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Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4(RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA–RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism.

Humans with obesity differ in their susceptibility to developing insulin resistance and type 2 diabetes (T2D). This variation may relate to the extent of adipose tissue (AT) inflammation that develops as their obesity progresses. The state of macrophage activation has a central role in determining the degree of AT inflammation and thus its dysfunction, and these states are driven by epigenomic alterations linked to gene expression. The underlying mechanisms that regulate these alterations, however, are poorly defined. Here we demonstrate that a co-repressor complex containing G protein pathway suppressor 2 (GPS2) crucially controls the macrophage epigenome during activation by metabolic stress. The study of AT from humans with and without obesity revealed correlations between reduced GPS2 expression in macrophages, elevated systemic and AT inflammation, and diabetic status. The causality of this relationship was confirmed by using macrophage-specific Gps2-knockout (KO) mice, in which inappropriate co-repressor complex function caused enhancer activation, pro-inflammatory gene expression and hypersensitivity toward metabolic-stress signals. By contrast, transplantation of GPS2-overexpressing bone marrow into two mouse models of obesity (ob/ob and diet-induced obesity) reduced inflammation and improved insulin sensitivity. Thus, our data reveal a potentially reversible disease mechanism that links co-repressor-dependent epigenomic alterations in macrophages to AT inflammation and the development of T2D.

Chromosome conformation capture combined with high-throughput sequencing (4C-seq) is a powerful tool to map genomic DNA regions that communicate with a specific locus of interest such as functional single-nucleotide polymorphism (SNPs)-containing regions. This protocol describes detailed steps to perform 4C-seq in mouse macrophage RAW264.7 cells, starting from the primer design based on cistrome and epigenome data, sample processing, and to the bioinformatics analysis.

In search of therapeutic agents for estrogen-related pathologies, phytoestrogens are being extensively explored. In contrast to naringenin, 8-prenylnaringenin is a potent hop-derived estrogenic compound, highlighting the importance of the prenyl group for hormonal activity. We investigated the effects of substituting the prenyl group at C(8) with alkyl chains of varying lengths and branching patterns on estrogen receptor (ER) subtype ER- and ER-binding affinities and transcriptional activities. In addition, features of the ligand-induced receptor conformations were explored using a set of specific ER-binding peptides. The new 8-alkylnaringenins were found to span an activity spectrum ranging from full agonism to partial agonism to antagonism. Most strikingly, 8-(2,2-dimethylpropyl)naringenin exhibited full agonist character on ER, but pronounced antagonist character on ER. Knowledge on how ER-subtype-selective activities can be designed provides valuable information for future drug or tool compound discovery.

Liver receptor homologue 1 (LRH-1) is a member of the nuclear receptor superfamily originally found in liver cells. LRH-1 participates in regulation of cholesterol metabolism and bile acid synthesis. Recent studies have shown that LRH-1 is even more highly expressed in the ovary, and LRH-1 has been implicated as a key transcriptional regulator of cytochrome P450 aromatase (P450arom) in vitro. In the present study, we investigated the spatiotemporal expression patterns of LRH-1 using in situ hybridization and immunohistochemistry in ovaries from rats with a 4-day estrous cycle, from pregnant rats, from immature rats treated with eCG to stimulate follicular development, and from eCG-treated rats that were subsequently given hCG to stimulate ovulation and luteinization. To establish a potential connection between the expression of LRH-1 and that of the steroidogenic genes in vivo, we directly compared the localization patterns of LRH-1 and P450aromtranscripts in consecutive ovarian sections from these animals. LRH-1 mRNA and protein were primarily localized to granulosa cells and luteinized follicles or newly formed corpora lutea (CLs) of immature and adult rats, and the levels of expression increased during eCG-hCG-induced follicular development and ovulation. In the functional CLs of pregnant rats, a biphasic change in LRH-1 mRNA content occurred throughout the gestation process, whereas LRH-1 protein was persistently detected during the entire pregnancy. In the consecutive ovarian sections, expression of LRH-1 was approximately colocalized with that of P450aromin both tertiary and Graafian follicles and the functional CLs of pregnant rats. LRH-1 mRNA and protein expression preceded those of P450aromduring early follicular development. Stage-specific expression of LRH-1 in rat granulosa and luteal cells suggests a role for LRH-1 in the regulation of ovarian function. The overlapping but distinct expression patterns of LRH-1 and P450aromcircumstantially support the recent finding that LRH-1 serves as a critical upstream regulator of P450aromgene expression in ovarian cells, but LRH-1 also may be a multifunctional steroidogenic factor in ovarian physiology.

