H3K27 were found (lower in brain than in tail fin). This first in vivo analysis of the association of histone modifications and TR binding/gene activation during vertebrate development for any nuclear receptor indicate that chromatin context of thyroid-responsive elements loci controls the capacity to bind TR through variations in histone H3K4 methylation, and that the histone code, notably H3, contributes to the fine tuning of gene expression that underlies complex physiological T(3) responses.

Molecular characterization of thyroid hormone-inhibited atrial L-type calcium channel expression: implication for atrial fibrillation in hyperthyroidism

Atrial fibrillation (AF) is a common complication in hyperthyroidism. Earlier studies demonstrate that thyroid hormone decreases L-type calcium channel (LCC) current expression with resultant shortening of action potential duration (APD), providing a substrate for AF. The aim of this study was to investigate the potential mechanism underlying the regulatory effect of thyroid hormone on LCC. In a hyperthyroid rat model, thyroid hormone (triiodothyronine [T3]) administration down-regulated atrial LCC expression. In vitro, treatment of murine atrial myocytes (HL-1) with T3 decreased the expression of LCC and its current, resulting in abbreviation of APD. Furthermore, T3 inhibited the activation of cyclic AMP response element (CRE)-binding protein (CREB), including phosphorylation at Ser133 and its nuclear translocation. Transient transfection studies in HL-1 cells indicated that T3 reduced LCC promoter activity. Deletion and mutation analysis of the LCC promoter region along with chromatin immunoprecipitation using anti-CREB antibody showed that CRE was essential for T3-mediated LCC gene expression. Transfection of dominant-negative CREB (mutated Ser133) and mutant thyroid hormone receptor (TR, mutated Cys51) abolished the T3-dependent effects, suggesting an association between both transcriptional factors. Co-immunoprecipitation documented an increased binding of TR with CREB after T3 treatment. The transcriptional cross-talk 3 between TR and CREB bound to CRE mediates T3-inhibited CREB activity and LCC expression. Thyroid hormone-induced TR binding of CREB inhibits CREB activity and LCC current expression, which may contribute to AF. These findings provide an important mechanistic insight into hyperthyroidism-induced AF.

A Novel Cytoplasmic Adaptor for Retinoic Acid Receptor (RAR) and Thyroid Receptor Functions as a Derepressor of RAR in the Absence of Retinoic Acid*

In most mammalian cells, the retinoic acid receptor (RAR) is nuclear rather than cytoplasmic, regardless of its cognate ligand, retinoic acid (RA). In testis Sertoli cells, however, RAR is retained in the cytoplasm and moves to the nucleus only when RA is supplied. This led us to identify a protein that regulates the translocation of RAR. From yeast two-hybrid screening, we identified a novel RAR-interacting protein called CART1 (cytoplasmic adaptor for RAR and TR). Systematic interaction assays using deletion mutants showed that the C-terminal CoRNR box of CART1 was responsible for the interaction with the NCoR binding region of RAR and TR. Such interaction was impaired in the presence of ligand RA, as further determined by GST pulldown assays in vitro and immunoprecipitation assays in vivo. Fluorescence microscopy showed that unliganded RAR was captured by CART1 in the cytoplasm, whereas liganded RAR was liberated and moved to the nucleus. Overexpression of CART1 blocked the transcriptional repressing activity of unliganded apoRAR, mediated by corepressor NCoR in the nucleus. CART1 siRNA treatment in a mouse Sertoli cell line, TM4, allowed RAR to move to the nucleus and blocked the derepressing function of CART1, suggesting that CART1 might be a cytoplasmic, testis-specific derepressor of RAR.

Modular Insulators: Genome Wide Search for Composite CTCF/Thyroid Hormone Receptor Binding Sites

The conserved 11 zinc-finger protein CTCF is involved in several transcriptional mechanisms, including insulation and enhancer blocking. We had previously identified two composite elements consisting of a CTCF and a TR binding site at the chicken lysozyme and the human c-myc genes. Using these it has been demonstrated that thyroid hormone mediates the relief of enhancer blocking even though CTCF remains bound to its binding site. Here we wished to determine whether CTCF and TR combined sites are representative of a general feature of the genome, and whether such sites are functional in regulating enhancer blocking. Genome wide analysis revealed that about 18% of the CTCF regions harbored at least one of the four different palindromic or repeated sequence arrangements typical for the binding of TR homodimers or TR/RXR heterodimers. Functional analysis of 10 different composite elements of thyroid hormone responsive genes was performed using episomal constructs. The episomal system allowed recapitulating CTCF mediated enhancer blocking function to be dependent on poly (ADP)-ribose modification and to mediate histone deacetylation. Furthermore, thyroid hormone sensitive enhancer blocking could be shown for one of these new composite elements. Remarkably, not only did the regulation of enhancer blocking require functional TR binding, but also the basal enhancer blocking activity of CTCF was dependent on the binding of the unliganded TR. Thus, a number of composite CTCF/TR binding sites may represent a subset of other modular CTCF composite sites, such as groups of multiple CTCF sites or of CTCF/Oct4, CTCF/Kaiso or CTCF/Yy1 combinations.

