Role of the mediator complex in nuclear hormone receptor signaling.

Mediator is an evolutionarily conserved multisubunit protein complex that plays a key role in regulating transcription by RNA polymerase II. The complex functions by serving as a molecular bridge between DNA-bound transcriptional activators and the basal transcription apparatus. In humans, Mediator was first characterized as a thyroid hormone receptor (TR)-associated protein (TRAP) complex that facilitates ligand-dependent transcriptional activation by TR. More recently, Mediator has been established as an essential coactivator for a broad range of nuclear hormone receptors (NRs) as well as several other types of gene-specific transcriptional activators. A single subunit of the complex, MED1/TRAP220, is required for direct ligand-dependent interactions with NRs. Mediator coactivates NR-regulated gene expression by facilitating the recruitment and activation of the RNA polymerase II-associated basal transcription apparatus. Importantly, Mediator acts in concert with other NR coactivators involved in chromatin remodeling to initiate transcription of NR target genes in a multistep manner. In this review, we summarize the functional role of Mediator in NR signaling pathways with an emphasis on the underlying molecular mechanisms by which the complex interacts with NRs and subsequently facilitates their action. We also focus on recent advances in our understanding of TRAP/Mediator's pathophysiological role in mammalian disease and development.

Generation of a mammalian cell line stably expressing a tetracycline-regulated epitope-tagged human androgen receptor: implications for steroid hormone receptor research.

The androgen receptor (AR) is hormone-activated transcription factor that regulates the expression of genes involved in differentiation, development, and maintenance of male reproductive functions. To establish a useful model system for studying molecular mechanisms of AR action, we generated a HeLa-derived cell line (termed E19) that stably expresses human AR. Because overexpression of AR in cultured cells can be cytotoxic, we placed AR expression under the control of a tetracycline-regulated promoter. The stably expressed AR also contains an N-terminal FLAG-epitope tag (f:AR) that provides an advantageous method for immunopurification. We show that f:AR expression in E19 cells can be precisely modulated by varying the concentration of tetracycline or its chemical derivative doxycycline in the growth media. The functional activity of E19-expressed f:AR is demonstrated in vivo by its ability to activate transiently transfected AR reporter genes in an androgen-dependent manner, and in vitro by its ability to specifically bind AR-response elements using DNA-mobility shift assays. We further show that f:AR in androgen-stimulated E19 cells is markedly phosphorylated and coimmunopurifies with the transcriptional coactivator CREB-binding protein (CBP). The implications of these findings on steroid receptor research and the identification of receptor coregulatory factors will be discussed.

Temporal formation of distinct thyroid hormone receptor coactivator complexes in HeLa cells.

Thyroid hormone receptors (TRs) regulate transcription by recruiting distinct coregulatory complexes to target gene promoters. Coactivators implicated in ligand-dependent activation by TR include p300, the CREB-binding protein (CBP), members of the p160/SRC family, and the multisubunit TR-associated protein (TRAP) complex. Using a stable TR-expressing HeLa cell line, we show that interaction of TR with members of the p160/SRC family, CBP, and the p300/CBP-associated factor (PCAF) occurs rapidly (approximately 10 min) following addition of thyroid hormone (T3). In close agreement with these observations, we find that TR is associated with potent histone acetyltransferase activity rapidly following T3-treatment. By contrast, we observe that formation of TR-TRAP complexes occurs significantly later (approximately 3 h) post T3 treatment. An examination of the kinetics of T3-induced gene expression in HeLa cells reveals bimodal or delayed activation on specific T3-responsive promoters. Taken together, our data are consistent with the hypothesis that T3-dependent activation at specific target promoters may involve the regulated action of multiple TR-coactivator complexes.

Specific structural motifs determine TRAP220 interactions with nuclear hormone receptors.

