Sex- and species-specific contribution of CD99 to T cell costimulation during multiple sclerosis.

BACKGROUND: Differences in immune responses between women and men are leading to a strong sex bias in the incidence of autoimmune diseases that predominantly affect women, such as multiple sclerosis (MS). MS manifests in more than twice as many women, making sex one of the most important risk factor. However, it is incompletely understood which genes contribute to sex differences in autoimmune incidence. To address that, we conducted a gene expression analysis in female and male human spleen and identified the transmembrane protein CD99 as one of the most significantly differentially expressed genes with marked increase in men. CD99 has been reported to participate in immune cell transmigration and T cell regulation, but sex-specific implications have not been comprehensively investigated. METHODS: In this study, we conducted a gene expression analysis in female and male human spleen using the Genotype-Tissue Expression (GTEx) project dataset to identify differentially expressed genes between women and men. After successful validation on protein level of human immune cell subsets, we assessed hormonal regulation of CD99 as well as its implication on T cell regulation in primary human T cells and Jurkat T cells. In addition, we performed in vivo assays in wildtype mice and in Cd99-deficient mice to further analyze functional consequences of differential CD99 expression. RESULTS: Here, we found higher CD99 gene expression in male human spleens compared to females and confirmed this expression difference on protein level on the surface of T cells and pDCs. Androgens are likely dispensable as the cause shown by in vitro assays and ex vivo analysis of trans men samples. In cerebrospinal fluid, CD99 was higher on T cells compared to blood. Of note, male MS patients had lower CD99 levels on CD4+ T cells in the CSF, unlike controls. By contrast, both sexes had similar CD99 expression in mice and Cd99-deficient mice showed equal susceptibility to experimental autoimmune encephalomyelitis compared to wildtypes. Functionally, CD99 increased upon human T cell activation and inhibited T cell proliferation after blockade. Accordingly, CD99-deficient Jurkat T cells showed decreased cell proliferation and cluster formation, rescued by CD99 reintroduction. CONCLUSIONS: Our results demonstrate that CD99 is sex-specifically regulated in healthy individuals and MS patients and that it is involved in T cell costimulation in humans but not in mice. CD99 could potentially contribute to MS incidence and susceptibility in a sex-specific manner.

The immune system protects us from bacterial and viral infections and impacts the outcome of many diseases. Thus, understanding immunological processes is crucial to unravel pathogenic mechanisms and to develop new therapeutic treatment options. Sex is a biological variable affecting immunity and it is known that females and males differ in their immunological responses. Women mount stronger immune responses leading to more rapid control of infections and greater vaccine efficacy compared to men. However, this enhanced immune responsiveness is accompanied by female preponderance and susceptibility to autoimmune diseases like systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis (MS). MS sex ratio varies around 2:1 to 3:1 with a steadily increasing incidence in female MS patients making sex one of the top risk factors for developing MS. However, the underlying biological mechanisms including sex hormones as well as genetic and epigenetic factors and their complex interplay remain largely unknown. Here, we discovered the gene and its encoded protein CD99 to be differentially expressed between women and men with men showing increased expression on many immune cell subsets including T cells. Since T cells are key contributors to MS pathogenesis, we examined the role of CD99 on T cells of healthy individuals and MS patients. We were able to identify CD99-mediated T cell regulation, which might contribute to sex differences in MS susceptibility and incidence indicating the importance to include sex as a biological variable. Of note, these differences were not reproduced in mice showing the necessity of functional research in humans.

Lack of a protective effect of the Tmem106b "protective SNP" in the Grn knockout mouse model for frontotemporal lobar degeneration.

Genetic variants in TMEM106B are a common risk factor for frontotemporal lobar degeneration and the most important modifier of disease risk in patients with progranulin (GRN) mutations (FTLD-GRN). TMEM106B is encoding a lysosomal transmembrane protein of unknown molecular function. How it mediates its disease-modifying function remains enigmatic. Several TMEM106B single nucleotide polymorphisms (SNPs) are significantly associated with disease risk in FTLD-GRN carriers, of which all except one are within intronic sequences of TMEM106B. Of note, the non-coding SNPs are in high linkage disequilibrium with the coding SNP rs3173615 located in exon six of TMEM106B, resulting in a threonine to serine change at amino acid 185 in the minor allele, which is protective in FTLD-GRN carriers. To investigate the functional consequences of this variant in vivo, we generated and characterized a knockin mouse model harboring the Tmem106bT186S variant. We analyzed the effect of this protective variant on FTLD pathology by crossing Tmem106bT186S mice with Grn-/- knockout mice, a model for GRN-mediated FTLD. We did not observe the amelioration of any of the investigated Grn-/- knockout phenotypes, including transcriptomic changes, lipid alterations, or microgliosis in Tmem106bT186S/T186S × Grn-/- mice, indicating that the Tmem106bT186S variant is not protective in the Grn-/- knockout mouse model. These data suggest that effects of the associated SNPs not directly linked to the amino acid exchange in TMEM106B are critical for the modifying effect.

