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The liver receptor homologue 1 (LRH-1 (NR5A2)) belongs to the orphan nuclear receptor family, indicating that initially no ligand was known. Although recent studies have shown that ligand binding can be obtained, the biological relevance remains elusive. Here, we modify the observed X-ray ligand into a biologically more significant phospholipid (phosphatidylserine, PS) present in human, to study, by molecular dynamics (MD) simulations, the impact of the ligand on the receptor and the interaction with different cofactor peptides. Furthermore, we characterize the interactions between receptor and the cofactor peptides of DAX-1 (NR0B1), Prox1 and SHP LXXLL box 1 and 2 (NR0B2) in terms of specificity. Our MD simulation results show different interaction patterns for the SHP box2 compared to DAX-1, PROX1 and SHP box1. SHP box2 shows specific interactions at its more C-terminal end while the other investigated peptides show specific interactions at several positions but particularly at the +2 site. The peptide +2 side chain interacts with a charged amino acid of the receptor, in hLRH-1 Asp372. Together with the charge clamp residues Arg361 and Glu534, Asp372 forms a triangle shaped charge clamp responsible for peptide orientation and increased affinity. The binding of the PS ligand causes no overall structural changes of the receptor but affects the interactions with cofactor peptides. The cofactor peptides from SHP decrease its interaction with the receptor upon ligand binding while DAX-1 and PROX1 are unchanged or increase. The diverse ligand binding response of the cofactor provides an opportunity for drug design with the possibility to create agonist ligands to modify cofactor interaction.

The selenoprotein thioredoxin reductase (TrxR1) is an integral part of the thioredoxin system. It serves to transfer electrons from NADPH to thioredoxin leading to its reduction. Interestingly, recent work has indicated that thioredoxin reductase can regulate the activity of transcription factors such as p53, hypoxia-inducible factor, and AP-1. Here, we describe that an alternative splicing variant of thioredoxin reductase (TrxR1b) containing an LXXLL peptide motif, is implicated in direct binding to nuclear receptors. In vitro interaction studies revealed direct interaction of the TrxR1b with the estrogen receptors alpha and beta. Confocal microscopy analysis showed nuclear colocalization of the TrxR1b with both estrogen receptor alpha and beta in estradiol-17beta-treated cells. Transcriptional studies demonstrated that TrxR1b can affect estrogen-dependent gene activation differentially at classical estrogen response elements as compared with AP-1 response elements. Based on these results, we propose a model where thioredoxin reductase directly influences the estrogen receptor-coactivator complex assembly on non-classical estrogen response elements such as AP-1. In summary, our results suggest that TrxR1b is an important modulator of estrogen signaling.

In contrast with the class A heat stress transcription factors (HSFs) of plants, a considerable number of HSFs assigned to classes B and C have no evident function as transcription activators on their own. However, in the following article, we provide evidence that tomato (Lycopersicon peruvianum) HsfB1 represents a novel type of coactivator cooperating with class A HSFs (e.g., with tomato HsfA1). Provided the appropriate promoter architecture, the two HSFs assemble into an enhanceosome-like complex, resulting in strong synergistic activation of reporter gene expression. Moreover, HsfB1 also cooperates in a similar manner with other activators, for example, with the ASF1/2 enhancer binding proteins of the 35S promoter of Cauliflower mosaic virus or with yet unidentified activators controlling housekeeping gene expression. By these effects, HsfB1 may help to maintain and/or restore expression of certain viral or housekeeping genes during ongoing heat stress. The coactivator function of HsfB1 depends on a histone-like motif in its C-terminal domain with an indispensable Lys residue in the center (GRGKMMK). This motif is required for recruitment of the plant CREB binding protein (CBP) ortholog HAC1. HsfA1, HsfB1, and HAC1/CBP form ternary complexes in vitro and in vivo with markedly enhanced efficiency in promoter recognition and transcription activation in plant and mammalian (COS7) cells. Using small interfering RNA-mediated knock down of HAC1 expression in Arabidopsis thaliana mesophyll protoplasts, the crucial role for the coactivator function of HsfB1 was confirmed., (Copyright 2004 American Society of Plant Biologists)