SHP (NROB2) is an atypical orphan nuclear receptor that lacks a DNA-binding domain but contains a putative ligand-binding domain. Previous studies have revealed that SHP interacts with a variety of nuclear receptors and inhibits their transcriptional activity, thereby acting as a corepressor. In this report we identify the glucocorticoid receptor (GR) as a novel downstream target receptor for SHP inhibition. SHP potently inhibits dexamethasone-induced transcriptional GR activity in mammalian cells, and the inhibition involves a functional second NR-box within SHP. Interestingly, this motif shows a high homology with the NR-box in the glucocorticoid and cAMP-inducible GR coactivator PGC-1, indicating similar binding specificity and shared target receptors. We show that SHP antagonizes PGC-1 coactivation and, in addition, we identify the PGC- 1-regulated phospho(enol)pyruvate carboxykinase (PEPCK) promoter as a novel target promoter for SHP inhibition. This implies a physiologically relevant role for SHP in modulating hepatic glucocorticoid action. Furthermore, when coexpressing green fluorescent protein-tagged GR together with SHP, an intranuclear redistribution of GR was observed. As inhibition-deficient SHP mutants were unable to induce this redistribution, intranuclear tethering of target receptors may represent yet another, previously uncovered, aspect of SHP inhibition.

The activation function 2/ligand-dependent interaction between nuclear receptors and their coregulators is mediated by a short consensus motif, the so-called nuclear receptor (NR) box. Nuclear receptors exhibit distinct preferences for such motifs depending both on the bound ligand and on the NR box sequence. To better understand the structural basis of motif recognition, we characterized the interaction between estrogen receptor alpha and the NR box regions of the p160 coactivator TIF2. We have determined the crystal structures of complexes between the ligand-binding domain of estrogen receptor alpha and 12-mer peptides from the Box B2 and Box B3 regions of TIF2. Surprisingly, the Box B3 module displays an unexpected binding mode that is distinct from the canonical LXXLL interaction observed in other ligand-binding domain/NR box crystal structures. The peptide is shifted along the coactivator binding site in such a way that the interaction motif becomes LXXYL rather than the classical LXXLL. However, analysis of the binding properties of wild type NR box peptides, as well as mutant peptides designed to probe the Box B3 orientation, suggests that the Box B3 peptide primarily adopts the "classical" LXXLL orientation in solution. These results highlight the potential difficulties in interpretation of protein-protein interactions based on co-crystal structures using short peptide motifs.

Estrogen receptors (ERs) associate with distinct transcriptional coactivators to mediate activation of target genes in response to estrogens. Previous work has provided multiple evidence for a critical role of p160 coactivators and associated histone acetyltransferases in estrogen signaling. In contrast, the involvement of the mammalian mediator complex remains to be established. Further, although the two subtypes ERα and ERβ appear to be similar in regard to principles of LXXLL-mediated coactivator binding to the AF-2 activation domain, there are indications that the context-dependent transcriptional activation profiles of the two ERs can be quite distinct. Potentially, this could be attributed to differences with regard to coregulator recruitment. We have here studied the interactions of the nuclear receptor-binding subunit of the mammalian mediator complex, referred to as TRAP220, with ERα and ERβ. In comparison to the p160 coactivator TIF2, we find that TRAP220 displays ERβ preference. Here, we show that this is a feature of the binding specificity of the TRAP220 LXXLL motifs and demonstrate that the ER subtype-specific F-domain influences TRAP220 interaction. Such differences with regard to coactivator recruitment indicate that the relative importance of individual coregulators in estrogen signaling could depend on the dominant ER subtype.

We have discovered that the orphan receptor DAX-1 (NROB1) interacts with the estrogen receptors ERα and ERβ. Interaction occurs with ligand-activated ERs in solution and on DNA and is mediated by the unique DAX-1 N-terminal repeat domain. Each of the three repeats contains a leucine-rich receptor-binding motif, known as the LXXLL motif, which is usually found in nuclear receptor coactivators. We have demonstrated that DAX-1 functions as an inhibitor of ER activation in mammalian cells and suggest a mechanism involving two sequential events, occupation of the ligand-induced coactivator-binding surface and subsequent recruitment of corepressors. Accordingly, we propose that DAX-1 itself acts as a corepressor for ERs. Because DAX-1 is coexpressed with ERs in reproductive tissues, these interactions could play significant roles by influencing estrogen signaling pathways. Our results point at functional similarities between DAX-1 and the orphan receptor SHP (NROB2) in that they have acquired features of transcriptional coregulators that are unique for members of the nuclear receptor family.

