GPS2 is required for cholesterol efflux by triggering histone demethylation, LXR recruitment, and coregulator assembly at the ABCG1 locus.

Transcriptional coregulators, rather than ligand signals, are suspected to confer context and pathway specificity to nuclear receptor signaling, but the identity of such specifying coregulators and the underlying molecular mechanisms remain largely enigmatic. Here we address this issue in metabolic oxysterol receptor LXR pathways and describe the selective requirement of GPS2 for ABCG1 cholesterol transporter gene transcription and cholesterol efflux from macrophages. We implicate GPS2 in facilitating LXR recruitment to an ABCG1-specific promoter/enhancer unit upon ligand activation and identify functional links to histone H3K9 demethylation. We further describe fundamental differences between ABCG1 and ABCA1 with regard to GPS2 in relation to other coregulators, which are likely to apply to additional LXR-regulated genes. Our work identifies a coregulator-dependent epigenetic mechanism governing the access of a nuclear receptor to communicating regulatory regions in the genome. The pathway and coregulator selectivity of this mechanism implies pharmacological possibilities for the development of selective LXR agonists.

Pancreatic exocrine insufficiency in LXRbeta-/- mice is associated with a reduction in aquaporin-1 expression.

Liver X receptors (LXRs) alpha and beta are nuclear oxysterol receptors with a key role in cholesterol, triglyceride, and glucose metabolism. In LXRbeta(-/-) mice on a normal diet, there is a reduction in size of perigonadal fat pad and, on high-fat diet there is resistance to obesity. In the present study, we investigated the reason for the resistance of LXRbeta(-/-) mice to weight gain. In LXRbeta(-/-) mice we found pancreatic exocrine insufficiency with reduced serum levels of amylase and lipase, reduced proteolytic activity in feces, chronic inflammatory infiltration, and, in the ductal epithelium, an increased apoptosis without compensatory proliferation. Electron microscopy revealed ductal dilatation with intraductal laminar structures characteristic of cystic fibrosis. To investigate the relationship between LXRbeta and pancreatic secretion, we studied the expression of LXRbeta and the water channel, aquaporin-1 (AQP1), in the ductal epithelium of the pancreas. In WT mice, ductal epithelial cells expressed LXRbeta in the nuclei and AQP1 on the plasma membrane. In LXRbeta(-/-) mice neither LXRbeta nor AQP1 was detectable. Moreover, in WT mice the LXR agonist (T2320) increased AQP1 gene expression. These data demonstrate that in LXRbeta(-/-) mice dietary resistance to weight gain is caused by pancreatic insufficiency and that LXRbeta regulates pancreatic exocrine secretion through the control of AQP1 expression. Pancreatic exocrine insufficiency is the main cause of malabsorption syndrome responsible for weight loss in adults and growth failure in children. Several genes are known to be involved in the pathogenesis and susceptibility to pancreatic insufficiency. LXRbeta should be included in that list.

Estrogen receptor beta2 negatively regulates the transactivation of estrogen receptor alpha in human breast cancer cells.

Estrogens, by binding to and activating two estrogen receptors (ERalpha and ERbeta), are critically involved in the development of the mammary gland and breast cancer. An isoform of ERbeta, ERbeta2 (also called ERbetacx), with an altered COOH-terminal region, is coexpressed with ERalpha in many human breast cancers. In this study, we generated a stable cell line from MCF7 breast cancer cells expressing an inducible version of ERbeta2, along with endogenous ERalpha, and examined the effects of ERbeta2 on the ERalpha protein levels and function. We showed that ERbeta2 inhibited ERalpha-mediated transactivation via estrogen response element and activator protein-1 sites of reporter constructs as well as the endogenous genes pS2 and MMP-1. Chromatin immunoprecipitation assays revealed that ERbeta2 expression caused a significant reduction in the recruitment of ERalpha to both the pS2 and MMP-1 promoters. Furthermore, ERbeta2 expression induced proteasome-dependent degradation of ERalpha. The inhibitory effects of ERbeta2 on ERalpha activity were further confirmed in HEK293 cells that lack functional endogenous ERs. We also showed that ERbeta2 can interact with ERalpha both in vitro and in mammalian cells, which is compatible with a model where ERbeta2/ERalpha heterodimers are targeted to the proteasome. Finally, in human breast cancer samples, we observed that expression of ERbeta2 significantly correlated with ERalpha-negative phenotype. Our data suggest that ERbeta2 could influence ERalpha-mediated effects relevant for breast cancer development, including hormone responsiveness.

Structure of the retinoid X receptor α-liver X receptor ß (RXRα-LXRß) heterodimer on DNA.

Nuclear receptors (NRs) are conditional transcription factors with common multidomain organization that bind diverse DNA elements. How DNA sequences influence NR conformation is poorly understood. Here we report the crystal structure of the human retinoid X receptor α-liver X receptor ß (RXRα-LXRß) heterodimer on its cognate element, an AGGTCA direct repeat spaced by 4 nt. The complex has an extended X-shaped arrangement, with DNA- and ligand-binding domains crossed, in contrast to the parallel domain arrangement of other NRs that bind an AGGTCA direct repeat spaced by 1 nt. The LXRß core binds DNA via canonical contacts and auxiliary DNA contacts that enhance affinity for the response element. Comparisons of RXRα-LXRßs in the crystal asymmetric unit and with previous NR structures reveal flexibility in NR organization and suggest a role for RXRα in adaptation of heterodimeric complexes to DNA.