Nuclear receptors (NRs) comprise a family of ligand inducible transcription factors. To achieve transcriptional activation of target genes, DNA-bound NRs directly recruit general transcription factors (GTFs) to the preinitiation complex or bind intermediary factors, so-called coactivators. These coactivators often constitute subunits of larger multiprotein complexes that act at several functional levels, such as chromatin remodeling, enzymatic modification of histone tails, or modulation of the preinitiation complex via interactions with RNA polymerase II and GTFs. The binding of NR to coactivators is often mediated through one of its activation domains. Many NRs have at least two activation domains, the ligand-independent activation function (AF)-1, which resides in the N-terminal domain, and the ligand-dependent AF-2, which is localized in the C-terminal domain. In this review, we summarize and discuss current knowledge regarding the molecular mechanisms of AF-1- and AF-2-mediated gene activation, focusing on AF-1 and AF-2 conformation and coactivator binding.

TRAP220 (thyroid hormone receptor-associated protein) is a recently cloned nuclear receptor coactivator, which interacts with several nuclear receptors in a ligand-dependent manner and stimulates transcription by recruiting the TRAP mediator complex to hormone responsive promoter regions. TRAP220 has been shown to interact with thyroid hormone receptors, vitamin D receptors, peroxisome proliferator-activated receptors, retinoic acid receptors and oestrogen receptors. Thyroid hormone and retinoic acid play very important roles in brain development and they also influence adult brain. Using in situ hybridization we have examined expression of TRAP220 mRNA in the central nervous system during development and in adult rat and mouse brain. Expression of TRAP220 was seen already during early embryonic development in the epithelium of neural tube at E9 in mouse and at E12 in rat. At later stages of development the strongest signal was seen in different layers of cerebral neocortex, external germinal layer of cerebellum, differentiating fields of hippocampus and neuroepithelium, and a moderate signal was detected in basal ganglia, different areas of diencephalon and midbrain. In adult rat brain the signal was more restricted than during development. TRAP220 expression occurred mostly in the granular layer of cerebellar cortex, piriform cortex and hippocampal formation. The signal was found predominantly in neurons. Our work supports the assumption that TRAP220 plays an important role in growth and differentiation of central nervous system and may have a function in certain areas of adult brain.

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.

DAX-1 (NROB1) is an atypical member of the nuclear receptor family that is predominantly expressed in mammalian reproductive tissues. While a receptor function of DAX-1 remains enigmatic, previous work has indicated that DAX-1 inhibits the activity of the orphan receptor steroidogenic factor 1 and the estrogen receptors (ERs), presumably via direct occupation of the coactivator-binding surface and subsequent recruitment of additional corepressors. In vivo evidence points at a particular role of DAX-1 for the development and maintenance of male reproductive functions. In this study, we have identified the androgen receptor (AR) NR3C4 as a novel target for DAX-1. We show that DAX-1 potently inhibits ligand-dependent transcriptional activation as well as the interaction between the N- and C-terminal activation domains of AR. We provide evidence for direct interactions of the two receptors that involve the N-terminal repeat domain of DAX-1 and the C-terminal ligand-binding and activation domain of AR. Moreover, DAX-1, known to shuttle between the cytoplasm and the nucleus, is capable of relocalizing AR in both cellular compartments, suggesting that intracellular tethering is associated with DAX-1 inhibition. These results implicate novel inhibitory mechanisms of DAX-1 action with particular relevance for the modulation of androgen-dependent gene transcription in the male reproductive system.

Highlights: [•] Nutrient excess leads to the expression of inflammatory molecules and the recruitment of immune cells. [•] Transcription factors control the regulation of inflammatory genes. [•] Transcriptional coregulators act as epigenomic checkpoints of inflammatory gene expression. [ABSTRACT FROM AUTHOR]

SHP (short heterodimer partner, NROB2) is an atypical orphan member of the mammalian nuclear receptor family that consists only of a putative ligand-binding domain and thus cannot bind DNA. Instead, SHP acts as a transcriptional coregulator by inhibiting the activity of various nuclear receptors (downstream targets) via occupation of the coactivator-binding surface and active repression. However, repression mechanisms have remained elusive and may involve coinhibitory factors (upstream targets) distinct from known nuclear receptor corepressors. Here, we describe the isolation of mouse E1A-like inhibitor of differentiation 1 (EID1) as a candidate coinhibitor for SHP. We characterize the interactions between SHP and EID1 and identify two repression-defective SHP mutations that have lost the ability to bind EID1. We suggest histone acetyltransferases and histones as targets for EID1 action and propose that SHP inhibition of transcription involves EID1 antagonism of CBP/p300-dependent coactivator functions. [ABSTRACT FROM AUTHOR]