The adaptor protein PICK1 targets the sorting receptor SorLA.

SorLA is a member of the Vps10p-domain (Vps10p-D) receptor family of type-I transmembrane proteins conveying neuronal endosomal sorting. The extracellular/luminal moiety of SorLA has a unique mosaic domain composition and interacts with a large number of different and partially unrelated ligands, including the amyloid precursor protein as well as amyloid-ß. Several studies support a strong association of SorLA with sporadic and familial forms of Alzheimer's disease (AD). Although SorLA seems to be an important factor in AD, the large number of different ligands suggests a role as a neuronal multifunctional receptor with additional intracellular sorting capacities. Therefore, understanding the determinants of SorLA's subcellular targeting might be pertinent for understanding neuronal endosomal sorting mechanisms in general. A number of cytosolic adaptor proteins have already been demonstrated to determine intracellular trafficking of SorLA. Most of these adaptors and several ligands of the extracellular/luminal moiety are shared with the Vps10p-D receptor Sortilin. Although SorLA and Sortilin show both a predominant intracellular and endosomal localization, they are targeted to different endosomal compartments. Thus, independent adaptor proteins may convey their differential endosomal targeting. Here, we hypothesized that Sortilin and SorLA interact with the cytosolic adaptors PSD95 and PICK1 which have been shown to bind the Vps10p-D receptor SorCS3. We observed only an interaction for SorLA and PICK1 in mammalian-two-hybrid, pull-down and cellular recruitment experiments. We demonstrate by mutational analysis that the C-terminal minimal PDZ domain binding motif VIA of SorLA mediates the interaction. Moreover, we show co-localization of SorLA and PICK1 at vesicular structures in primary neurons. Although the physiological role of the interaction between PICK1 and SorLA remains unsolved, our study suggests that PICK1 partakes in regulating SorLA's intracellular itinerary.

Revisiting the proteolytic processing of cell adhesion molecule L1.

The important functions of cell adhesion molecule L1 in the nervous system depend on diverse proteolytic enzymes which generate different L1 fragments. It has been reported that cleavage in the third fibronectin type III (FNIII) homologous domain generates the fragments L1-80 and L1-140, while cleavage in the first FNIII domain yields the fragments L1-70 and L1-135. These results raised questions concerning the L1 cleavage sites. We thus generated gene-edited mice expressing L1 with mutations of the cleavage sites either in the first or third FNIII domain. By immunoprecipitations and immunoblot analyses using brain homogenates and different L1 antibodies, we show that L1-70 and L1-135 are generated in wild-type mice, but not or only to a low extent in L1 mutant mice. L1-80 and L1-140 were not detected in wild-type or mutant mice. Mass spectrometry confirmed the results from immunoprecipitations and immunoblot analyses. Based on these observations, we propose that L1-70 and L1-135 are the predominant fragments in the mouse nervous system and that the third FNIII domain is decisive for generating these fragments. Treatment of cultured cerebellar neurons with trypsin or plasmin, which were both proposed to generate L1-80 and L1-140 by cleaving in the third FNIII domain, showed by immunoprecipitations and immunoblot analyses that both proteases lead to the generation of L1-70 and L1-135, but not L1-80 and L1-140. We discuss previous observations on the basis of our new results and propose a novel view on the molecular features that render previous and present observations compatible.

A variant in IL6ST with a selective IL-11 signaling defect in human and mouse.

The GP130 cytokine receptor subunit encoded by IL6ST is the shared receptor for ten cytokines of the IL-6 family. We describe a homozygous non-synonymous variant in IL6ST (p.R281Q) in a patient with craniosynostosis and retained deciduous teeth. We characterize the impact of the variant on cytokine signaling in vitro using transfected cell lines as well as primary patient-derived cells and support these findings using a mouse model with the corresponding genome-edited variant Il6st p.R279Q. We show that human GP130 p.R281Q is associated with selective loss of IL-11 signaling without affecting IL-6, IL-27, OSM, LIF, CT1, CLC, and CNTF signaling. In mice Il6st p.R279Q lowers litter size and causes facial synostosis and teeth abnormalities. The effect on IL-11 signaling caused by the GP130 variant shows incomplete penetrance but phenocopies aspects of IL11RA deficiency in humans and mice. Our data show that a genetic variant in a pleiotropic cytokine receptor can have remarkably selective defects.

