The estrogen-related receptor ␣ (ERR␣) is one of the first orphan nuclear receptors identified. Still, we know little about the mechanisms that regulate its expression and its activity. In this study, we show that the transcriptional coactivator PGC-1, which is implicated in the control of energy metabolism, regulates ERR␣ at two levels. First, PGC-1 induces the expression of ERR␣. Consistent with this induction, levels of ERR␣ mRNA in vivo are highest in PGC-1 expressing tissues, such as heart, kidney, and muscle, and up-regulated in response to signals that induce PGC-1, such as exposure to cold. Second, PGC-1 interacts physically with ERR␣ and enables it to activate transcription. Strikingly, we find that PGC-1 converts ERR␣ from a factor with little or no transcriptional activity to a potent regulator of gene expression, suggesting that ERR␣ is not a constitutively active nuclear receptor but rather one that is regulated by protein ligands, such as PGC-1. Our findings suggest that the two proteins act in a common pathway to regulate processes relating to energy metabolism. In support of this hypothesis, adenovirus-mediated delivery of small interfering RNA for ERR␣, or of PGC-1 mutants that interact selectively with different types of nuclear receptors, shows that PGC-1 can induce the fatty acid oxidation enzyme MCAD (medium-chain acyl-coenzyme A dehydrogenase) in an ERR␣-dependent manner.The nuclear receptor ERR␣ 1 was identified in 1988 as a protein that shares significant sequence similarity to known steroid receptors, such as the estrogen receptor (1). ERR␣ and its relatives ERR␤ and ERR␥ form a small family of orphan nuclear receptors that are evolutionarily related to the estrogen receptors ER␣ and ER␤, and whose in vivo function is still unclear (Refs. 1 and 2 and reviewed in Ref.3). The three ERRs recognize and bind similar DNA sequences, which include estrogen response elements (EREs) recognized by ERs, as well as extended ERE half-sites that have been termed ERR response elements (4 -7). Despite their high similarity to ligand-dependent receptors, ERRs seem to regulate transcription in the absence of known natural lipophilic agonist ligands. Searches for ligands have so far identified only synthetic antagonists. 4-Hydroxytamoxifen, which binds ERR␤ and ERR␥ but not ERR␣, and diethylstilbestrol, which binds all three ERRs, inhibit the ability of ERRs to activate transcription (8, 9). In support of the pharmacological data, elucidation of the crystal structure of the ERR␥ LBD suggests that the ERRs assume the conformation of ligand-activated nuclear receptors in the absence of ligand (10) and that agonist ligands may not be required. These findings raise the question of how the activity of these nuclear receptors is regulated.One way to control orphan receptor activity is to express the receptors in a temporally and spatially restricted manner. ERR␣ is expressed widely; however, particularly high ERR␣ mRNA levels have been noted at sites of ossification during development, and in heart, kidney, brown...

How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in insulin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.

The lack of methods for proteome-scale detection of arginine methylation restricts our knowledge of its relevance in physiological and pathological processes. Here we show that most tryptic peptides containing methylated arginine(s) are highly basic and hydrophilic. Consequently, they could be considerably enriched from total cell extracts by simple protocols using either one of strong cation exchange chromatography, isoelectric focusing, or hydrophilic interaction liquid chromatography, the latter being by far the most effective of all. These methods, coupled with heavy methyl-stable isotope labeling by amino acids in cell culture and mass spectrometry, enabled in T cells the identification of 249 arginine methylation sites in 131 proteins, including 190 new sites and 93 proteins not previously known to be arginine methylated. By extending considerably the number of known arginine methylation sites, our data reveal a novel proline-rich consensus motif and identify for the first time arginine methylation in proteins involved in cytoskeleton rearrangement at the immunological synapse and in endosomal trafficking.