A recent study in Molecular Cell () uncovered parallel yet distinct SUMOylation pathways that provide insights into molecular mechanisms of transrepression mediated by activated nuclear receptors LXRα/β and PPARγ, thereby clarifying receptor, signal, and gene specificity of transrepression in inflammatory processes. [Copyright &y& Elsevier]

Defects in adipocyte lipolysis drive multiple aspects of cardiometabolic disease, but the transcriptional framework controlling this process has not been established. To address this, we performed a targeted perturbation screen in primary human adipocytes. Our analyses identified 37 transcriptional regulators of lipid mobilization, which we classified as (i) transcription factors, (ii) histone chaperones, and (iii) mRNA processing proteins. On the basis of its strong relationship with multiple readouts of lipolysis in patient samples, we performed mechanistic studies on one hit, ZNF189, which encodes the zinc finger protein 189. Using mass spectrometry and chromatin profiling techniques, we show that ZNF189 interacts with the tripartite motif family member TRIM28 and represses the transcription of an adipocyte-specific isoform of phosphodiesterase 1B (PDE1B2). The regulation of lipid mobilization by ZNF189 requires PDE1B2, and the overexpression of PDE1B2 is sufficient to attenuate hormone-stimulated lipolysis. Thus, our work identifies the ZNF189-PDE1B2 axis as a determinant of human adipocyte lipolysis and highlights a link between chromatin architecture and lipid mobilization. [ABSTRACT FROM AUTHOR]

Lung cancer progression relies on angiogenesis, which is a response to hypoxia typically coordinated by hypoxia-inducible transcription factors (HIFs), but growing evidence indicates that transcriptional programs beyond HIFs control tumor angiogenesis. Here, we show that the redox-sensitive transcription factor BTB and CNC homology 1 (BACH1) controls the transcription of a broad range of angiogenesis genes. BACH1 is stabilized by lowering ROS levels; consequently, angiogenesis gene expression in lung cancer cells, tumor organoids, and xenograft tumors increased substantially following administration of vitamins C and E and ^-acetylcysteine in a BACH1-dependent fashion under normoxia. Moreover, angiogenesis gene expression increased in endogenous BACH1-overexpressing cells and decreased in BACH7-knockout cells in the absence of antioxidants. BACH1 levels also increased upon hypoxia and following administration of prolyl hydroxylase inhibitors in both H/F7A-knockout and WT cells. BACH1 was found to be a transcriptional target of HIF1a, but BACH1's ability to stimulate angiogenesis gene expression was HIF1a independent. Antioxidants increased tumor vascularity in vivo in a BACH1-dependent fashion, and overexpressing BACH1 rendered tumors sensitive to antiangiogenesis therapy. BACH1 expression in tumor sections from patients with lung cancer correlated with angiogenesis gene and protein expression. We conclude that BACH1 is an oxygen- and redox-sensitive angiogenesis transcription factor. [ABSTRACT FROM AUTHOR]

Genetic and experimental evidence points to a critical involvement of the atypical mammalian orphan receptor DAX-1 in reproductive development and steroidogenesis. Unlike conventional nuclear receptors, DAX-1 appears not to function as a DNA-bound transcription factor. Instead, it has acquired the capability to act as a transcriptional corepressor of steroidogenic factor 1 (SF-1). The interplay of DAX-1 and SF-1 is considered a central, presumably ligand-independent element of adrenogonadal development and function that requires tight regulation. This raises a substantial interest in identifying its modulators and the regulatory signals involved. Here, we uncover molecular mechanisms that link DAX-1 to the ubiquitin modification system via functional interaction with the E3 ubiquitin ligase RNF31. We demonstrate that RNF31 is coexpressed with DAX-1 in steroidogenic tissues and participates in repressing steroidogenic gene expression. We provide evidence for the in vivo existence of a corepressor complex containing RNF31 and DAX-1 at the promoters of the StAR and CYP19 genes. Our data suggest that RNF31 functions to stabilize DAX-1, which might be linked to DAX-1 monoubiquitination. In conclusion, RNF31 appears to be required for DAX-1 to repress transcription, provides means to regulate DAX-1 in ligand-independent ways, and emerges as a relevant coregulator of steroidogenic pathways governing physiology and disease. [ABSTRACT FROM AUTHOR]

