Interactive Fly, Drosophila

Thyroid hormone receptor coactivators

Nuclear hormone receptors are ligand-regulated transcription factors that play critical roles in metazoan homeostasis, development, and reproduction. Many nuclear hormone receptors exhibit bimodal transcriptional properties and can either repress or activate the expression of a given target gene. Repression appears to require a physical interaction between a receptor and a corepressor complex containing either the SMRT/TRAC or N-CoR/RIP13 polypeptides. Different receptors are found to interact with different domains in the SMRT and N-CoR corepressors and these divergent interactions may therefore contribute to distinct repression phenotypes. Intriguingly, different isoforms of a single nuclear hormone receptor class also differ markedly in their interactions with corepressors, indicative of their nonidentical actions in cellular regulation. Evidence is presented that combinatorial interactions between different receptors can, through the formation of heterodimeric receptors, result in novel receptor-corepressor interactions not observed for homomeric receptors (Wong, 1998).

SMRT contains within its C-terminal region at least two subdomains, denoted RID-1 and RID-2, that are independently able to confer physical and functional interactions with a defined subset of the nuclear hormone receptor family. Intriguingly, there is no extensive amino acid relatedness between RID-1 and RID-2, and different receptors display different abilities to interact with these two SMRT subdomains. T3Ralpha interacts with both SMRT RID-1 and RID-2 in vitro and in two-hybrid assays in vivo in both yeast and mammalian cells. T3Ralpha also interacts with both of the analogous RIDs of N-CoR; these interaction domains of N-CoR are related but are not identical in sequence to the corresponding interaction domains of SMRT. Perhaps reflecting this nonidentity of the SMRT and N-CoR RIDs, RARalpha interacts almost exclusively with RID-1 of SMRT but interacts moderately well with both RID-1 and RID-2 of N-CoR. Thus, different receptors make different patterns of contact with the SMRT and N-CoR corepressors, and these distinct patterns of contact may potentially be manifested as differences in transcriptional regulation (Wong, 1998).

Unexpectedly, not all isoforms within a given receptor family interact equally well with a corepressor; specifically, RARbeta interacts very poorly with SMRT and N-CoR, whereas RARalpha and RARgamma interact quite well with both corepressors. These different RAR isoforms are thought to perform distinct functions in development and differentiation; the determination that they possess distinct corepressor interaction properties suggests at least one biochemical basis for their nonidentical physiological roles. The divergent corepressor association properties of the RARbeta isoform map to a small cluster of amino acids within the D domain of the receptor that differ from the equivalent sequences in RARalpha and RARgamma Preliminary analysis suggests that changing individual nonconserved amino acids from the RARbeta sequence to that of RARalpha (such as an A175P or a T181I substitution) fails to confer strong corepressor association; apparently more subtle, or multiple, amino acid divergences within this small cluster contribute to the isoform specificity. Notably, this amino acid cluster is proximal to the N-CoR box, a domain previously implicated in corepressor binding by RARs and T3Rs. Recently, it was proposed that the N-CoR box may itself play only an indirect role in the receptor-corepressor interaction, perhaps by stabilizing the conformation of the receptor rather than by providing the actual amino acid contacts involved in the binding of the corepressor. Consistent with this view, conservation of the N-CoR box itself is not necessary for corepressor binding; COUP-TF, RXRs, and PPARs, for example, all lack a detectable N-CoR box but, nonetheless, tether SMRT and N-CoR. However, whether by direct or indirect means, the amino acids within and immediately flanking the N-CoR box play a critical role in defining the ability of RARs and T3Rs to associate with corepressors (Wong, 1998).

Nuclear hormone receptors have been shown to repress transcription in the absence of ligand. This repression is mediated by a corepressor complex that contains the Sin3A protein (see Drosophila Sin3A) and histone deacetylases (HDAC1 and 2). Studies by several groups demonstrate that this complex is recruited to nuclear receptors through the highly related corepressors SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor). This paper describes the cloning, characterization, and chromosomal mapping of forms of human and mouse SMRT that include a 1,000-aa extension, which reveals striking homology to the amino terminus of N-CoR. Structure and function studies of wild-type and natural splicing variants suggest the presence of 3-4 amino terminal domains that repress in a cooperative as well as mechanistically distinct fashion (Ordentlich, 1999).