The TRAP coactivator complex is a large, multisubunit complex of nuclear proteins which associates with nuclear hormone receptors (NRs) in the presence of cognate ligand and stimulates NR-mediated transcription. A single subunit, TRAP220, is thought to target the entire complex to a liganded receptor through a domain containing two of the signature LXXLL motifs shown previously in other types of coactivator proteins to be essential for mediating NR binding. In this work, we demonstrate that each of the two LXXLL-containing regions, termed receptor binding domains 1 and 2 (RBD-1 and RBD-2), is differentially preferred by specific NRs. The retinoid X receptor (RXR) displays a weak yet specific activation function 2 (AF2)-dependent preference for RBD-1, while the thyroid hormone receptor (TR), vitamin D(3) receptor (VDR), and peroxisome proliferator-activated receptor all exhibit a strong AF2-dependent preference for RBD-2. Using site-directed mutagenesis, we show that preference for RBD-2 is due to the presence of basic-polar residues on the amino-terminal end of the core LXXLL motif. Furthermore, we show that the presence and proper spacing of both RBD-1 and RBD-2 are required for an optimal association of TRAP220 with RXR-TR or RXR-VDR heterodimers bound to DNA and for TRAP220 coactivator function. On the basis of these results, we suggest that a single molecule of TRAP220 can interact with both subunits of a DNA-bound NR heterodimer.

Identification of mouse TRAP100: a transcriptional coregulatory factor for thyroid hormone and vitamin D receptors.

Nuclear hormone receptors (NRs) regulate transcription in part by recruiting distinct transcriptional coregulatory complexes to target gene promoters. The thyroid hormone receptor (TR) was recently purified from thyroid hormone-cultured HeLa cells in association with a complex of novel nuclear proteins termed TRAPs (thyroid hormone receptor-associated proteins) ranging in size from 20 to 240 kDa. The TRAP complex markedly enhances TR-mediated transcription in vitro, suggesting a coactivator role for one or more of the TRAP components. Here we present the mouse cDNA for the 100-kDa component of the TRAP complex (mTRAP100). The mTRAP100 protein contains seven LxxLL motifs thought to be potential binding surfaces for liganded NRs, yet surprisingly fails to interact with TR and other NRs in vitro. By contrast, mTRAP100 coprecipitates in vivo with another component of the TRAP complex (TRAP220), which directly contacts TR and the vitamin D receptor in a ligand-dependent manner. Our findings thus suggest that TRAP100 is targeted to NRs in association with TRAP complexes specifically containing TRAP220. Transient overexpression of mTRAP100 in mammalian cells further enhances ligand-dependent transcription by both TR and the vitamin D receptor, revealing a functional role for mTRAP100 in NR-mediated transactivation. The presence of an intrinsic mTRAP100 transactivation function is suggested by the ability of mTRAP100 to activate transcription constitutively when tethered to the GAL4 DNA-binding domain. Collectively, these findings suggest that TRAP100, in concert with other TRAPs, plays an important functional role in mediating transactivation by specific NRs.

Identity between TRAP and SMCC complexes indicates novel pathways for the function of nuclear receptors and diverse mammalian activators.

The human thyroid hormone receptor-associated protein (TRAP) complex, an earlier described coactivator for nuclear receptors, and an SRB- and MED-containing cofactor complex (SMCC) that mediates activation by Gal4-p53 are shown to be virtually the same with respect to specific polypeptide subunits, coactivator functions, and mechanisms of action (activator interactions). In parallel with ligand-dependent interactions of nuclear receptors with the TRAP220 subunit, p53 and VP16 activation domains interact directly with a newly cloned TRAP80 subunit. These results indicate novel pathways for the function of nuclear receptors and other activators (p53 and VP16) through a common coactivator complex that is likely to target RNA polymerase II. Identification of the TRAP230 subunit as a previously predicted gene product also suggests a coactivator-related transcription defect in certain disease states.

Thyroid hormone receptor-associated proteins and general positive cofactors mediate thyroid hormone receptor function in the absence of the TATA box-binding protein-associated factors of TFIID.

