The binding of lipophilic hormones, retinoids and vitamins to members of the nuclear-receptor superfamily modifies the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes. Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160, TIF-2/GRIP-1 and CBP/p300 which function as co-activators of transcription, and RIP-140, TIF-1 and TRIP-1/SUG-1 whose functions are unclear. Here we report that a short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors. We show that the ability of SRC-1 to bind the oestrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the oestrogen receptor that are required for its ligand-induced activation function. We propose that the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins.

No abstract

Regulatory T cells (Tregs) are a specific subset of lymphocytes that are critical for the maintenance of self-tolerance. Expression levels of the transcription factor Foxp3 have been causally associated with Treg differentiation and function. Recent studies show that Foxp3 can also be transiently expressed in effector T cells; however, stable Foxp3 expression is required for development of a functional Treg suppressor phenotype. Here, we demonstrate that Foxp3 is acetylated, and this can be reciprocally regulated by the histone acetyltransferase p300 and the histone deacetylase SIRT1. Hyperacetylation of Foxp3 prevented polyubiquitination and proteasomal degradation, therefore dramatically increasing stable Foxp3 protein levels. Moreover, using mouse splenocytes, human peripheral blood mononuclear cells, T cell clones, and skin-derived T cells, we demonstrate that treatment with histone deacetylase inhibitors resulted in significantly increased numbers of functional Treg cells. Taken together, our data demonstrate that modulation of the acetylation state of Foxp3 provides a novel molecular mechanism for assuring rapid temporal control of Foxp3 levels in T cells, thereby regulating Treg numbers and functionality. Manipulating Foxp3 acetylation levels could therefore provide a new therapeutic strategy to control inappropriate (auto)immune responses.

Steroid receptor co-activator (SRC1) is one of a number of transcriptional co-activators that are capable of potentiating the activity of nuclear receptors including the oestrogen receptor (ER). Here we report that two isoforms, SRC1a and SRC1e, which diverge at their C-termini, are functionally distinct as they differ in their abilities to enhance the activity of the ER in intact cells. SRC1e enhanced the ability of the ER to stimulate transcription to a greater extent than SRC1a, which had negligible effects on certain promoters. To elucidate the basis of this functional difference, we compared the nuclear receptor-binding properties and mapped the transcriptional activation domains in the two SRC1 isoforms. Both isoforms share a triplet of nuclear receptor-binding motifs (LXXLL motifs) for binding to functional ER dimers, and an activation domain which co-localizes with the CBP-binding domain, while SRC1a contains a unique LXXLL motif in its C-terminus. Although this LXXLL motif increases the affinity for the ER in vitro, it does not appear to be responsible for the functional difference between the two isoforms. This difference is due to a second activation domain that is CBP independent and is suppressed in the SRC1a isoform. Thus, SRC1 exists as functionally distinct isoforms which are likely to play different roles in ER-mediated transcription.

The development of resistance to chemotherapy is a major obstacle for lasting effective treatment of cancer. Here, we demonstrate that endogenous mesenchymal stem cells (MSCs) become activated during treatment with platinum analogs and secrete factors that protect tumor cells against a range of chemotherapeutics. Through a metabolomics approach, we identified two distinct platinum-induced polyunsaturated fatty acids (PIFAs), 12-oxo-5,8,10-heptadecatrienoic acid (KHT) and hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)), that in minute quantities induce resistance to a broad spectrum of chemotherapeutic agents. Interestingly, blocking central enzymes involved in the production of these PIFAs (cyclooxygenase-1 and thromboxane synthase) prevents MSC-induced resistance. Our findings show that MSCs are potent mediators of resistance to chemotherapy and reveal targets to enhance chemotherapy efficacy in patients.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors controlling the expression of genes involved in lipid homeostasis. PPARs activate gene transcription in response to a variety of compounds including hypolipidemic drugs as well as natural fatty acids. From the plethora of PPAR activators, Scatchard analysis of receptor-ligand interactions has thus far identified only four ligands. These are the chemotactic agent leukotriene B4 and the hypolipidemic drug Wy 14,643 for the alpha-subtype and a prostaglandin J2 metabolite and synthetic antidiabetic thiazolidinediones for the gamma-subtype. Based on the hypothesis that ligand binding to PPAR would induce interactions of the receptor with transcriptional coactivators, we have developed a novel ligand sensor assay, termed coactivator-dependent receptor ligand assay (CARLA). With CARLA we have screened several natural and synthetic candidate ligands and have identified naturally occurring fatty acids and metabolites as well as hypolipidemic drugs as bona fide ligands of the three PPAR subtypes from Xenopus laevis. Our results suggest that PPARs, by their ability to interact with a number of structurally diverse compounds, have acquired unique ligand-binding properties among the superfamily of nuclear receptors that are compatible with their biological activity.

SUMMARY Stable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function.

IFN-γ directly triggers degranulation and death of Paneth cells, which do not respond to microbial stimulation in primary culture.