Networks of N-isopropylacrylamide (NIPAM) copolymers, coupled to spherical phospholipid bilayers, are suitable as a model for the study of the interaction between the cytoskeleton and cellular membranes, as well as for promising new drug delivery systems with triggerable drug release properties and improved stability. In this article, we describe a simple preparation technique for liposomes from egg phosphatidyl choline (EPC) encapsulating a cross-linked NIPAMminus signTEGDM copolymer skeleton (tetraethylene glycol dimethacrylate, TEGDM) which is coupled only to the inner monolayer by a novel membrane anchor monomer. Polymerization in the lipid vesicles was initiated at the inner membrane surface by the radical initiator 2,2-diethoxy-acetophenone (DEAP) permeating through the membrane from the outside. The effects of photopolymerization and polymer formation on vesicle shape and membrane integrity were studied by transmission electron microscopy (TEM), cryo-TEM, and atomic force microscopy (AFM). Upon UV irradiation, approximately 100% of the vesicles contained a polymer gel and only occasional changes in the spherical shape of the liposomes were observed. The architecture of the polymer network inside the liposomal compartment was determined by the conditions of the photopolymerization. Composite structures of polymer hollow spheres or solid spheres, respectively, tethered to spherical membrane vesicles were produced. The increased stability of the polymer-tethered lipid bilayers against solubilization by sodium cholate, compared to pure EPC vesicles, was determined by radiolabeling the lipid membrane.

VP16 has been widely used to unravel the mechanisms underlying gene transcription. Much of the previous work has been conducted in reconstituted in vitro systems. Here we study the formation of transcription complexes at stable reporters under the control of an inducible Tet-VP16 activator in living cells. In this simplified model for gene activation VP16 recruits the general factors and the cofactors Mediator, GCN5, CBP, and PC4, within minutes to the promoter region. Activation is accompanied by only minor changes in histone acetylation and H3K4 methylation but induces a marked promoter-specific increase in H3K79 methylation. Mediated through contacts with VP16 several subunits of the cleavage and polyadenylation factor (CPSF/ CstF) are concentrated at the promoter region. We provide in vitro and in vivo evidence that VP16 activates transcription through a specific MED25-associated Mediator, which is deficient in CDK8.The herpes simplex virus-encoded protein VP16 is a potent activator that controls the transcription of immediate early viral genes through the interaction with host cell factors (1). VP16 harbors a strong activation domain that functions well in both yeast and human cells when tethered to independent DNA-binding domains (2-4). Based on these properties, fusion proteins of VP16 with GAL4 or DNA-binding domains of other transactivators have been widely used as models to investigate the basic principles of transcription activation.In conjunction with molecular interactions involving the general transcription factors TFIIA, 3 TBP, TFIIB, and the TBPassociated factors (5-9), VP16 increases the number of functional pre-initiation complexes in vitro. Furthermore, VP16 stimulates open complex formation, which relates to interactions with the p62 subunit of TFIIH and the general cofactor PC4 (10).Comparative binding studies and functional experiments pointed to a key role for the cofactors CBP, STAGA/TFTC, and Mediator (11-16) in activation by VP16 (17). CBP interacts specifically with the C-terminal (H2) region of VP16, whereas Mediator binds tightly to both the N-terminal (H1) and C-terminal (H2) regions of the activation domain (18, 19). The histone acetyltransferase activity of CBP (p300) is critical for activation by VP16 in chromatin (20, 21). The other major histone acetyltransferase, GCN5, which is part of the SAGA-STAGA/ TFTC complex, is also recognized by VP16 (17,22,23). Further related to chromatin accessibility, the remodeling Swi/Snf complex interacts with VP16 (24).VP16 binds Mediator tightly and specifically through the MED17 and the MED25 subunits (19,25). Overexpression of the MED25 Mediator-binding domain has a dominant negative effect on VP16 activation, which substantiates the critical influence of a MED25-associated Mediator (19). Furthermore, Mediator apparently functions synergistically with other target proteins such as the histone acetyltransferases, CBP and GCN5, that are also necessary for activation by VP16 in living cells (17).Here, we use an inducible Tet-VP16 in combination...