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. Here we show that 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 were 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, and they further 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.

A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation.

A novel human complex that can either repress activator-dependent transcription mediated by PC4, or, at limiting TFIIH, act synergistically with PC4 to enhance activator-dependent transcription has been purified. This complex contains homologs of a subset of yeast mediator/holoenzyme components (including SRB7, SRB10, SRB11, MED6, and RGR1), homologs of other yeast transcriptional regulatory factors (SOH1 and NUT2), and, significantly, some components (TRAP220, TRAP170/hRGR1, and TRAP100) of a human thyroid hormone receptor-associated coactivator complex. The complex shows direct activator interactions but, unlike yeast mediator, can act independently of the RNA polymerase II CTD. These findings demonstrate both positive and negative functional capabilities for the human complex, emphasize novel (CTD-independent) regulatory mechanisms, and link the complex to other human coactivator complexes.

The TRAP220 component of a thyroid hormone receptor- associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion.

Cognate cDNAs are described for 2 of the 10 thyroid hormone receptor-associated proteins (TRAPs) that are immunopurified with thyroid hormone receptor alpha (TRalpha) from ligand-treated HeLa (alpha-2) cells. Both TRAP220 and TRAP100 contain LXXLL domains found in other nuclear receptor-interacting proteins and both appear to reside in a single complex with other TRAPs (in the absence of TR). However, only TRAP220 shows a direct ligand-dependent interaction with TRalpha, and these interactions are mediated through the C terminus of TRalpha and (at least in part) the LXXLL domains of TRAP220. TRAP220 also interacts with other nuclear receptors [vitamin D receptor, retinoic acid receptor alpha, retinoid X receptor alpha, peroxisome proliferation-activated receptor (PPAR) alpha, PPARgamma and, to a lesser extent, estrogen receptor] in a ligand-dependent manner, whereas TRAP100 shows only marginal interactions with estrogen receptor, retinoid X receptor alpha, PPARalpha, and PPARgamma. Consistent with these results, TRAP220 moderately stimulates human TRalpha-mediated transcription in transfected cells, whereas a fragment containing the LXXLL motifs acts as a dominant negative inhibitor of nuclear receptor-mediated transcription both in transfected cells (TRalpha) and in cell free transcription systems (TRalpha and vitamin D receptor). These studies indicate that TRAP220 plays a major role in anchoring other TRAPs to TRalpha during the function of the TRalpha-TRAP complex and, further, that TRAP220 (possibly along with other TRAPs) may be a global coactivator for the nuclear receptor superfamily.

Retinoid X receptor:vitamin D3 receptor heterodimers promote stable preinitiation complex formation and direct 1,25-dihydroxyvitamin D3-dependent cell-free transcription.

The numerous members of the steroid/nuclear hormone receptor superfamily act as direct transducers of circulating signals, such as steroids, thyroid hormone, and vitamin or lipid metabolites, and modulate the transcription of specific target genes, primarily as dimeric complexes. The receptors for 9-cis retinoic acid and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], RXR and VDR, respectively, as members of this superfamily, form a heterodimeric complex and bind cooperatively to vitamin D responsive elements (VDREs) to activate or repress the transcription of a multitude of genes which regulate a variety of physiological functions. To directly investigate RXR- and VDR-mediated transactivation, we developed a cell-free transcription system for 1,25(OH)2D3 signaling by utilizing crude nuclear extracts and a G-free cassette-based assay. Transcriptional enhancement in vitro was dependent on purified, exogenous RXR and VDR and was responsive to physiological concentrations of 1,25(OH)2D3. We found that RXR and VDR transactivated selectively from VDRE-linked templates exclusively as a heterodimeric complex, since neither receptor alone enhanced transcription in vitro. By the addition of low concentrations of the anionic detergent Sarkosyl to limit cell-free transcription to a single round and the use of agarose gel mobility shift experiments to assay factor complex assembly, we observed that 1,25(OH)2D3 enhanced RXR:VDR-mediated stabilization or assembly of preinitiation complexes to effect transcriptional enhancement from VDRE-linked promoter-containing DNA.

Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex.

Transcriptional regulation by nuclear hormone receptors is thought to involve interactions with putative cofactors that may potentiate receptor function. Here we show that human thyroid hormone receptor alpha purified from HeLa cells grown in the presence of thyroid hormone (T3) is associated with a group of distinct nuclear proteins termed thyroid hormone receptor-associated proteins (TRAPs). In an in vitro system reconstituted with general initiation factors and cofactors (and in the absence of added T3), the "liganded" thyroid hormone receptor (TR)/TRAP complex markedly activates transcription from a promoter template containing T3-response elements. Moreover, whereas the retinoid X receptor is not detected in the TR/TRAP complex, its presence is required for the function of the complex. In contrast, human thyroid hormone receptor alpha purified from cells grown in the absence of T3 lacks the TRAPs and effects only a low level of activation that is dependent on added ligand. These findings demonstrate the ligand-dependent in vivo formation of a transcriptionally active TR-multisubunit protein complex and suggest a role for TRAPs as positive coactivators for gene-specific transcriptional activation.

Unliganded thyroid hormone receptor alpha can target TATA-binding protein for transcriptional repression.

Unliganded human thyroid hormone receptor alpha (hTR alpha) can repress transcription by inhibiting the formation of a functional preinitiation complex (PIC) on promoters bearing thyroid hormone receptor (TR)-binding elements. Here we demonstrate that hTR alpha directly contacts the TATA-binding protein (TBP) and that preincubation of hTR alpha with TBP completely alleviates TR-mediated repression in vitro. Using stepwise preassembled PICs, we show that hTR alpha targets either the TBP/TFIIA or the TBP/TFIIA/TFIIB steps of PIC assembly for repression. We also show that the repression domain of hTR alpha maps to the C-terminal ligand-binding region and that direct TR-TBP interactions can be inhibited by thyroid hormone. Together, these results suggest a model in which unliganded hTR alpha contacts promoter-bound TBP and interferes with later steps in the initiation of transcription.

Control of transcription by Krüppel through interactions with TFIIB and TFIIE beta.

The zinc-finger protein Krüppel (Kr) is an integral part of the Drosophila segmentation gene cascade and is essential in organogenesis during later embryonic development. In tissue culture, Kr regulates transcription. Monomeric Kr can act as a transcriptional activator, whereas Kr dimers formed at high concentrations cause repression. Here we show that Kr-dependent control of transcription involves functional interactions with components of the basal RNA polymerase II transcription machinery, which includes the initiation factors TFIIA, B, E, F, H and I (refs 10, 11) as well as the TATA-binding protein (TBP) and TBP-associated factors (TAFs) contained in the multisubunit TFIID (ref. 12). Our results indicate that when acting from a site close to a basal promoter, monomeric Kr interacts with TFIIB to activate transcription, whereas an interaction of the Kr dimer with TFIIE beta, a subunit of TFIIE, results in transcriptional repression.

Unliganded thyroid hormone receptor inhibits formation of a functional preinitiation complex: implications for active repression.

The thyroid hormone receptor (TR) belongs to the steroid/nuclear receptor superfamily of ligand-inducible transcription factors. Numerous studies using transient transfection assays have demonstrated that in the absence of thyroid hormone (T3), unliganded TR acts as a constitutive repressor of transcription on genes bearing TR-response elements. We examined the molecular mechanism of TR repression in vitro using both HeLa nuclear extracts and purified basal factors. Here, we show that unliganded TR is an active transcriptional repressor, distinct from passive repressors that compete with activators for DNA binding. Repression by TR can be relieved by adding the T3 analog triiodothyroactic acid, suggesting that liganded TR undergoes a conformational change that masks or disrupts the repressor function. Repression by TR is mediated through the basal transcription machinery and can occur independently of previously characterized TATA-binding protein-associated cofactors thought to be involved in either basal repression or activator-dependent transcription. TR inhibits transcription at an early step during preinitiation complex (PIC) assembly, as preassembled PICs are refractory to the inhibitory effects of TR.