Dynamic Allostery Controls Coat Protein Conformer Switching during MS2 Phage Assembly

Previously, an RNA stem–loop (TR) encompassing 19 nt of the genome of bacteriophage MS2 was shown to act as an allosteric effector of conformational switching in the coat protein during in vitro capsid assembly. TR RNA binding to symmetric coat protein dimers results in conformational changes, principally at the FG-loop connecting the F and G β-strands in each subunit, yielding an asymmetric structure. The FG-loops define the quasi-equivalent conformers of the coat protein subunit (A, B, and C) in the T = 3 capsid. Efficient assembly of this capsid in vitro requires that both symmetrical and asymmetrical forms of the coat protein dimer be present in solution, implying that they closely resemble the quasi-equivalent dimers (A/B and C/C) seen in the final capsid. Experiments show that assembly can be triggered by a number of RNA stem–loops unrelated to TR in sequence and detailed secondary structure, suggesting that there is little sequence specificity to the allosteric effect. Since the stem–loop binding site on the coat protein dimer is distal to the FG-loops the mechanism of this switching effect needs to be investigated. We have analyzed the vibrational modes of both TR-bound and RNA-free coat protein dimers using an all-atom normal-mode analysis. The results suggest that asymmetric contacts between the A-duplex RNA phosphodiester backbone and the EF-loop in one coat protein subunit result in the FG-loop of that subunit becoming more dynamic, whilst the equivalent loop on the other monomer decreases its mobility. The increased dynamic behaviour occurs in the FG-loop of the subunit required to undergo the largest conformational change when adopting the quasi-equivalent B conformation. The free energy barrier on the pathway to form this new structure would consequently be reduced compared to the unbound subunit. Our results also imply that the allosteric effect should be independent of the base sequence of the bound stem–loop, as observed experimentally. As a test of this model, we also examined the vibrational modes of a known assembly mutant, W82R, which cannot assemble beyond dimer. This mutation leads to an increased mobility of the DE-loop rather than the FG-loop after TR binding, consistent with the non-assembling phenotype of this mutant protein.

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 (TRβ) 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 TRβ 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.

The functional relationship between corepressor N‐CoR and SMRT in mediating transcriptional repression by thyroid hormone receptor alpha

A central issue in mediating repression by nuclear hormone receptors is the distinct or redundant function between corepressors N-CoR and SMRT. To address the functional relationship between SMRT and N-CoR in TR-mediated repression, we have identified multiple TR-target genes, including BCL3 (B-Cell Lymphoma 3-encoded protein), Spot14 (thyroid hormone-inducible hepatic protein), FAS (fatty acid synthase), and ADRB2 (beta-adrenergic receptor). We demonstrated that siRNA treatment against either NCoR or SMRT is sufficient for the derepression of multiple TR-target genes. By the combination of sequence mining and physical association as determined by chromatin immunoprecipitation (ChIP) assays, we mapped the putative TREs (TR binding sites) in BCL3, Spot14, FASN, and ADRB2 genes. Our data clearly shows that the SMRT and N-CoR are independently recruited to various TR target genes. We also present evidence that overexpression of N-CoR can restore repression of endogenous genes after knocking-down SMRT. Finally, unliganded, corepressor-free TR is defective in repression and interacts with coactivator, p300. Collectively, these results suggest that both SMRT and N-CoR are limited in cells and that knocking down either of them results in corepressor-free TR and consequently derepression of TR target genes. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea gorvenment (MOST) (No. R13-2002-054-04002-0) & (KOSEF 2007-8-1158)

Regulation of Fibroblast Growth Factor Receptor-1 (Fgfr1) by Thyroid Hormone: Identification of a Thyroid Hormone Response Element in the Murine Fgfr1 Promoter