SMRT (silencing mediator for retinoid and thyroid hormone receptors) and N-CoR (nuclear receptor copressor) mediate transcriptional repression of important regulators that are involved in many signaling pathways. SMRT and N-CoR are related proteins that form complexes with mSin3A/B and histone deacetylases to induce local chromatin condensation and transcriptional repression. However, SMRT is substantially smaller than N-CoR, lacking an N-terminal domain of approximately 1,000 aa that are present in N-CoR. The identification of SMRT-extended (SMRTe), which contains an N-terminal sequence that shows striking similarity with N-CoR, is described. As in N-CoR, this SMRTe-N-terminal domain also represses basal transcription. SMRTe expression is regulated during cell cycle progression and SMRTe transcripts are present in many embryonic tissues. These data redefine a structurally and functionally more related nuclear receptor corepressor family and suggest an additional role for SMRTe in the regulation of cycle-specific gene expression in diverse signaling pathways (Park, 1999).

TRAM-1, a thyroid hormone receptor activator molecule, is a ~160-kDa protein homologous with SRC-1/TIF2. TRAM-1 binds to thyroid hormone receptor (TR) and other NRs in a ligand-dependent manner and enhances the ligand-induced transcriptional activity of TR. The AF-2 region in NRs has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivators. Surprisingly, TRAM-1 retains strong ligand-dependent interaction with an AF-2 mutant of TR (E457A), while SRC-1 fails to interact with this mutant. A critical TRAM-1 binding site exists in rat TRbeta1 outside of AF-2, as TRAM-1 shows weak ligand-dependent interaction with a helix 3 ligand binding domain TR mutant (K288A), compared with SRC-1. These results suggest that TRAM-1 is a coactivator that may exhibit its activity by interacting with subdomains of NRs other than the AF-2 region, in contrast to SRC-1/TIF2 (Takeshita, 1997).

Steroid receptors and coactivator proteins are thought to stimulate gene expression by facilitating the assembly of basal transcription factors into a stable preinitiation complex. What is not clear, however, is how these transcription factors gain access to transcriptionally repressed chromatin to modulate the transactivation of specific gene networks in vivo. The available evidence indicates that acetylation of chromatin in vivo is coupled to transcription and that specific histone acetyltransferases (HATs) target histones bound to DNA and overcome the inhibitory effect of chromatin on gene expression. SRC-1 possesses intrinsic histone acetyltransferase activity; it also interacts with another HAT, p300/CBP-associated factor (PCAF). The HAT activity of SRC-1 maps to its carboxy-terminal region and is primarily specific for histones H3 and H4. Acetylation by SRC-1 and PCAF of histones bound at specific promoters may result from ligand binding to steroid receptors and could be a mechanism by which the activation functions of steroid receptors and associated coactivators enhance formation of a stable preinitiation complex, thereby increasing transcription of specific genes from transcriptionally repressed chromatin templates. (Spencer, 1997).

The role of the transcriptional coactivator p300 in gene activation by thyroid hormone receptor (TR) upon the addition of ligand has been investigated. The ligand-bound TR targets chromatin disruption, independent of gene activation. Exogenous p300 facilitates transcription from a disrupted chromatin template, but does not itself disrupt chromatin in the presence or absence of ligand-bound receptor. Nevertheless, the acetyltransferase activity of p300 is required to facilitate transcription from a disrupted chromatin template. Expression of E1A prevents aspects of chromatin remodeling and transcriptional activation dependent on TR and p300. E1A selectively inhibits the acetylation of non-histone substrates. E1A does not prevent the assembly of a DNase I-hypersensitive site induced by TR, but does inhibit topological alterations and the loss of canonical nucleosome arrays dependent on the addition of ligand. Mutants of E1A incompetent for interaction with p300 partially inhibit chromatin disruption but still allow nuclear receptors to activate transcription. It is concluded that p300 has no essential role in chromatin disruption, but makes use of acetyltransferase activity to stimulate transcription at a subsequent step (Li, 1999).