Ligand-induced transcriptional activation of gene expression by nuclear receptors is dependent on recruitment of coactivators as intermediary factors. The present work describes the cloning and characterization of RAP250, a novel human nuclear receptor coactivator. The results of in vitroand invivoexperiments indicate that the interaction of RAP250 with nuclear receptors is ligand-dependent or ligand-enhanced depending on the nuclear receptor and involves only one short LXXLL motif called nuclear receptor box. Transient transfection assays further demonstrate that RAP250 has a large intrinsic glutamine-rich activation domain and can significantly enhance the transcriptional activity of several nuclear receptors, acting as a coactivator. Interestingly, Northern blot and in situhybridization analyses reveal that RAP250 is widely expressed with the highest expression in reproductive organs (testis, prostate and ovary) and brain. Together, our data suggest that RAP250 may play an important role in mammalian gene expression mediated by nuclear receptor.

ABSTRACTSHP (short heterodimer partner) is an unusual orphan nuclear receptor consisting only of a ligand-binding domain, and it exhibits unique features of interaction with conventional nuclear receptors. While the mechanistic basis of these interactions has remained enigmatic, SHP has been suggested to inhibit nuclear receptor activation by at least three alternatives; inhibition of DNA binding via dimerization, direct antagonism of coactivator function via competition, and possibly transrepression via recruitment of putative corepressors. We now show that SHP binds directly to estrogen receptors via LXXLL-related motifs. Similar motifs, referred to as NR (nuclear receptor) boxes, are usually critical for the binding of coactivators to the ligand-regulated activation domain AF-2 within nuclear receptors. In concordance with the NR box dependency, SHP requires the intact AF-2 domain of agonist-bound estrogen receptors for interaction. Mutations within the ligand-binding domain helix 12, or binding of antagonistic ligands, which are known to result in an incomplete AF-2 surface, abolish interactions with SHP. Supporting the idea that SHP directly antagonizes receptor activation via AF-2 binding, we demonstrate that SHP variants, carrying either interaction-defective NR box mutations or a deletion of the repressor domain, have lost the capacity to inhibit agonist-dependent transcriptional estrogen receptor activation. Furthermore, our studies indicate that SHP may function as a cofactor via the formation of ternary complexes with dimeric receptors on DNA. These novel insights provide a mechanistic explanation for the inhibitory role of SHP in nuclear receptor signaling, and they may explain how SHP functions as a negative coregulator or corepressor for ligand-activated receptors, a novel and unique function for an orphan nuclear receptor.

ABSTRACTNuclear hormone receptors exert transcriptional activation of target genes upon hormone induction via interactions with the basal transcription machinery. This interaction is mediated by cofactors which physically bind to receptors, thereby acting as coactivators or corepressors leading to activation or repression, respectively. Here we report the screening for and cloning of a peroxisome proliferator receptor-interacting protein, the rat homolog of TIF2. By sequence comparison with the related coactivator SRC-1, we identified three short conserved motifs (NR boxes) in both proteins which are the putative binding sites of TIF2 to nuclear hormone receptors. We demonstrate here by generation of amino acid exchanges within the NR boxes that all three boxes located in the receptor interaction domain of TIF2 are necessary and sufficient for interaction. The three boxes individually can bind to hormone receptors but display preferences in binding for certain receptors. In addition, we show that the interaction domain of TIF2 can compete with other AF-2-dependent cofactors for binding to receptors. Finally, we demonstrate cooperative binding of two TIF2 molecules to a heterodimeric nuclear receptor complex even in the presence of only one cognate ligand, indicating an allosteric effect on the heterodimeric partner upon coactivator binding.

SHP (short heterodimer partner) is an unusual orphan nuclear receptor that contains a putative ligand-binding domain but lacks a conserved DNA-binding domain. Although no conventional receptor function has yet been identified, SHP has been proposed to act as a negative regulator of nuclear receptor signaling pathways, because it interacts with and inhibits DNA binding and transcriptional activity of various nonsteroid receptors, including thyroid hormone and retinoid receptors. We show here that SHP interacts directly with agonist-bound estrogen receptors, ERα and ERβ, and inhibits ER-mediated transcriptional activation. SHP specifically targets the ligand-regulated activation domain AF-2 and competes for binding of coactivators such as TIF2. Thus, SHP may represent a new category of negative coregulators for ligand-activated nuclear receptors. SHP mRNA is widely expressed in rat tissues including certain estrogen target tissues, and subcellular localization studies demonstrate that SHP is a nuclear protein, suggesting a biological significance of the SHP interactions with ERs. Taken together, these results identify ERs as novel SHP targets and suggest that competition for coactivator-binding is a novel mechanism by which SHP may inhibit nuclear receptor activation.

