V-erbA generates ribosomes devoid of RPL11 and regulates translational activity in avian erythroid progenitors.

The v-erbA oncogene transforms chicken erythrocytic progenitors (T2EC) by blocking their differentiation and freezing them in a state of self-renewal. Transcriptomes of T2EC, expressing either v-erbA or a non-transforming form of v-erbA (S61G), were compared using serial analysis of gene expression and some, but not all, mRNA-encoding ribosomal proteins were seen to be affected by v-erbA. These results suggest that this oncogene could modulate the composition of ribosomes. In the present study, we demonstrate, using two-dimensional difference in gel electrophoresis, that v-erbA-expressing cells have a lower amount of RPL11 associated with the ribosomes. The presence of ribosomes devoid of RPL11 in v-erbA-expressing cells was further confirmed by immunoprecipitation. In order to assess the possible impact of these specialized ribosomes on the translational activity, we analyzed proteomes of either v-erbA or S61G-expressing cells using 2D/mass spectrometry, and identified nine proteins present in differing amounts within these cells. Among these proteins, we focused on HSP70 because of its involvement in erythroid differentiation. Our results indicate that, in v-erbA-expressing cells, hsp70 is not only transcribed but also translated more efficiently, as shown by polyribosome fractionation experiments. We demonstrate here, for the first time, the existence of ribosomes with different protein components, notably ribosomes devoid of RPL11, and a regulation of mRNA translation depending on v-erbA oncogene expression.

Transcription factor NF-Y is a functional regulator of the transcription of core clock gene Bmal1.

The circadian clock enables organisms to adjust to daily environmental changes and synchronize multiple molecular, biochemical, physiological, and behavioral processes accordingly. In mammalian clock work, Bmal1 is the most important core clock gene, which works with another core clock gene Clock to drive the expression of other clock genes and clock-controlled genes. However, the regulation of Bmal1 has not been fully understood. This work was aimed at identifying the positive regulator(s) of Bmal1 transcription. A series of 5' deletion reporter constructs was generated, and binding site mutations of mouse Bmal1 promoter fragments were cloned into pGL3-basic and pGL3(R2.1)-basic plasmids and transfected into NIH 3T3 cells. Luciferase activity was either measured 48 h after transfection or recorded for 4 days after serum shock. DNA affinity precipitation assay was used to detect the transcription factors binding to Bmal1 promoter. Small interfering RNA against nuclear factor Y, subunit A (NF-YA) and dominant negative NF-YA were employed to study the role of NF-Y in Bmal1 transcription regulation. Deletion and mutation analyses identified two clusters of CCAAT/GC-boxes at the proximal region of Bmal1 promoter as the activating cis-elements. Bmal1 promoter activity was up-regulated by NF-Y and/or Sp1 and repressed by dominant negative NF-YA or siRNA against NF-YA. The activation of Bmal1 promoter activity by NF-Y and Sp1 was inhibited by Rev-Erbα. DNA affinity precipitation assay showed that NF-Y and Sp1 bound to the two CCAAT/GC clusters of Bmal1 promoter. These results indicate that NF-Y is a functional activator of Bmal1 transcription and it cooperates with Sp1 and Rev-Erbα to generate the daily cycle of Bmal1 expression.

Multiple novel signals mediate thyroid hormone receptor nuclear import and export.

Thyroid hormone receptor (TR) is a member of the nuclear receptor superfamily that shuttles between the cytosol and nucleus. The fine balance between nuclear import and export of TR has emerged as a critical control point for modulating thyroid hormone-responsive gene expression; however, sequence motifs of TR that mediate shuttling are not fully defined. Here, we characterized multiple signals that direct TR shuttling. Along with the known nuclear localization signal in the hinge domain, we identified a novel nuclear localization signal in the A/B domain of thyroid hormone receptor α1 that is absent in thyroid hormone receptor ß1 and inactive in the oncoprotein v-ErbA. Our prior studies showed that thyroid hormone receptor α1 exits the nucleus through two pathways, one dependent on the export factor CRM1 and the other CRM1-independent. Here, we identified three novel CRM1-independent nuclear export signal (NES) motifs in the ligand-binding domain as follows: a highly conserved NES in helix 12 (NES-H12) and two additional NES sequences spanning helix 3 and helix 6, respectively. Mutations predicted to disrupt the α-helical structure resulted in a significant decrease in NES-H12 activity. The high degree of conservation of helix 12 suggests that this region may function as a key NES in other nuclear receptors. Furthermore, our mutagenesis studies on NES-H12 suggest that altered shuttling of thyroid hormone receptor ß1 may be a contributing factor in resistance to thyroid hormone syndrome. Taken together, our findings provide a detailed mechanistic understanding of the multiple signals that work together to regulate TR shuttling and transcriptional activity, and they provide important insights into nuclear receptor function in general.