Thyroid hormone receptors (TR) function as part of multiprotein complexes that also include retinoid X receptor (RXR) and transcriptional coregulators. Both the Thyroid receptor CoR box and ninth heptad domains are required for interaction with RXR, and in turn, both domains are required for interaction with corepressor proteins N-CoR and SMRT. Remarkably, the recruitment of RXR to the repression-defective CoR box and ninth-heptad mutants via a heterologous dimerization interface restores both corepressor interaction and repression. The addition of thyroid hormone obviates the CoR box requirement for RXR interaction, provided that the AF2 activation helix at the C terminus of TR is intact. These results indicate that RXR differentially recognizes the unliganded and liganded conformations of TR and that these differences appear to play a major role in the recruitment of corepressors to TR-RXR heterodimers (Zhang, 1997).

Coactivators previously implicated in ligand-dependent activation functions by thyroid hormone receptor (TR) include p300 and CREB-binding protein (CBP), the steroid receptor coactivator-1 (SRC-1)-related family of proteins, and the multicomponent TR-associated protein (TRAP) complex. Two positive cofactors (PC2 and PC4) derived from the upstream stimulatory activity (USA) cofactor fraction act synergistically to mediate thyroid hormone (T3)-dependent activation either by TR or by a TR-TRAP complex in an in vitro system reconstituted with purified factors and DNA templates. Significantly, the TRAP-mediated enhancement of activation by TR does not require the TATA box-binding protein-associated factors of TFIID. Furthermore, neither the pleiotropic coactivators CBP and p300 nor members of the SRC-1 family are detected in either the TR-TRAP complex or the other components of the in vitro assay system. These results show that activation by TR at the level of naked DNA templates is enhanced by cooperative functions of the TRAP coactivators and the general coactivators PC2 and PC4. These results also indicate a potential functional redundancy between TRAPs and TATA box-binding protein-associated factors in TFIID. In conjunction with earlier studies on other nuclear receptor-interacting cofactors, the present study also suggests a multistep pathway, involving distinct sets of cofactors, for activation of hormone responsive genes (Fondell, 1999).

Transcriptional repression by nuclear hormone receptors is thought to result from a unison of targeting chromatin modification and disabling the basal transcriptional machinery. Xenopus oocytes have been used to compare silencing effected by the thyroid hormone receptor (TR) and its mutated version, the oncoprotein v-ErbA, on partly and fully chromatinized TR-responsive templates in vivo. Repression by v-ErbA is not as efficient as that mediated by TR, is significantly more sensitive to histone deacetylase (HDAC) inhibitor treatment and, unlike TR, v-ErbA requires mature chromatin to effect repression. Both v-ErbA and TR can recruit the corepressor N-CoR, but, in contrast to existing models, both show a concomitant enrichment for HDAC3 that occurs without an association with Sin3, HDAC1/RPD3, Mi-2 or HDAC5. A requirement for chromatin infrastructure in N-CoR/HDAC3-effected repression is proposed and it is suggested that the inability of v-ErbA to silence on partly chromatinized templates may stem from its impaired capacity to interfere with basal transcriptional machinery function. In support of this notion, v-ErbA is found to be to be less competent than TR for binding to TFIIB in vitro and in vivo (Urnov, 2000).

Adaptive thermogenesis is an important component of energy homeostasis and a metabolic defense against obesity. A novel transcriptional coactivator of nuclear receptors, termed PGC-1, has been cloned from a brown fat cDNA library. PGC-1 mRNA expression is dramatically elevated upon cold exposure of mice in both brown fat and skeletal muscle, key thermogenic tissues. PGC-1 greatly increases the transcriptional activity of PPARgamma and the thyroid hormone receptor on the uncoupling protein (UCP-1) promoter. Ectopic expression of PGC-1 in white adipose cells activates expression of UCP-1 and key mitochondrial enzymes of the respiratory chain, and increases the cellular content of mitochondrial DNA. These results indicate that PGC-1 plays a key role in linking nuclear receptors to the transcriptional program of adaptive thermogenesis (Puigserver, 1998).