Transcription of germ line V alpha segments correlates with ongoing T-cell receptor alpha-chain rearrangement.

M14T is a virally transformed immature T-cell line which continues to rearrange its T-cell antigen receptor (TCR) alpha-chain genes in vitro and thus represents a dynamic system for studying TCR assembly. In an effort to investigate whether the TCR alpha locus is accessible for V(D)J rearrangement events, we examined M14T cells for the presence of germ line TCR alpha transcripts. Several unrearranged V alpha segments were found to be transcriptionally active in M14T cells. By comparison, germ line V alpha transcripts are absent in nonlymphoid and pro-T-cell lines and barely detectable in mature T-cell lines, suggesting that this phenomenon is likely stage and tissue specific. We demonstrate a perfect correlation between transcriptionally active V alpha segments and their involvement in ongoing V alpha-to-J alpha rearrangements. In addition, data suggesting that the unrearranged J alpha locus is also transcriptionally active in the M14T line are presented. Furthermore, the recombination-activating genes RAG-1 and RAG-2 are differentially expressed, with RAG-2 detectable only by polymerase chain reaction, implying that very low levels of one of these gene products are sufficient to complement the other to facilitate VJ rearrangements. These findings provide the first direct evidence for an accessibility model of antigen receptor rearrangement in T cells.

On the mechanism of non-allelically excluded V alpha-J alpha T cell receptor secondary rearrangements in a murine T cell lymphoma.

We had previously demonstrated that several subclones derived from a CD3+, CD4-/CD8-, TCR-alpha beta+ murine T cell line have undergone secondary V alpha-J alpha rearrangements at the TCR-alpha locus (1). In an effort to examine the molecular mechanism responsible for these V alpha-J alpha replacements, the structures of TCR-alpha cDNA prepared from both the parental and subcloned T cell lines have been determined. Here we report that: 1) the mechanism whereby the secondary rearrangements occur is a precise deletion event that involves germ-line V alpha genes 5' to the preexisting V alpha-J alpha complex joining to J alpha segments 3' of the preexisting complex deleting the region in between, 2) preexisting productive V alpha-J alpha rearrangements of the parental line do not allelically exclude productive and nonproductive secondary rearrangements, 3) both productively rearranged TCR-alpha alleles of the parental cell line can undergo secondary rearrangements, 4) the presence of unrearranged germline V alpha transcripts in the parental line support an "accessibility" model of regulated lymphocyte receptor gene rearrangement. In addition, we present data which suggests that one of the subcloned lines has undergone a third rearrangement of one of its TCR-alpha alleles. One interpretation of these results is that T cells may have the ability to circumvent allelic exclusion at the TCR-alpha locus early in their ontogeny. This could provide T cells with an additional mechanism for generating an Ag receptor repertoire which is not found in B cells.

The joining of germ-line V alpha to J alpha genes replaces the preexisting V alpha-J alpha complexes in a T cell receptor alpha, beta positive T cell line.

To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCR alpha and beta genes in the progeny of the CD3+, CD4-/CD8- M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing V alpha-J alpha rearrangements by joining an upstream V alpha gene to a downstream J alpha segment. Both the productively and nonproductively rearranged alleles in the M14T line can undergo secondary rearrangements while its TCR beta genes are stable. These secondary recombinations are usually productive, and new forms of TCR alpha polypeptides are expressed in these cells in association with the original C beta chain. Developmental control of this V alpha-J alpha replacement phenomenon could play a pivotal role in the thymic selection of the T cell repertoire.