Ligand-dependent degradation of retinoid X receptors does not require transcriptional activity or coactivator interactions.

Cells utilize ubiquitin-mediated proteolysis to regulate the activity of numerous proteins involved in signal transduction, cell cycle control, and transcriptional regulation. For a number of transcription factors, there appears to be a direct correlation between transcriptional activity and protein instability, suggesting that cells use targeted destruction as one method to down-regulate or attenuate gene expression. In this report we demonstrate that retinoid X receptors (RXRs) which function as versatile mediators of nuclear hormone-dependent gene expression are marked for destruction upon binding agonist ligands. Interestingly, when RXR serves as a heterodimeric partner for retinoic acid (RAR) or thyroid hormone (TR) receptors, binding of agonists by RAR or TR leads to degradation of both the transcriptionally active RAR or TR subunits as well as the transcriptionally inactive RXR subunit. Furthermore, using a series of mutants in the ligand-dependent activation domain (activation function 2), we demonstrate that agonist-stimulated degradation of RXR does not require corepressor release, coactivator binding, or transcriptional activity. Taken together, the data suggest a model for targeted destruction of transcription factors based on structural or conformational signals as opposed to functional coupling with gene transcription.

Three amino acids specify coactivator choice by retinoid X receptors.

Binding of agonists to nuclear receptors results in a conformational change in receptor structure that promotes interaction between activated receptors and coactivators. Receptor-coactivator interactions are mediated by the agonist-dependent formation of a hydrophobic pocket on the part of receptors, and short leucine-rich sequences termed LxxLL motifs or nuclear receptor boxes present in coactivators. RXR-PPARgamma (retinoid X receptor-peroxisome proliferator-activated receptor-gamma) heterodimers play important roles in adipocyte and macrophage differentiation and have been implicated as therapeutic targets in diabetes, atherosclerosis, and cancer. Analysis of interactions between RXR-PPARgamma heterodimers and coactivator nuclear receptor boxes suggests that RXR and PPARgamma can distinguish among coactivators by recognizing distinct structural features of nuclear receptor boxes. The results also indicate that coactivator choice by RXR is mediated by three nonconserved amino acids of the nuclear receptor box. The ability of an optimized seven-amino acid nuclear receptor box to specifically interact with RXR and function as a selective inhibitor suggests the coactivator-binding pocket may serve as a new target for drug discovery.

Transactivation by retinoid X receptor-peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimers: intermolecular synergy requires only the PPARgamma hormone-dependent activation function.

The ability of DNA sequence-specific transcription factors to synergistically activate transcription is a common property of genes transcribed by RNA polymerase II. The present work characterizes a unique form of intermolecular transcriptional synergy between two members of the nuclear hormone receptor superfamily. Heterodimers formed between peroxisome proliferator-activated receptor gamma (PPARgamma), an adipocyte-enriched member of the superfamily required for adipogenesis, and retinoid X receptors (RXRs) can activate transcription in response to ligands specific for either subunit of the dimer. Simultaneous treatment with ligands specific for both PPARgamma and RXR has a synergistic effect on the transactivation of reporter genes and on adipocyte differentiation in cultured cells. Mutation of the PPARgamma hormone-dependent activation domain (named tauc or AF-2) inhibits the ability of RXR-PPARgamma heterodimers to respond to ligands specific for either subunit. In contrast, the ability of RXR- and PPARgamma-specific ligands to synergize does not require the hormone-dependent activation domain of RXR. The results of in vitro and in vivo experiments indicate that binding of ligands to RXR alters the conformation of the dimerization partner, PPARgamma, and modulates the activity of the heterodimer in a manner independent of the RXR hormone-dependent activation domain.

Retinoid receptors in development and disease.