Mitochondrial number and function are altered in response to external stimuli in eukaryotes. While several transcription/replication factors directly regulate mitochondrial genes, the coordination of these factors into a program responsive to the environment is not understood. PGC-1, a cold-inducible coactivator of nuclear receptors, stimulates mitochondrial biogenesis and respiration in muscle cells through an induction of uncoupling protein 2 (UCP-2) and through regulation of the nuclear respiratory factors (NRFs). PGC-1 stimulates a powerful induction of NRF-1 and NRF-2 gene expression; in addition, PGC-1 binds to and coactivates the transcriptional function of NRF-1 on the promoter for mitochondrial transcription factor A (mtTFA), a direct regulator of mitochondrial DNA replication/transcription. These data elucidate a pathway that directly links external physiological stimuli to the regulation of mitochondrial biogenesis and function (Wu, 1999).

Chromatin mediated activation and repression of thyroid hormone responsive genes

The thyroid hormone-inducible promoter of the Xenopus thyroid hormone receptor (TR)beta A gene has been assembled into chromatin using replication-coupled and -independent assembly pathways in vivo. Heterodimers of TR and 9-cis retinoic acid receptors (RXR) can bind to their recognition sites within chromatin both in vivo and in vitro and alternately repress or activate transcription depending on the absence or presence of thyroid hormone. Maximal transcriptional repression requires the presence of unliganded TR/RXR heterodimers during replication-coupled chromatin assembly. An increase in transcription directed by the TR beta A promoter of over two orders of magnitude occurs in vivo, following the addition of thyroid hormone. This increase in transcription involves the relief of the repressed state that is established by the unliganded TR/RXR heterodimer during replication-coupled chromatin assembly. The association of thyroid hormone with the chromatin-bound TR/RXR heterodimer leads to the disruption of local chromatin structure in a transcription-independent process. Thus, chromatin structure has multiple roles in the regulation of TR beta A gene expression in vivo: The TR/RXR heterodimer recognizes the response element within chromatin, TR/RXR makes use of the chromatin assembly process to silence transcription more efficiently, and TR/RXR directs the disruption of local chromatin structure in response to thyroid hormone (Wong, 1995b).

Chromatin disruption and transcriptional activation are both thyroid hormone-dependent processes regulated by the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR-RXR). In the absence of hormone, the TR-RXR dimer binds to nucleosomal DNA, locally disrupts histone-DNA contacts and generates a DNase I-hypersensitive site. Chromatin-bound unliganded TR-RXR silences transcription of the Xenopus TRbetaA gene within a canonical nucleosomal array. On addition of hormone, the receptor directs the extensive further disruption of chromatin structure over several hundred base pairs of DNA and activates transcription. A domain of the TR protein, the C-terminal nine amino acids, is necessary for directing this extensive hormone-dependent chromatin disruption. Particular TR-RXR heterodimers containing mutations in this domain are able to bind both hormone and their thyroid hormone receptor recognition element (TRE) within chromatin, yet are unable to direct the extensive hormone-dependent disruption of chromatin or to activate transcription. The hormone-dependent disruption of chromatin and transcriptional activation are independently regulated events, distinguished through the mutagenesis of basal promoter elements and by altering the position and number of TREs within the TRbetaA promoter. Chromatin disruption alone on a minichromosome is shown to be insufficient for transcriptional activation of the TRbetaA gene (Wong, 1997a).

Histone deacetylase and chromatin assembly contribute to the control of transcription of the Xenopus TRbetaA gene promoter by the heterodimer of Xenopus thyroid hormone receptor and 9-cis retinoic acid receptor (TR-RXR). Addition of the histone deacetylase inhibitor Trichostatin A (TSA) relieves repression of transcription due to chromatin assembly following microinjection of templates into Xenopus oocyte nuclei, and eliminates regulation of transcription by TR-RXR. Expression of Xenopus RPD3p (see Drosophila Rpd3), the catalytic subunit of histone deacetylase, represses the TRbetaA promoter, but only after efficient assembly of the template into nucleosomes. In contrast, the unliganded TR-RXR represses templates only partially assembled into nucleosomes; addition of TSA also relieves this transcriptional repression. This result indicates the distinct requirements for chromatin assembly in mediating transcriptional repression by the deacetylase alone, compared with those needed in the presence of unliganded TR-RXR. In addition, whereas hormone-bound TR-RXR targets chromatin disruption as assayed through changes in minichromosome topology and loss of a regular nucleosomal ladder on micrococcal nuclease digestion, addition of TSA relieves transcriptional repression but does not disrupt chromatin. Thus, TR-RXR can facilitate transcriptional repression in the absence of hormone through mechanisms in addition to recruitment of deacetylase, and disrupt chromatin structure through mechanisms in addition to the inhibition or release of deacetylase (Wong, 1998).