Development of phosphatase inhibitor-1 peptides acting as indirect activators of phosphatase 1.

Phosphatase inhibitor-1 (I-1) inhibits the catalytic subunit of protein phosphatase type 1 (PP1c) in its protein kinase A (PKA)-phosphorylated form (I-1(P)). It thereby amplifies PKA signaling, which, in the heart, mediates both beneficial (acute) and adverse (chronic) effects of catecholamines. Genetic deletion of I-1 was associated with protection against catecholamine toxicity, making the PP1c-I-1(P) complex a potential therapeutic target for chronic heart disease. Here, we sought to define targetable interaction sites of I-1 and PP1c, concentrating on the N-terminal domain of I-1 which includes the PP1c binding motif ((9)KIQF(12)) as well as a poly-Arg stretch. Substitution of (9)KIQ(11) residues for analogous amino acids, (9)RLN(11), resulted in doubling of the IC50 values, deletion of (9)KIQF(12) prevented I-1 PKA-dependent phosphorylation and thus activation. Mutation of the Arg residues preceding the PKA phosphorylation site (Thr35) to Ala (R/A(30-33)) abolished I-1 phosphorylation and its binding to and inhibition of PP1c. A series of synthetic peptides (4-11 residues) indicated that the KIQF motif as well as the surrounding anchoring residues was essential for interfering with the inhibitory effect of I-1(P) on PP1c, whereas the four Arg residues were not. Unexpectedly, the most effective nonapeptide (SPRKIQFTV) also antagonized the inhibitory effect of the non-conditional PP1 inhibitor-2 with similar affinity. Incubation of neonatal rat cardiac myocytes with a poly-Arg-modified SPRKIQFTV (10 µM) reduced catecholamine-induced phosphorylation of phospholamban, a well-known PKA downstream target sensitive to PP1c. Our data reiterate the importance of the KIQF motif and provide a tool for antagonizing I-1 inhibitory effects on PP1c, i.e., activating PP1 in vivo.

A multiple sclerosis-associated variant of CBLB links genetic risk with type I IFN function.

Multiple sclerosis (MS) is an autoimmune disease of the CNS, and autoreactive CD4(+) T cells are considered important for its pathogenesis. The etiology of MS involves a complex genetic trait and environmental triggers that include viral infections, particularly the EBV. Among the risk alleles that have repeatedly been identified by genome-wide association studies, three are located near the Casitas B-lineage lymphoma proto-oncogene b gene (CBLB). The CBLB protein (CBL-B) is a key regulator of peripheral immune tolerance by limiting T cell activation and expansion and hence T cell-mediated autoimmunity through its ubiquitin E3-ligase activity. In this study, we show that CBL-B expression is reduced in CD4(+) T cells from relapsing-remitting MS (RR-MS) patients during relapse. The MS risk-related single nucleotide polymorphism of CBLB rs12487066 is associated with diminished CBL-B expression levels and alters the effects of type I IFNs on human CD4(+) T cell proliferation. Mechanistically, the CBLB rs12487066 risk allele mediates increased binding of the transcription factor C/EBPß and reduced CBL-B expression in human CD4(+) T cells. Our data suggest a role of the CBLB rs12487066 variant in the interactions of a genetic risk factor and IFN function during viral infections in MS.

Impact of methionine oxidation as an initial event on the pathway of human prion protein conversion.