Palmitate alters the rhythmic expression of molecular clock genes and orexigenic neuropeptide Y mRNA levels within immortalized, hypothalamic neurons.

The control of energy homeostasis within the hypothalamus is under the regulated control of homeostatic hormones, nutrients and the expression of neuropeptides that alter feeding behavior. Elevated levels of palmitate, a predominant saturated fatty acid in diet and fatty acid biosynthesis, alter cellular function. For instance, a key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. To determine these mechanisms we used an immortalized, clonal, hypothalamic cell line, mHypoE-44, to demonstrate that palmitate directly alters the expression of molecular clock components, by increasing Bmal1 and Clock, or by decreasing Per2, and Rev-erbα, their mRNA levels and altering their rhythmic period within individual neurons. We found that these neurons endogenously express the orexigenic neuropeptides NPY and AgRP, thus we determined that palmitate administration alters the mRNA expression of these neuropeptides as well. Palmitate treatment causes a significant increase in NPY mRNA levels and significantly alters the phase of rhythmic expression. We explored the link between AMPK and the expression of neuropeptide Y using the AMPK inhibitor compound C and the AMP analog AICAR. AMPK inhibition decreased NPY mRNA. AICAR also elevated basal NPY, but prevented the palmitate-mediated increase in NPY mRNA levels. We postulate that this palmitate-mediated increase in NPY and AgRP synthesis may initiate a detrimental positive feedback loop leading to increased energy consumption.

Recruitment of the oncoprotein v-ErbA to aggresomes.

Aggresome formation, a cellular response to misfolded protein aggregates, is linked to cancer and neurodegenerative disorders. Previously we showed that Gag-v-ErbA (v-ErbA), a retroviral variant of the thyroid hormone receptor (TRα1), accumulates in and sequesters TRα1 into cytoplasmic foci. Here, we show that foci represent v-ErbA targeting to aggresomes. v-ErbA colocalizes with aggresomal markers, proteasomes, hsp70, HDAC6, and mitochondria. Foci have hallmark characteristics of aggresomes: formation is microtubule-dependent, accelerated by proteasome inhibitors, and they disrupt intermediate filaments. Proteasome-mediated degradation is critical for clearance of v-ErbA and T(3)-dependent TRα1 clearance. Our studies highlight v-ErbA's complex mode of action: the oncoprotein is highly mobile and trafficks between the nucleus, cytoplasm, and aggresome, carrying out distinct activities within each compartment. Dynamic trafficking to aggresomes contributes to the dominant negative activity of v-ErbA and may be enhanced by the viral Gag sequence. These studies provide insight into novel modes of oncogenesis across multiple cellular compartments.

The effect of oncoprotein v-erbA on thyroid hormone-regulated genes in hepatocytes and their potential role in hepatocellular carcinoma.

Mutant forms of thyroid hormone receptor (TR) with dominant negative activity are frequently found in human hepatocellular carcinoma (HCC). Interestingly, the v-erbA oncogene, known to exert a dominant-negative effect on the expression of thyroid hormone (T3)-responsive genes, led to the development of HCC in a transgenic mouse model. Thus it is possible that the oncogenic activity of v-erbA in hepatocytes may be mediated by its dominant negative activity on T3-responsive genes. Microarray analysis was used to identify genes differentially expressed in murine hepatocytes in culture (AML12 cells) stably transfected with v-erbA and exposed to T3. The Affymetrix GeneChip Mouse Genome 430 2.0 array consisted of over 39,000 transcripts representing well-known genes. We have identified twenty T3-responsive genes that are negatively regulated by v-erbA at 3 h, and eighteen genes at 24 h, such as follistatin, activin ßC, thrombomodulin, Six1, Rasgrp3 and Ndrg2, as well as genes that are regulated by v-erbA only, such as angiopoietin 1 and Igfr2. We have identified T3 responsive genes that are dysregulated by v-erbA. These genes are known to be involved in carcinogenesis. Our studies may provide insight into the potential role of mutant forms of TR in the pathogenesis of HCC.

Association of v-ErbA with Smad4 disrupts TGF-beta signaling.