Thyroid hormone (T3) and retinoic acid (RA) receptors regulate transcription of the rat growth hormone (GH) gene through binding to a common hormone response element (HRE) in the promoter. The effect of histone acetylation has been investigated on hormone-dependent expression of the rat GH gene. The effects of butyrate, which induces histone hyperacetylation, and trichostatin A (TSA), a highly specific inhibitor of histone deacetylases have been studied. GH-mRNA levels are significantly increased in pituitary GH4C1 cells incubated with T3 and RA, and this response is further stimulated in the presence of 1 mM butyrate. The effect of butyrate was mimicked by TSA. TSA produces a dose-dependent increase of activity in the absence of ligands, and potentiates the effect of T3 and RA. With butyrate, basal activity of the GH promoter increases by more than 10-fold and the effect of T3 and RA is no longer observed. Overexpression of T3 receptors, mimicking the absence of ligand, is able to counteract the stimulation of basal expression caused by butyrate. Thus, in the absence of ligand, the T3 receptor acts as a constitutive repressor of gene expression. Upon binding of the hormone, the T3 receptor is converted into an activator. These findings suggest that histone acetylation, which alters chromatin structure, may play an important role in hormone-mediated transcriptional regulation (Garcia-Villalba, 1997).

Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor. The proteins encoded by the ski proto-oncogene family directly bind to N-CoR/SMRT and mSin3A, and form a complex with HDAC. c-Ski and its related gene product Sno are required for transcriptional repression by Mad and thyroid hormone receptor (TRbeta). The oncogenic form, v-Ski, which lacks the mSin3A-binding domain, acts in a dominant-negative fashion, and abrogates transcriptional repression by Mad and TRbeta. In ski-deficient mouse embryos, the ornithine decarboxylase gene, whose expression is normally repressed by Mad-Max, is expressed ectopically. These results show that Ski is a component of the HDAC complex and that Ski is required for the transcriptional repression mediated by this complex. The involvement of c-Ski in the HDAC complex indicates that the function of the HDAC complex is important for oncogenesis (Nomura, 1999).

The protein associations and enzymatic requirements were investigated for the Xenopus histone deacetylase catalytic subunit RPD3 to direct transcriptional repression in Xenopus oocytes. Endogenous Xenopus RPD3 is present in nuclear and cytoplasmic pools, whereas RbAp48 and SIN3 are predominantly nuclear. Xenopus RbAp48 and SIN3 have been cloned and it has been shown that expression of RPD3, but not RbAp48 or SIN3, leads to an increase in nuclear and cytoplasmic histone deacetylase activity and transcriptional repression of the TRbetaA promoter. This repression requires deacetylase activity and nuclear import of RPD3 mediated by a carboxy-terminal nuclear localization signal. Exogenous RPD3 is not incorporated into oocyte deacetylase and ATPase complexes but cofractionates with a component of the endogenous RbAp48 in the oocyte nucleus. RPD3 associates with RbAp48 through N- and C-terminal contacts and RbAp48 also interacts with SIN3. Xenopus RbAp48 selectively binds to the segment of the N-terminal tail immediately proximal to the histone fold domain of histone H4 in vivo. Exogenous RPD3 may be targeted to histones through interaction with endogenous RbAp48 to direct transcriptional repression of the Xenopus TRbetaA promoter in the oocyte nucleus. However, the exogenous RPD3 deacetylase functions to repress transcription in the absence of a requirement for association with SIN3 or other targeted corepressors (Vermaak, 1999).

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