Nuclear receptors comprise a large family of ligand-dependent transcription factors that display considerable specificity in and selectivity in regulating the genetic programs they ultimately influence. The response to retinoic acid (RA) is mediated by two families of transcription factors which include the retinoic acids receptors (RARs) and the retinoid X receptors (RXRs). In human acute promyelocytic leukemia (APL), RAR alpha becomes an activated oncogene as a consequence of its fusion to the PML locus. Because patients with APL can be induced into remission with high dose RA therapy, we propose that PML-RAR is a new class of dominant negative oncogene that disrupts a structure that includes at least five other proteins. This mega-complex, referred to as a "POD", is disrupted in leukemic cells expressing the oncoprotein and is reassembled following high dose RA therapy in both cell culture an in patients.

The phantom ligand effect: allosteric control of transcription by the retinoid X receptor.

Regulation of gene expression via allosteric control of transcription is one of the fundamental concepts of molecular biology. Studies in prokaryotes have illustrated that binding of small molecules or ligands to sequence-specific transcription factors can produce conformational changes at a distance from the binding site. These ligand-induced changes can dramatically alter the DNA binding and/or trans-activation abilities of the target transcription factors. In this work, analysis of trans-activation by members of the steroid and thyroid hormone receptor superfamily identifies a unique form of allosteric control, the phantom ligand effect. Binding of a novel ligand (LG100754) to one subunit (RXR) of a heterodimeric transcription factor results in a linked conformational change in the second noncovalently bound subunit of the heterodimer (RAR). This conformational change results in both the dissociation of corepressors and association of coactivators in a fashion mediated by the activation function of the non-liganded subunit. Without occupying the RAR hormone binding pocket, binding of LG100754 to RXR mimics exactly the effects observed when hormone is bound to RAR. Thus, LG100754 behaves as a phantom ligand.

Activation of specific RXR heterodimers by an antagonist of RXR homodimers.

Retinoid X receptor (RXR) plays a central role in the regulation of many intracellular receptor signalling pathways and can mediate ligand-dependent transcription, acting as a homodimer or as a heterodimer. Here we identify an antagonist towards RXR homodimers which also functions as an agonist when RXR is paired as a heterodimer to specific partners, including peroxisome proliferator-activated receptor and retinoic acid receptor. This dimer-selective ligand confers differential interactions on the transcription machinery: the antagonist promotes association with TAF110 (TATA-binding protein (TBP)-associated factor 110) and the co-repressor SMRT, but not with TBP, and these properties are distinct from pure RXR agonists. This unique class of RXR ligands will provide a means to control distinct target genes at the level of transcription and allow the development of retinoids with a new pharmacological action.

Role of CBP/P300 in nuclear receptor signalling.

The nuclear receptor superfamily includes receptors for steroids, retinoids, thyroid hormone and vitamin D, as well as many related proteins. An important feature of the action of the lipophilic hormones and vitamins is that the maintenance of homeostatic function requires both intrinsic positive and negative regulation. Here we provide in vitro and in vivo evidence that identifies the CREB-binding protein (CBP) and its homologue P300 (refs 6,7) as cofactors mediating nuclear-receptor-activated gene transcription. The role of CBP/P300 in the transcriptional response to cyclic AMP, phorbol esters, serum, the lipophilic hormones and as the target of the E1A oncoprotein suggests they may serve as integrators of extracellular and intracellular signalling pathways.

Activation and repression by nuclear hormone receptors: hormone modulates an equilibrium between active and repressive states.

Transactivation-defective retinoid X and thyroid hormone receptors have been used to examine mechanisms of hormonal activation. Activation and repression of transcription by retinoid X and thyroid hormone receptors are shown to be mediated by physically distinct and functionally independent regions of the hormone binding domain. Nevertheless, the ability of receptors to respond to hormone requires communication between both functional domains. Deletion of the hormone-dependent transactivation function of the retinoid X receptor, the common subunit of heterodimeric nuclear receptors, significantly impairs hormone-dependent transcription by retinoic acid, thyroid hormone, and vitamin D receptors. The results indicate that receptors do not exist in static off and on conformations but that hormone alters an equilibrium between inactive and active states.

Interactions between the retinoid X receptor and a conserved region of the TATA-binding protein mediate hormone-dependent transactivation.

