Many cofactors bind the hormone-activated estrogen receptor (ER), yet the specific regulators of endogenous ER-mediated gene transcription are unknown. Using chromatin immunoprecipitation (ChIP), we find that ER and a number of coactivators rapidly associate with estrogen responsive promoters following estrogen treatment in a cyclic fashion that is not predicted by current models of hormone activation. Cycles of ER complex assembly are followed by transcription. In contrast, the anti-estrogen tamoxifen (TAM) recruits corepressors but not coactivators. Using a genetic approach, we show that recruitment of the p160 class of coactivators is sufficient for gene activation and for the growth stimulatory actions of estrogen in breast cancer supporting a model in which ER cofactors play unique roles in estrogen signaling.

N-CoR and SMRT are transcriptional corepressors that associate with nuclear hormone receptors (NRs) in the absence of ligand. This interaction is the molecular target of differentiation therapy for acute promyelocytic leukaemia, wherein retinoic acid dissociates corepressor from leukaemogenic receptor fusion proteins. Binding of ligand to NRs induces a conformation that attracts coactivator proteins containing an Leu-x-x-Leu-Leu motif (the 'NR box'). Here we show that N-CoR and SMRT contain sequences that are similar to the NR box and are repeated in each of two NR interaction domains. We show that this CoRNR ('corner') box is required for NR interaction, and that CoRNR box peptides specifically block corepressor interaction in vitro and repression in vivo. Sequences flanking the CoRNR box determine NR specificity. Thus, the key feature of hormone action, differential recognition of unliganded and liganded NRs by coactivators and corepressors, is due to very subtle differences between CoRNR and NR boxes. The molecular mechanisms of repression and activation by NRs are thus linked in an unexpected manner.

The three Drosophila EcR isoforms differ only at their N termini;thus, they share the conserved ligand-binding domain transcriptional activation function (AF2) and only differ in the unconserved A/B region, which contains a second, isoform-specific, activation function (AF1). We have developed a dominant-negative mutant EcR (EcR-DN), expressed it in flies with the GAL4/UAS system, and used it to block ecdysone signaling in eight tissues or groups of tissues. Localized EcR-DN arrests ecdysone-dependent development in the target cells and often — because of a molting checkpoint —arrests development globally. Simultaneously expressing individual wild-type EcR isoforms in the same target tissues suppresses the EcR-DN phenotype and identifies the rescuing isoform as sufficient to support the development of the target. Every isoform, and even an N-terminal truncated EcR that lacks any AF1, supports development in the fat body, eye discs, salivary glands,EH-secreting neurosecretory cells and in the dpp expression domain,implying that AF1 is dispensable in these tissues. By contrast, only EcR-A is able to support development in the margins of the wing discs, and only EcR-B2 can do so in the larval epidermis and the border cells of the developing egg chamber. In light of our results, the simplest explanations for the widespread spatial and temporal variations in EcR isoform titers appear untenable.

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.

No abstract

Retinoic acid receptor-related orphan receptor γ (RORγt) controls the differentiation of naive CD4(+) T cells into the TH17 lineage, which are critical cells in the pathogenesis of autoimmune diseases. Here we report that during TH17 differentiation, cholesterol biosynthesis and uptake programs are induced, whereas their metabolism and efflux programs are suppressed. These changes result in the accumulation of the cholesterol precursor, desmosterol, which functions as a potent endogenous RORγ agonist. Generation of cholesterol precursors is essential for TH17 differentiation as blocking cholesterol synthesis with chemical inhibitors at steps before the formation of active precursors reduces differentiation. Upon activation, metabolic changes also lead to production of specific sterol-sulfate conjugates that favor activation of RORγ over the TH17-inhibiting sterol receptor LXR. Thus, TH17 differentiation is orchestrated by coordinated sterol synthesis, mobilization and metabolism to selectively activate RORγ.

The ecdysone receptor is a heterodimer of the two nuclear receptors EcR and ultraspiracle (USP). We have identified the regions of Drosophila EcR and USP responsible for transcriptional activation of a semisynthetic Eip71CD promoter in Kc cells. The isoform-specific A/B domains of EcR-B1 and B2, but not those of EcR-A or USP, exhibit strong activation activity [activation function 1 (AF1)], both in isolation and in the context of the intact receptor. AF1 activity in isoform B1 derives from dispersed elements; the B2-specific AF1 consists of a 17-residue amphipathic helix. AF2 function was studied using a two-hybrid assay in Kc cells, based on the observation that potent hormone-dependent activation by the EcR/USP ligand-binding domain heterodimer requires the participation of both partners. Mutagenesis reveals that AF2 function depends on EcR helix 12, but not on the cognate USP region. EcR helix 12 mutants (F645A and W650A) exhibit a dominant negative phenotype. Thus, in the setting tested, the ecdysone receptor can activate transcription using the AF1 regions of EcR-B1 or -B2 and the AF2 region of EcR. USP acts as an allosteric effector for EcR, but does not contribute any intrinsic function.

Resistin, also known as Adipocyte Secreted Factor (ADSF) and Found in Inflammatory Zone 3 (FIZZ3), is a mouse protein with potential roles in insulin resistance and adipocyte differentiation. The resistin gene is expressed almost exclusively in adipocytes. Here we show that a proximal 264-base pair fragment of the mouse resistin promoter is sufficient for expression in adipocytes. Ectopic expression of the adipogenic transcription factor CCAAT/enhancer-binding protein (C/EBP␣) was sufficient for expression in non-adipogenic cells. C/EBP␣ binds specifically to a site that is essential for expression of the resistin promoter. Chromatin immunoprecipitation studies of the endogenous gene demonstrated adipocyte-specific association of C/EBP␣ with the proximal resistin promoter in adipocytes but not preadipocytes. C/EBP␣ binding was associated with the recruitment of coactivators p300 and CREB-binding protein and a dramatic increase in histone acetylation in the vicinity of the resistin promoter. The antidiabetic thiazolidinedione (TZD) drug rosiglitazone reduced resistin expression with an ED 50 similar to its K d for binding to peroxisome proliferator activated receptor ␥ (PPAR␥). Other TZD-and non-TZD PPAR␥ ligands also down-regulated resistin expression. However, no functional PPAR␥ binding site was found within 6.2 kb of the transcriptional start site, suggesting that if PPAR␥ is involved, it is either acting at a long distance from the start site, in an intron, or indirectly. Nevertheless, rosiglitazone treatment selectively decreased histone acetylation at the resistin promoter without a change in occupation by C/EBP␣, CREB-binding protein, or p300. Thus, adipocyte specificity of resistin gene expression is because of C/EBP␣ binding, leading to the recruitment of transcriptional coactivators and histone acetylation that is characteristic of an active chromatin environment. TZD reduces resistin gene expression at least in part by reducing histone acetylation associated with the binding of C/EBP␣ in mature adipocytes.