Abstract.: Similar to heat-stress transcription factors (HSFs) from non-plant sources, HSFA1 and HSFA2 from tomato (Lycopersicon esculentum Mill) contain two conserved clusters of basic amino acid residues (K/R1 and K/R2) which might serve as nuclear localization signal (NLS) motifs. Mutation of either one of them and functional testing of the corresponding proteins in a transient expression assay using tobacco (Nicotiana plumbaginifolia L.) protoplasts gave the following results. Whereas K/R1, positioned in all HSFs at the C-terminus of the DNA-binding domain, had no influence on nuclear import, the K/R1 mutants were impaired in their interaction with the DNA (band-shift assays). In contrast to this, mutants of the K/R2 motif, found 15–20 amino acid residues C-terminal of the oligomerization domain (HR-A/B region), had wild-type activity in DNA-binding but were defective in nuclear import. Thus, for the related tomato HSFA1 and HSFA2 the K/R2 cluster represents the only NLS motif, and in this function it cannot be replaced by K/R1.

Glucose homeostasis is maintained through organ crosstalk that regulates secretion of insulin to keep blood glucose levels within a physiological range. In type 2 diabetes, this coordinated response is altered, leading to a deregulation of beta cell function and inadequate insulin secretion. Reprogramming of white adipose tissue has a central role in this deregulation, but the critical regulatory components remain unclear. Here, we demonstrate that expression of the transcriptional coregulator GPS2 in white adipose tissue is correlated with insulin secretion rate in humans. The causality of this relationship is confirmed using adipocyte-specific GPS2 knockout mice, in which inappropriate secretion of insulin promotes glucose intolerance. This phenotype is driven by adipose-tissue-secreted factors, which cause increased pancreatic islet inflammation and impaired beta cell function. Thus, our study suggests that, in mice and in humans, GPS2 controls the reprogramming of white adipocytes to influence pancreatic islet function and insulin secretion.

Non-alcoholic fatty liver disease (NAFLD) is a very common indication for liver transplantation. How fat-rich diets promote progression from fatty liver to more damaging inflammatory and fibrotic stages is poorly understood. Here, we show that disrupting phosphorylation at Ser196 (S196A) in the liver X receptor alpha (LXRα, NR1H3) retards NAFLD progression in mice on a high-fat-high-cholesterol diet. Mechanistically, this is explained by key histone acetylation (H3K27) and transcriptional changes in pro-fibrotic and pro-inflammatory genes. Furthermore, S196A-LXRα expression reveals the regulation of novel diet-specific LXRα-responsive genes, including the induction of Ces1f, implicated in the breakdown of hepatic lipids. This involves induced H3K27 acetylation and altered LXR and TBLR1 cofactor occupancy at the Ces1f gene in S196A fatty livers. Overall, impaired Ser196-LXRα phosphorylation acts as a novel nutritional molecular sensor that profoundly alters the hepatic H3K27 acetylome and transcriptome during NAFLD progression placing LXRα phosphorylation as an alternative anti-inflammatory or anti-fibrotic therapeutic target.

Hypertrophic white adipose tissue (WAT) represents a maladaptive mechanism linked to the risk for developing type 2 diabetes in humans. However, the molecular events that predispose WAT to hypertrophy are poorly defined. Here, we demonstrate that adipocyte hypertrophy is triggered by loss of the corepressor GPS2 during obesity. Adipocyte-specific GPS2 deficiency in mice (GPS2 AKO) causes adipocyte hypertrophy, inflammation, and mitochondrial dysfunction during surplus energy. This phenotype is driven by HIF1A activation that orchestrates inadequate WAT remodeling and disrupts mitochondrial activity, which can be reversed by pharmacological or genetic HIF1A inhibition. Correlation analysis of gene expression in human adipose tissue reveals a negative relationship between GPS2 and HIF1A, adipocyte hypertrophy, and insulin resistance. We propose therefore that the obesity-associated loss of GPS2 in adipocytes predisposes for a maladaptive WAT expansion and a pro-diabetic status in mice and humans.