Disruption of the transforming growth factor-beta (TGF-beta) pathway is observed in the majority of cancers. To further understand TGF-beta pathway inactivation in cancer, we stably expressed the v-ErbA oncoprotein in TGF-beta responsive cells. v-ErbA participates in erythroleukemic transformation of cells induced by the avian erythroblastosis virus (AEV). Here we demonstrate that expression of v-ErbA was sufficient to antagonize TGF-beta-induced cell growth inhibition and that dysregulation of TGF-beta signaling required that v-ErbA associate with the Smad4 which sequesters Smad4 in the cytoplasm. We also show that AEV-transformed erythroleukemia cells were resistant to TGF-beta-induced growth inhibition and that TGF-beta sensitivity could be recovered by reducing v-ErbA expression. Our results reveal a novel mechanism for oncogenic disruption of TGF-beta signaling and provide a mechanistic explanation of v-ErbA activity in AEV-induced erythroleukemia.

Multiple mutations contribute to repression by the v-Erb A oncoprotein.

The v-Erb A oncoprotein of avian erythroblastosis virus is derived from c-Erb A, a hormone-activated transcription factor. Notably, v-Erb A has sustained multiple mutations relative to c-Erb A and functions as a constitutive transcriptional repressor. We report here an analysis of the contributions of these different mutations to v-Erb A function. Our experiments demonstrate that two amino-acid differences between v-Erb A and c-Erb A, located in the 'I-box,' alter the dimerization properties of the viral protein, resulting in more stable homodimer formation, increased corepressor binding, and increased target gene repression. An additional amino-acid difference between v- and c-Erb A, located in helix 3 of the hormone binding domain, renders corepressor binding by the viral protein more resistant to release by thyroid hormone. Finally, we report that a C-terminal truncation in v-Erb A not only inhibits exchange of corepressor and coactivator, as previously noted, but also permits v-Erb A to recruit both SMRT and N-CoR corepressors, whereas c-Erb A is selective for N-CoR. The latter two mutations in v-Erb A also impair its ability to suppress c-Jun function in response to T3 hormone. We propose that the acquisition of oncogenic potential by the v-Erb A protein was a multistep process involving a series of mutations that alter the transcriptional repressive properties of the viral protein through multiple mechanisms.

Cancer promoted by the oncoprotein v-ErbA may be due to subcellular mislocalization of nuclear receptors.

The retroviral v-ErbA oncoprotein is a highly mutated variant of the thyroid hormone receptor alpha (TRalpha), which is unable to bind T(3) and interferes with the action of TRalpha in mammalian and avian cancer cells. v-ErbA dominant-negative activity is attributed to competition with TRalpha for T(3)-responsive DNA elements and/or auxiliary factors involved in the transcriptional regulation of T(3)-responsive genes. However, competition models do not address the altered subcellular localization of v-ErbA and its possible implications in oncogenesis. Here, we report that v-ErbA dimerizes with TRalpha and the retinoid X receptor and sequesters a significant fraction of the two nuclear receptors in the cytoplasm. Recruitment of TRalpha to the cytoplasm by v-ErbA can be partially reversed in the presence of ligand and when chromatin is disrupted by the histone deacetylase inhibitor trichostatin A. These results define a new mode of action of v-ErbA and illustrate the importance of cellular compartmentalization in transcriptional regulation and oncogenesis.

The orphan receptor Rev-erbalpha gene is a target of the circadian clock pacemaker.

Rev-erbalpha is a ubiquitously expressed orphan nuclear receptor which functions as a constitutive transcriptional repressor and is expressed in vertebrates according to a robust circadian rhythm. We report here that two Rev-erbalpha mRNA isoforms, namely Rev-erbalpha1 and Rev-erbalpha 2, are generated through alternative promoter usage and that both show a circadian expression pattern in an in vitro system using serum-shocked fibroblasts. Both promoter regions P1 (Rev-erbalpha1) and P2 (Rev-erbalpha2) contain several E-box DNA sequences which function as response elements for the core circadian-clock components: CLOCK and BMAL1. The CLOCK-BMAL1 heterodimer stimulates the activity of both P1 and P2 promoters in transient transfection assay by 3-6-fold. This activation was inhibited by the overexpression of CRY1, a component of the negative limb of the circadian transcriptional loop. Critical E-box elements were mapped within both promoters. This regulation is conserved in vertebrates since we found that the CLOCK-BMAL1 heterodimer also regulates the zebrafish Rev-erbalpha gene. In line with these data Rev-erbalpha circadian expression was strongly impaired in the livers of Clock mutant mice and in the pineal glands of zebrafish embryos treated with Clock and Bmal1 antisense oligonucleotides. Together these data demonstrate that CLOCK is a critical regulator of Rev-erbalpha circadian gene expression in evolutionarily distant vertebrates and suggest a role for Rev-erbalpha in the circadian clock output.