T(3) is essential for normal skeletal development, acting mainly via the TRalpha1 nuclear receptor. Nevertheless, the mechanisms of T(3) action in bone are poorly defined. Fibroblast growth factor receptor-1 (FGFR1) is also essential for bone formation. Fgfr1 expression and activity are positively regulated by T(3) in osteoblasts, and in mice that harbor a dominant negative PV mutation targeted to TRalpha1 or TRbeta, Fgfr1 expression is sensitive to skeletal thyroid status. To investigate mechanisms underlying T(3) regulation of FGFR1, we obtained primary calvarial osteoblasts from wild-type and TRbeta(PV/PV) littermate mice. T(3) treatment increased Fgfr1 expression 2-fold in wild-type cells, but 8-fold in TRbeta(PV/PV) osteoblasts. The 4-fold increased T(3) sensitivity of TRbeta(PV/PV) osteoblasts was associated with a markedly increased ratio of TRalpha1:TRbeta1 expression that resulted from reduced TRbeta1 expression in TRbeta(PV/PV) osteoblasts compared with wild-type. Bioinformatics and gel shift studies, and mutational analysis, identified a specific TR binding site 279-264 nucleotides upstream of the murine Fgfr1 promoter transcription start site. Transient transfection analysis of a series of Fgfr1 promoter 5'-deletion constructs, of a mutant reporter construct, and a series of heterologous promoter constructs, confirmed that this region of the promoter mediates a TR-dependent transcriptional response to T(3). Thus, in addition to indirect regulation of FGFR1 expression by T(3) reported previously, T(3) also activates the Fgfr1 promoter directly via a thyroid hormone response element located at positions -279/-264.

Proteomic Analysis of the Kaposi's Sarcoma-Associated Herpesvirus Terminal Repeat Element Binding Proteins

Terminal repeat (TR) elements of Kaposi's sarcoma-associated herpesvirus (KSHV), the potential origin sites of KSHV replication, have been demonstrated to play important roles in viral replication and transcription and are most likely also critical for the segregation of the KSHV genome to daughter cells. To search for the cellular proteins potentially involved in KSHV genome maintenance, we performed affinity chromatography analysis, using KSHV TR DNA as the affinity ligand. Proteomic analysis was then carried out to identify the TR-interacting proteins. We identified a total of 123 proteins from both KSHV-positive and -negative cells, among which most were identified exclusively from KSHV-positive cells. These proteins were categorized as proliferation/cell cycle regulatory proteins, proteins involved in spliceosome components, such as heterogeneous nuclear ribonuclear proteins, the DEAD/H family, the switch/sucrose nonfermenting protein family, splicing factors, RNA binding proteins, transcription regulation proteins, replication factors, modifying enzymes, and a number of proteins that could not be broadly categorized. To support the proteomic results, the presence of four candidate proteins, ATR, BRG1, NPM1 and PARP-1, in the elutions was further characterized in this study. The binding and colocalization of these proteins with the TR were verified using chromatin immunoprecipitation and immunofluorescence in situ hybridization analysis. These newly identified TR binding proteins provide a number of clues and potential links to understanding the mechanisms regulating the replication, transcription, and genome maintenance of KSHV. This study will facilitate the generation and testing of new hypotheses to further our understanding of the mechanisms involved in KSHV persistence and its associated pathogenesis.

Blue news: NTP binding properties of the blue-light sensitive YtvA protein from Bacillus subtilis

The blue-light sensitive protein YtvA from Bacillus subtilis is built of a photoactive, flavin-binding LOV (Light, Oxygen and Voltage) domain and a STAS domain with unknown function. Here we show that YtvA binds a fluorescent derivative of guanosine triphosphate (GTP TR) that can be displaced by both GTP or ATP. Unspecific NTP (N = G or A) binding is supported by the molecular model of YtvA-STAS. Blue-light activation of YtvA results in small and dark-reversible spectroscopic changes for GTP TR, suggesting that light-driven conformational changes are transmitted from the LOV core to the GTP TR binding site. These results support the idea that STAS domains may have a general NTP binding role and open a way to investigate the molecular functionality of YtvA-STAS.

Direct interactions between corepressors and coactivators permit the integration of nuclear receptor-mediated repression and activation.

The unliganded thyroid hormone receptor beta (TRbeta) represses the basal transcriptional activity of target genes, in part through interactions with the nuclear receptor corepressor (N-CoR). In this study we have identified a rather unexpected interaction between N-CoR and the nuclear receptor coactivator ACTR. We have demonstrated in vitro and in intact cells that N-CoR directly associates with ACTR and that the interaction surfaces on N-CoR and ACTR are distinct from those required for TR binding. The significance of this finding was demonstrated by showing that N-CoR facilitates an interaction between unliganded-TRbeta and ACTR. One possible consequence of the formation of the trimeric complex of N-CoR/ACTR/unliganded-TR is that N-CoR may raise the local concentration of ACTR at target gene promoters. In support of this hypothesis it was demonstrated that the presence of N-CoR can enhance TRbeta-mediated transcriptional activation. It is proposed, therefore, that TRbeta- mediated activation and repression are integrally linked in a manner that is not predicted by the current models of nuclear receptor action.