SHP has been implicated as a pleiotropic regulator of diverse biological functions by its ability to inhibit numerous nuclear receptors. Recently, we reported that SHP inhibits transcription of CYP7A1, a key gene in bile acid biosynthesis, by recruiting histone deacetylases (HDACs) and a Swi/Snf-Brm complex. To further delineate the mechanism of this inhibition, we have examined whether methylation of histones is also involved and whether a functional interplay between chromatin-modifying enzymes occurs. The histone methyltransferase G9a, but not SUV39, was colocalized with SHP in the nucleus and directly interacted with SHP in vitro. G9a, which was coimmunoprecipitated with hepatic SHP, methylated Lys-9 of histone 3 (H3K9) in vitro. Expression of G9a enhanced inhibition of CYP7A1 transcription by SHP, while a catalytically inactive G9a dominant negative (DN) mutant reversed the SHP inhibition. G9a was recruited to and H3K9 was methylated at the CYP7A1 promoter in a SHP-dependent manner in bile acid-treated HepG2 cells. Expression of the G9a-DN mutant inhibited H3K9 methylation, blocked the recruitment of the Brm complex, and partially reversed CYP7A1 inhibition by bile acids. Inhibition of HDAC activity with trichostatin A blocked deacetylation and methylation of H3K9 at the promoter, and, conversely, inhibition of H3K9 methylation by G9a-DN partially blocked deacetylation. Hepatic expression of G9a-DN in mice fed cholic acid disrupted bile acid homeostasis, resulting in increased bile acid pools and partial de-repression of Cyp7a1 and Cyp8b1. Our studies establish a critical role for G9a methyltransferase, histone deacetylases, and the Swi/Snf-Brm complex in the SHP-mediated inhibition of hepatic bile acid synthesis via coordinated chromatin modification at target genes. [ABSTRACT FROM AUTHOR]

Small heterodimer partner (SHP; NR0B2), an exceptional member of the mammalian nuclear receptor family, directly modulates the activities of conventional nuclear receptors by acting as an inducible and tissue-specific corepressor. Recent progress in dissecting underlying molecular mechanisms, identifying target factors and target genes, and uncovering physiological functions points to the regulatory involvement of SHP in diverse metabolic and intracellular pathways that awaits future clarification. In this review, we carry out a comprehensive survey of all published data and discuss our current understanding of molecular mechanisms and physiological consequences governing SHP action. [Copyright &y& Elsevier]

While the role of transcription factors and coactivators in controlling enhancer activity and chromatin structure linked to gene expression is well established, the involvement of corepressors is not. Using inflammatory macrophage activation as a model, we investigate here a corepressor complex containing GPS2 and SMRT both genome-wide and at the Ccl2 locus, encoding the chemokine CCL2 (MCP-1). We report that corepressors co-occupy candidate enhancers along with the coactivators CBP (H3K27 acetylase) and MED1 (mediator) but act antagonistically by repressing eRNA transcription-coupled H3K27 acetylation. Genome editing, transcriptional interference, and cistrome analysis reveals that apparently related enhancer and silencer elements control Ccl2 transcription in opposite ways. 4C-seq indicates that corepressor depletion or inflammatory signaling functions mechanistically similarly to trigger enhancer activation. In ob/ob mice, adipose tissue macrophage-selective depletion of the Ccl2 enhancer-transcribed eRNA reduces metaflammation. Thus, the identified corepressor-eRNA-chemokine pathway operates in vivo and suggests therapeutic opportunities by targeting eRNAs in immuno-metabolic diseases. • Corepressor recruitment is a genome-wide feature of inflammatory enhancers • Corepressors control enhancer-silencer-promoter looping within the Ccl2 TAD • Corepressors antagonize eRNA transcription and CBP-mediated H3K27 acetylation • Ccl2 eRNA depletion in ob/ob mice supports eRNA function and targeting options Huang et al. dissect epigenetic mechanisms underlying the anti-inflammatory action of macrophage corepressors. By studying chromatin structure and histone modifications, transcription factor and coregulator dynamics, and eRNA transcription at the mouse Ccl2 locus, they uncover a role of corepressors in controlling enhancer and silencer remodeling linked to inflammatory gene expression. [ABSTRACT FROM AUTHOR]