The retinoid X receptor (RXR) participates in a wide array of hormonal signaling pathways, either as a homodimer or as a heterodimer, with other members of the steroid and thyroid hormone receptor superfamily. In this report the ligand-dependent transactivation function of RXR has been characterized, and the ability of RXR to interact with components of the basal transcription machinery has been examined. In vivo and in vitro experiments indicate the RXR ligand-binding domain makes a direct, specific, and ligand-dependent contact with a highly conserved region of the TATA-binding protein. The ability of mutations that reduce ligand-dependent transcription by RXR to disrupt the RXR-TATA-binding protein interaction in vivo and in vitro suggests that RXR makes direct contact with the basal transcription machinery to achieve activation.

Genetic dissection of centromere function.

A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation. Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex. Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined.

Cell-cell interactions trigger the rapid induction of a specific high mobility group-like protein during early stages of conjugation in Tetrahymena.

Conjugation in Tetrahymena represents an ordered developmental pathway which represents the sexual phase of the ciliate life cycle. This pathway is initiated when starved cells of opposite mating types are mixed and are allowed to make a series of cell-cell contacts (a period termed costimulation) which lead to the formation of mating pairs. Here, we demonstrate that two previously described abundant high mobility group (HMG)-like proteins, HMG B and HMG C, whose synthesis appeared to be coordinately regulated in vegetative cells, are not required during the same stages of conjugation. The level of mRNA for both HMG B and HMG C is high during vegetative growth and during the development of new macronuclei. However, specific induction of HMG B mRNA is observed soon after cells of opposite mating types are mixed. Thus, the genes which encode HMG B and HMG C in Tetrahymena can be controlled independently or coordinately. Nuclear run-on experiments show that a significant factor underlying the rapid induction of HMG B message early in the sexual cycle is an increase in the transcriptional activity of the HMG B gene. Experiments are presented which show that this induction of HMG B message requires protein synthesis and is dependent upon the cell-cell contacts made during costimulation. Essentially all of the HMG B protein, which is newly synthesized during this period, is targeted to parental macronuclei where it serves an as yet undetermined function(s).

Macronuclei and micronuclei in Tetrahymena thermophila contain high-mobility-group-like chromosomal proteins containing a highly conserved eleven-amino-acid putative DNA-binding sequence.

HMG (high-mobility-group protein) B and HMG C are abundant nonhistone chromosomal proteins isolated from Tetrahymena thermophila macronuclei with solubilities, molecular weights, and amino acid compositions like those of vertebrate HMG proteins. Genomic clones encoding each of these proteins have been sequenced. Both are single-copy genes that encode single polyadenylated messages whose amounts are 10 to 15 times greater in growing cells than in starved, nongrowing cells. The derived amino acid sequences of HMG B and HMG C contain a highly conserved sequence, the HMG 1 box, found in vertebrate HMGs 1 and 2, and we speculate that this sequence may represent a novel, previously unrecognized DNA-binding motif in this class of chromosomal proteins. Like HMGs 1 and 2, HMGs B and C contain a high percentage of aromatic amino acids. However, the Tetrahymena HMGs are small, are associated with nucleosome core particles, and can be specifically extracted from macronuclei by elutive intercalation, properties associated with vertebrate HMGs 14 and 17, not HMGs 1 and 2. Thus, it appears that these Tetrahymena proteins have features in common with both of the major subgroups of higher eucaryotic HMG proteins. Surprisingly, a linker histone found exclusively in transcriptionally inactive micronuclei also has several HMG-like characteristics, including the ability to be specifically extracted from nuclei by elutive intercalation and the presence of the HMG 1 box. This finding suggests that at least in T. thermophila, proteins with HMG-like properties are not restricted to regions of transcriptionally active chromatin.

Characterization of phosphorylation sites in histone H1 in the amitotic macronucleus of Tetrahymena during different physiological states.