V-erba homodimers mediate the potent dominant negative activity of v-erba on everted repeats.

The oncoprotein v-erbA is a mutated form of TRalpha1 that is unable to bind thyroid hormone (T3). V-erbA homodimerizes or heterodimerizes with retinoid X receptor (RXR) on core motifs arranged as direct, everted, or inverted repeats (DRs, ERs, or IRs). We created a series of v-erbA mutants in order to obtain a better understanding of the role of v-erbA homodimers versus v-erbA-RXR heterodimers in the dominant negative activity of v-erbA on ERs (the most potent v-erbA response elements). We found that one of these mutants, v-erbA mutant E325A, is able to homodimerize but unable to heterodimerize with RXR on ERs. Our data also suggest that v-erbA homodimers interact preferentially with the corepressor NCoR over SMRT and that the interaction with corepressors is stronger with v-erbA homodimers over v-erbA-RXR heterodimers. Furthermore, functional studies showed that v-erbA homodimers rather than v-erbA-RXR heterodimers mediate the dominant negative activity of v-erbA on ERs.

Repression of vascular endothelial growth factor A in glioblastoma cells using engineered zinc finger transcription factors.

Angiogenic factors are necessary for tumor proliferation and thus are attractive therapeutic targets. In this study, we have used engineered zinc finger protein (ZFP) transcription factors (TFs) to repress expression of vascular endothelial growth factor (VEGF)-A in human cancer cell lines. We create potent transcriptional repressors by fusing a designed ZFP targeted to the VEGF-A promoter with either the ligand-binding domain of thyroid hormone receptor alpha or its viral relative, vErbA. Moreover, this ZFP-vErbA repressor binds its intended target site in vivo and mediates the specific deacetylation of histones H3 and H4 at the targeted promoter, a result that emulates the natural repression mechanism of these domains. The potential therapeutic relevance of ZFP-mediated VEGF-A repression was addressed using the highly tumorigenic glioblastoma cell line U87MG. Despite the aberrant overexpression of VEGF-A in this cell line, engineered ZFP TFs were able to repress the expression of VEGF-A by >20-fold. The VEGF-A levels observed after ZFP TF-mediated repression were comparable to those of a nonangiogenic cancer line (U251MG), suggesting that the degree of repression obtained with the ZFP TF would be sufficient to suppress tumor angiogenesis. Thus, engineered ZFP TFs are shown to be potent regulators of gene expression with therapeutic promise in the treatment of disease.

Sequences required for the transition from monomeric to homodimeric forms of thyroid hormone receptor alpha and v-erbA.

Thyroid hormone receptor alpha (TRalpha) and the oncoprotein v-erbA (a mutated form of TRalpha incapable of binding T3) bind as heterodimers with retinoid X receptor (RXR) to DNA sequences with different orientations of AGGTCA half sites. v-erbA can also form homodimers, whereas, TRalpha1 homodimerizes poorly. Therefore, in order to obtain a better understanding for the distinct homodimerization properties between TRalpha1 and v-erbA, we created chimeras between these two receptors and tested their abilities to homodimerize on direct and everted repeats (DRs, ERs). We found that the enhanced homodimerization properties of v-erbA compared to TRalpha1 map to isoleucine at position 339 in conjunction with serine at position 351 and alanine at position 358. Our data indicate that the methyl group in isoleucine at position 339 plays an important role in v-erbA homodimerization, particularly on ER 6. Functional studies with I339V+S351P+A358T, a v-erbA mutant unable to homodimerize but still able to heterodimerize with RXR on ERs and DRs, indicate that v-erbA-RXR heterodimers mediate the dominant negative activity of v-erbA on DRs. However, the repressor activity of this mutant is weaker than that of the wild type v-erbA on ERs, suggesting that v-erbA homodimers rather than v-erbA-RXR heterodimers mediate the potent dominant negative activity of v-erbA on ERs.

Dimerization of v-erbA on inverted repeats.