Mouse estrogen receptor beta isoforms exhibit differences in ligand selectivity and coactivator recruitment.

Estrogens exert their physiological effects through two estrogen receptor (ER) subtypes, ERalpha and ERbeta. In mouse, the cloning of an alternative splice variant of the wild-type ERbeta (mERbeta1), mERbeta2, which contains an 18 amino acid insertion in the ligand binding domain, contributed an additional level of complexity to estrogen signaling. In this study we have assayed the interaction of several known ligands with mERbeta1 and mERbeta2. The binding affinity of estradiol was 14-fold higher for mERbeta1 than for mERbeta2. In contrast, raloxifene was dramatically (8-fold) mERbeta2 selective. The selectivity for mERbeta2 was abolished when the 2-arylbenzothiophene core of the raloxifene molecule was tested for binding affinity, demonstrating that the 3-aroyl side chain of raloxifene plays an important role in contributing to its mERbeta2 selectivity. The opposite isoform selectivity found for estradiol and raloxifene in our ligand binding assay was also reflected in the transactivation assay system. That is, mERbeta2 required 10-fold greater estradiol concentrations for maximal activation compared to mERbeta1, whereas raloxifene was more potent in antagonizing estradiol-induced gene expression via mERbeta2 than mERbeta1. The raloxifene core behaved as a pure agonist. Furthermore, mERbeta2 showed significantly decreased estradiol-induced maximal transcriptional activity as compared to mERbeta1. A pull-down assay indicated that the interactions of TIF2 and RAP250 with mERbeta2 were weaker than with mERbeta1. To assess TIF2 and RAP250 interactions with ERs more quantitatively, we examined the interaction of LXXLL containing peptides derived from TIF2 and RAP250 with mERbeta1 and mERbeta2 using surface plasmon resonance analysis. Our results indicate that mERbeta2 interacts with both coactivators with lower affinity, which may explain its reduced transcriptional activity. Taken together, these results suggest that ligand selectivity and coactivator recruitment of the ERbeta isoforms constitute additional levels of specificity that influence the transcriptional response in estrogen target cells.

Selection of DNA aptamers against rat liver X receptors.

Liver X receptors alpha and beta (LXRalpha; LXRbeta) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. LXRs play an important role in the reverse cholesterol transport and govern the expression of many of the proteins that are indispensable for the regulation of normal cholesterol levels in the body. SELEX, an in vitro selection technology, was used on a single stranded DNA library harboring a 12 randomized nucleotide sequence in order to isolate aptamers showing affinity for LXRalpha. Enzyme-linked assays and surface plasmon resonance measurements showed that the selected aptamers had strong affinities for LXRalpha with apparent dissociation constants, K(d)s, in nanomolar range. All clones carried CG-repeats, indicating a probability for a similar manner of binding to LXRalpha. Very high cross-reactivities were observed when testing the aptamers with LXRbeta (up to 700%) and RXRalpha (up to 50%). If instead we regard the aptamer sequences as selected against LXRbeta, the cross-reactivities decrease considerably, to 17% for LXRalpha and 7% for RXRalpha. Therefore, in the future we are planning to use the obtained aptamers as binders for LXRbeta.

A transcriptional inhibitor targeted by the atypical orphan nuclear receptor SHP.

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.

Purification of functional full-length liver X receptor beta produced in Escherichia coli.

Liver X receptor beta (LXRbeta) is a ligand dependent transcription factor that is a member of the nuclear receptor superfamily. LXRbeta and its isoform LXRalpha have recently been recognized as important regulators of lipid homeostasis in vertebrates. N-terminally hexahistidine-tagged rat LXRbeta was expressed in Escherichia coli as a full-length protein and purified in two chromatographic steps, immobilized metal affinity chromatography and gel filtration. From 10g of bacterial cells, 2.5mg of protein was recovered. The purified LXRbeta is functional with respect to ligand-, DNA-, and coactivator-binding. The synthetic ligand T0901317 bound to LXRbeta with high affinity yielding a K(d) of 2.7nM. Specific interaction with DR4 response elements, in the presence of RXR, was demonstrated with electrophoretic mobility shift assay. Furthermore, surface plasmon resonance analysis of LXRbeta binding to coactivator peptides revealed a ligand dependent interaction with the C-terminal nuclear receptor binding site of the coactivator RAP250. The purified LXRbeta constitutes an important tool for further functional and structural studies.

Identification of a functional variant of estrogen receptor beta in an African population.

In this study, we identified five novel polymorphisms in the estrogen receptor beta (ERbeta) gene in an African population. Interestingly, two of these variants are expected to change the amino acid sequence of the ERbeta protein. These changes correspond to an isoleucine to valine substitution at amino acid position 3 (I3V) and a valine to glycine substitution at position 320 (V320G), respectively. The functional consequences of these amino acid substitutions were determined in different in vitro assays. The I3V mutation displayed no differences with regard to transcriptional activity in a reporter assay, as compared with the wild-type receptor. The V320G mutation, however, showed significantly decreased maximal transcriptional activity in a reporter assay, although its binding affinity for 17beta-estradiol was not affected. A pull-down assay indicated that the interaction of full-length TIF2 with hERbetaV320G was weaker than with hERbetawt. Moreover, surface plasmon resonance analysis revealed reduced interaction of the V320G ERbeta variant with the NR box I and II modules of TIF2. To our knowledge, this represents the first identification of a functional polymorphism in the ERbeta gene. This novel polymorphism provides a tool for human genetic studies of diseases in the African population.