Histone H1 is highly phosphorylated in transcriptionally active, amitotic macronuclei of Tetrahymena during vegetative growth. However, the level of H1 phosphorylation changes dramatically in response to different physiological conditions. H1 is hyperphosphorylated in response to heat shock and during prezygotic stages of conjugation. Conversely, H1 is largely dephosphorylated during prolonged starvation and during elimination of parental macronuclei during conjugation. Mapping of phosphorylation sites within H1 indicates that phosphorylation occurs at multiple sites in the amino-terminal portion of the molecule, predominantly at threonine residues. Two of these sites have been identified by compositional analyses and microsequencing of tryptic peptides. Interestingly, two major sites contain the sequence Thr-Pro-Val-Lys similar to that contained in the sites recognized by growth-associated histone kinase in other organisms. No new sites are detected during the hyperphosphorylation of H1 which occurs during heat shock or in early stages of conjugation, and no sites are preferentially dephosphorylated during starvation or later stages of conjugation. Therefore, changes in the overall level of H1 phosphorylation, as opposed to phosphorylation or dephosphorylation at particular sites, appear to be important in the regulation of chromatin structure under these physiological conditions. Further, since no cell division or DNA replication occurs under these conditions, changes in the level of H1 phosphorylation are best correlated to changes in gene expression during heat shock, starvation, and conjugation. We suggest that, at least in Tetrahymena, H1 hyperphosphorylation is used as a rapid and transient mechanism for the cessation of transcription under conditions of cellular stress.

Tetrahymena contain two distinct and unusual high mobility group (HMG)-like proteins.

Previous studies have described the existence of high mobility group (HMG)-like proteins in macronuclei of the ciliated protozoan, Tetrahymena thermophila (Hamana, K., and K. Iwai, 1979, J. Biochem. [Tokyo], 69:1097-1111; Levy-Wilson, B., M. S. Denker, and E. Ito, 1983, Biochemistry, 22:1715-1721). In this report, two of these proteins, LG-1 and LG-2, have been further characterized. Polyclonal antibodies raised against LG-1 and LG-2 fail to cross react with each other or any other macronuclear polypeptide in immunoblotting analyses. As well, LG-1 and LG-2 antibodies do not react with calf thymus, chicken, or yeast HMG proteins. Consistent with these results, a 47 amino-terminal sequence of LG-1 has been determined that shows limited homology to both calf thymus HMGs 1 and 2 and HMGs 14 and 17. Two internal sequences of V8 protease-generated peptides from LG-2 have been determined, and these do not share any homology to the LG-1 sequence or any other sequenced HMG proteins. Comparison of the partial sequences of LG-1 and LG-2 with the complete amino acid sequence of the Tetrahymena histone H1 (Wu, M., C. D. Allis, R. Richman, R. G. Cook, and M. A. Gorovsky, 1986, Proc. Natl. Acad. Sci. USA, 83:8674-8678) rules out the possibility that LG-1 and LG-2 are proteolytically derived from H1, the other major macronuclear perchloric acid-soluble protein. Interestingly, however, both LG-1 and LG-2 are efficiently extracted from macronuclei by elutive intercalation (Schröter, H., G. Maier, H. Ponsting, and A. Nordheim, 1985, Embo (Eur. Mol. Biol. Organ.) J., 4:3867-3872), suggesting that both may share yet undetermined properties with HMGs 14 and 17 of higher eukaryotes. Examination of the pattern of LG-1 and LG-2 synthesis during the sexual phase of the life cycle, conjugation, demonstrates that the synthesis of LG-1 and LG-2 is coordinately increased from basal levels during the differentiation of new macronuclei (7-13 h), suggesting that both of these proteins play a role in determining a macronuclear phenotype. However, a specific induction of LG-2 synthesis is detected in early stages of conjugation (meiotic prophase, 1-4 h), leading to maximal synthesis of LG-2 at 3 h. Interestingly, the early induction of LG-2 synthesis closely parallels the hyperphosphorylation of histone H1. Taken together, these data suggest that LG-1 and LG-2 are not strongly related to each other or to higher eukaryotic HMG proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Nonrandom utilization of acetylation sites in histones isolated from Tetrahymena. Evidence for functionally distinct H4 acetylation sites.