Thyroid hormone receptors (TRs) and the oncoprotein v-erbA can heterodimerize with retinoid X receptor (RXR) on core motifs arranged as inverted repeats (IR0) which contain the consensus sequence AGGTCA. On this core motif, v-erbA can also form homodimers whereas TRs homodimerize very poorly. Therefore to obtain a better understanding of distinct homodimerization properties of TR alpha 1 as compared to those of v-erbA, we created chimeras between these two receptors and tested their abilities to homodimerize on IR0. We found that the enhanced homodimerization properties of v-erbA compared to those of TR alpha 1 on IR0 map to amino acids 107-156 in v-erbA/121-170 in TR alpha 1 (VT-2 chimera). Furthermore, functional studies on transient transfections showed that v-erbA-RXR heterodimers do not mediate the dominant negative activity of v-erbA on an inverted repeat response element. These data, in conjunction with our previous studies, indicate that v-erbA homodimers mediate the repressor activity of v-erbA on IR0.

The v-erbA oncogene blocks expression of alpha2/beta1 integrin a normal inhibitor of erythroid progenitor proliferation.

T2EC are chicken erythrocytic progenitors that balance between self-renewal and differentiation as a function of response to specific growth factors. Their transformation by the v-erbA oncogene locks them into the self-renewal program. We show here that the expression of the VLA-2 integrin alpha2 subunit mRNA is downregulated by v-erbA and that VLA-2 engagement and clustering, brought about by treatment with an alpha2-specific antibody or by culture on the VLA-2 ligand collagen I, inhibits T2EC proliferation. From competition studies using antibodies, VLA-2 was shown to be involved in the collagen-induced response. While engagement of VLA-2 inhibited proliferation, it was not sufficient to induce differentiation. The transformation of T2EC by v-erbA decreased their interaction with collagen I and the VLA-2 brake on cell proliferation, which may account for the increased proliferation potential of transformed erythrocytic progenitors and for their shedding into the blood of infected chickens. Our data suggest that the interaction between erythroid progenitors and collagen, mediated by VLA-2, play a major role in the control of erythropoiesis in vitro and that this pathway is a target of the v-erbA oncogene.

In vivo repression of an erythroid-specific gene by distinct corepressor complexes.

To assess the mechanisms of repression of the erythroid-specific carbonic anhydrase II (CAII) locus we used chromatin immunoprecipitation and show that an NCoR-histone deacetylase (HDAC)3 complex is recruited by the nuclear receptor v-ErbA to the intronic HS2 enhancer turning it into a potent silencer. Furthermore we demonstrate that efficient CAII silencing requires binding of a MeCP2-targeted HDAC-containing corepressor complex to the hypermethylated CpG-island at the promoter. Activation of transcription by either AZAdC or thyroid hormone results in loss of one of the two corepressor complexes. Thyroid hormone further replaces the enhancer-bound NCoR-corepressor complex by the TRAP220 coactivator. Treatment with the HDAC inhibitor trichostatin A (TSA) causes activation of CAII transcription and histone H3 and H4 hyperacetylation at the enhancer, apparently without affecting binding of the two corepressor complexes. Unexpectedly, histone H3 and H4 at the fully repressed promoter are already hyperacetylated despite the close apposition of the MeCP2-targeted HDAC complex. Acetylation of histone H4, but not H3, at the promoter is moderately increased following TSA treatment. Our data suggest that the hyperacetylated but repressed CAII promoter is (partially) remodeled and primed for activation in v-ErbA-transformed cells.

The v-ErbA oncoprotein quenches the activity of an erythroid-specific enhancer.

v-ErbA is a mutated variant of thyroid hormone receptor (TRalpha/NR1A1) borne by the Avian Erythroblastosis virus causing erythroleukemia. TRalpha is known to activate transcription of specific genes in the presence of its cognate ligand, T3 hormone, while in its absence it represses it. v-ErbA is unable to bind ligand, and hence is thought to contribute to leukemogenesis by actively repressing erythroid-specific genes such as the carbonic anhydrase II gene (CA II). In the prevailing model, v-ErbA occludes liganded TR from binding to its cognate elements and constitutively interacts with the corepressors NCoR/SMRT. We previously identified a v-ErbA responsive element (VRE) within a DNase I hypersensitive region (HS2) located in the second intron of the CA II gene. We now show that HS2 fulfils all the requirements for a genuine enhancer that functions independent of its orientation and position with a profound erythroid-specific activity in normal erythroid progenitors (T2ECs) and in leukemic erythroid cell lines. We find that the HS2 enhancer activity is governed by two adjacent GATA-factor binding sites. v-ErbA as well as unliganded TR prevent HS2 activity by nullifying the positive function of factors bound to GATA-sites. However, v-ErbA, in contrast to TR, does not convey active repression to silence the transcriptional activity intrinsic to a heterologous tk promoter. We propose that depending on the sequence and context of the binding site, v-ErbA contributes to leukemogenesis by occluding liganded TR as well as unliganded TR thereby preventing activation or repression, respectively.

Transcriptional repression by thyroid hormone receptors. A role for receptor homodimers in the recruitment of SMRT corepressor.

Nuclear hormone receptors, such as the thyroid hormone receptors (T3Rs) and retinoid X receptors (RXRs), are ligand-regulated transcription factors that control key aspects of metazoan gene expression. T3Rs can bind to DNA either as receptor homodimers or as heterodimers with RXRs. Once bound to DNA, nuclear hormone receptors regulate target gene expression by recruiting auxiliary proteins, denoted corepressors and coactivators. We report here that T3R homodimers assembled on DNA exhibit particularly strong interactions with the SMRT corepressor, whereas T3R.RXR heterodimers are inefficient at binding to SMRT. Mutants of T3R that exhibit enhanced repression properties, such as the v-Erb A oncoprotein or the T3Rbeta-Delta432 mutant found in human resistance to thyroid hormone syndrome, display enhanced homodimerization properties and exhibit unusually strong interactions with the SMRT corepressor. Significantly, the topology of a DNA binding site can determine whether that site recruits primarily homodimers or heterodimers and therefore whether corepressor is efficiently or inefficiently recruited to the resulting receptor-DNA complex. We suggest that T3R homodimers, and not heterodimers, may be important mediators of transcriptional repression and that the nature of the DNA binding site, by selecting for receptor homodimers or heterodimers, can influence the ability of the receptor to recruit corepressor.

A choice between transcriptional enhancement and repression by the v-erbA oncoprotein governed by one nucleotide in a thyroid hormone responsive half site.

The v-erbA oncoprotein (P75gag-v-erbA) can repress thyroid hormone receptor induced transcriptional activation of target genes. A central question is how hormone responsive elements in a target gene determine the transcriptional regulation mediated by P75gag-v-erbA. We addressed this with receptors chimeric between P75gag-v-erbA and thyroid hormone receptor (TR) by testing their regulatory activities on thyroid hormone response elements (TREs) differing in the sequence of the consensus core recognition motif AGGTCA. We report here that enhances, TR dependent transcriptional activation is conferred by P75gag-v-erbA when the thymidine in the half site recognition motif is exchanged for an adenosine. The enhancement was independent of the DNA binding region of P75gag-v-erbA, whereas increased expression of corepressor abolished the enhancing effect. The data indicate that the enhancement results from an impaired DNA binding by the oncoprotein combined with an effective scavenging of corepressors. Our data thus suggest the P75gag-v-erbA indirectly can contribute to enhancement of thyroid hormone induced gene expression.

Thyroid hormone receptor, v-ErbA, and chromatin.

The thyroid hormone receptor and the highly related viral oncoprotein v-erbA are found exclusively in the nucleus as stable constituents of chromatin. Unlike most transcriptional regulators, the thyroid hormone receptor binds with comparable affinity to naked and nucleosomal DNA. In vitro reconstitution experiments and in vivo genomic footprinting have delineated the chromatin structural features that facilitate association with the receptor. Chromatin bound thyroid hormone receptor and v-erbA generate Dnase I hypersensitive sites independent of ligand. The unliganded thyroid hormone receptor and v-erbA associate with a corepressor complex containing NCoR, SIN3, and histone deacetylase. The enzymatic activity of the deacetylase and a chromatin environment are essential for the dominant repression of transcription by both the unliganded thyroid hormone receptor and v-erbA. In the presence of ligand, the thyroid hormone receptor undergoes a conformational change that weakens interactions with the corepressor complex while facilitating the recruitment of transcriptional coactivators such as p300 and PCAF possessing histone acetyltransferase activity. The ligand-bound thyroid hormone receptor directs chromatin disruption events in addition to histone acetylation. Thus, the thyroid hormone receptor and v-erbA make very effective use of their stable association with chromatin and their capacity to alter the chromatin environment as a major component of the transcription regulation process. This system provides an exceptionally useful paradigm for investigating the structural and functional consequences of targeted chromatin modification.