Macro- and micronuclei of the ciliated protozoan Tetrahymena thermophila afford a unique opportunity to study histone acetylation under conditions where postsynthetic "transcription"-related acetylation and synthetic "deposition"-related acetylation are nonoverlapping. Recent studies have demonstrated that at least two general systems of acetylation operate in Tetrahymena. One is postsynthetic, macronuclear specific, and may be related to gene expression in that nucleus (Vavra, K. J., Allis, C. D., and Gorovsky, M. A. (1982) J. Biol. Chem. 257, 2591-2598). The other is synthetic, common to macro- and micronuclei, and is likely related to histone deposition during replication (Allis, C. D., Chicoine, L. G., Richman, R., and Schulman, I. G. (1985a) Proc. Natl. Acad. Sci. U. S. A., 82, 8048-8052). A unique feature of H3 and H4 in Tetrahymena is that neither are blocked at their amino termini. We have exploited this fact as well as the resolving capability of acid-urea gel electrophoresis and current microsequencing techniques to examine whether utilization of different NH2-terminal acetylation sites is random or nonrandom during the progression toward high acetylation states. Of the four acetylation sites which have been identified in H4 (in Tetrahymena these are lysines at positions 4, 7, 11, and 15), we find that lysine 7 is the exclusive site of postsynthetic acetylation in populations of monoacetylated H4 isolated from macronuclei. This site is retained in populations of diacetylated H4, which are acetylated exclusively at lysines 4 and 7. Our data also suggest that there is some preference for using lysine 11 (as compared to 15) as the third site of acetylation in triacetylated molecules. The data demonstrate that the postsynthetic acetylation-deacetylation process is surprisingly nonrandom for H4 in Tetrahymena macronuclei. We have also investigated the same question with macronuclear H3 (which contains acetylation sites at lysines 9, 14, 18, and 23). Our data demonstrate that unlike H4, lysines at position 9 or 14 are likely to be utilized as sites of acetylation within a population of monoacetylated H3. Both of these acetylation sites are retained in diacetylated H3 which suggests that if site 9 is used initially as the site of monoacetylation, 14 is used secondarily (and vice versa). Our data show, moreover, that there is a preference for utilizing lysine 18 as the third acetylation site (in triacetylated H3). Thus, these data show that H3 is also acetylated in a nonrandom fashion in macronuclei. Finally, we have determined which acetylation sites are utilized in macro- or micronuclear H4 when it is undergoing active synthesis and deposition.(ABSTRACT TRUNCATED AT 400 WORDS)

Deposition-related histone acetylation in micronuclei of conjugating Tetrahymena.

Macro- and micronuclei of the ciliated protozoan, Tetrahymena thermophila, afford a unique opportunity to study histone acetylation under conditions where acetylation associated with the regulation of transcription and acetylation associated with the deposition of histones on the DNA are separable. In this study we demonstrate that histone H3 and histone H4 synthesized in young (5 hr) conjugating Tetrahymena are deposited into micronuclei in acetylated forms. Most of the newly synthesized histone H3 migrates as a monoacetylated form while essentially all of the new histone H4 is deposited as a diacetylated species. Since micronuclei replicate rapidly during this stage of the life cycle, but are transcriptionally inactive, these data suggest that histone acetylation is related functionally to histone deposition and chromatin assembly. Pulse-chase experiments show that micronuclei also contain a butyrate-sensitive deacetylase activity(ies) which operates to remove the deposition-related acetate groups from newly synthesized and deposited H3 and H4. This enzymatic activity probably contributes to the steady state level of micronuclear histone acetylation that is low or nonexistent. Thus, evidence is emerging for at least two independent systems of histone acetylation in Tetrahymena. The first system is specific to macronuclei and may be related to gene expression. The second system is common to macro- or micronuclear histones (H3 and H4) and may be related to histone deposition during DNA replication.