Prion diseases comprise a group of fatal neurodegenerative disorders characterized by the autocatalytic conversion of the cellular prion protein PrP(C) into the infectious misfolded isoform PrP(Sc). Increasing evidence supports a specific role of oxidative stress in the onset of pathogenesis. Although the associated molecular mechanisms remain to be elucidated in detail, several studies currently suggest that methionine oxidation already detected in misfolded PrP(Sc) destabilizes the native PrP fold as an early event in the conversion pathway. To obtain more insights about the specific impact of surface-exposed methionine residues on the oxidative-induced conversion of human PrP we designed, produced, and comparatively investigated two new pseudosulfoxidation mutants of human PrP 121-231 that comprises the well-folded C-terminal domain. Applying circular dichroism spectroscopy and dynamic light scattering techniques we showed that pseudosulfoxidation of all surface exposed Met residues formed a monomeric molten globule-like species with striking similarities to misfolding intermediates recently reported by other groups. However, individual pseudosulfoxidation at the polymorphic M129 site did not significantly contribute to the structural destabilization. Further metal-induced oxidation of the partly unfolded pseudosulfoxidation mutant resulted in the formation of an oligomeric state that shares a comparable size and stability with PrP oligomers detected after the application of different other triggers for structural conversion, indicating a generic misfolding pathway of PrP. The obtained results highlight the specific importance of methionine oxidation at surface exposed residues for PrP misfolding, strongly supporting the hypothesis that increased oxidative stress could be one causative event for sporadic prion diseases and other neurodegenerative disorders.

Nuclear factor I-A represses expression of the cell adhesion molecule L1.

BACKGROUND: The neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression. RESULTS: We identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold. CONCLUSION: Our findings suggest that NFI-A, in particular its brain-specific isoform, represses L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF).

The murine AE4 promoter predominantly drives type B intercalated cell specific transcription.

AE4 is an anion exchanger almost exclusively expressed in the collecting ducts of the kidney. This very restricted expression prompted us to analyze its transcription in more detail. 5' RACE yielded alternative transcriptional start sites that are predicted to code for N-terminal protein variants. Comparison of the 5' genomic sequence between species identified a transcriptionally active region with three conserved spans. In transgenic mice beta-galactosidase expression driven by this fragment resembled endogenous AE4 expression and was predominantly restricted to type B intercalated cells. Hence this promoter could prove useful to target type B intercalated cells by genetic approaches.

Transcriptional ERRgamma2-mediated activation is regulated by sentrin-specific proteases.

Modification with SUMOs (small ubiquitin-related modifiers) has emerged as an important means of regulating the activity of transcription factors, often by repressing their activity. The ERRgamma [oestrogen receptor-related receptor gamma; ERR3 or NR3B3 (nuclear receptor subfamily 3, group B, gene3)] is a constitutively active orphan nuclear receptor. A PDSM, (phosphorylation-dependent sumoylation motif) is located in the close vicinity of the N-terminally located ERRgamma2-specific AF-1 (activation function-1). Its function can be replaced by an NDSM (negatively charged amino acid-dependent sumoylation motif). A mutational analysis reveals that ERRgamma2 activity is modulated through sumoylation of a lysine residue at position 40, which in turn is regulated by phosphorylation. Phosphorylation at the +5 position relative to the sumoylation target is directly visualized by a high-resolution EMSA (electrophoretic mobility-shift assay). Sumoylation represses the activity of ERRgamma both with and without forced expression of the PGC-1beta (peroxisome-proliferator-activated receptor gamma co-activator-1beta). Fusion proteins of a heterologous DNA-binding domain with the ERRgamma2 N-terminus demonstrate the function of the PDSM as the RF-1 (repression function-1) for the neighbouring AF-1. De-repression is achieved by co-expression of sentrin/SENP (sentrin-specific protease) family members. Together, our results demonstrate reversible phosphorylation-dependent sumoylation as a means to regulate the activity of an orphan nuclear receptor.

Germ cell nuclear factor is a repressor of CRIPTO-1 and CRIPTO-3.

The pluripotency of embryonic stem and embryonic carcinoma cells is maintained by the expression of a set of "stemness" genes. Whereas these genes are down-regulated upon induction of differentiation, the germ cell nuclear factor (GCNF) is transiently up-regulated and represses several pluripotency genes. CRIPTO-1, a co-receptor for the morphogen nodal, is strongly expressed in undifferentiated cells and is rapidly down-regulated during retinoic acid-induced differentiation. Although CRIPTO-1 is expressed at very low levels in adult tissues under normal conditions, it is found highly expressed in a broad range of tumors, where it acts as a potent oncogene. We show that expression of CRIPTO-1 is directly repressed by GCNF during differentiation of the human teratocarcinoma cell line, NT2. GCNF bound to a DR0 element of the CRIPTO-1 promoter in vitro, as shown by electrophoretic mobility shift assays, and in vivo, as demonstrated by chromatin immunoprecipitation. Reporter gene assays demonstrated that GCNF-mediated repression of the CRIPTO-1 promoter is dependent upon the DR0 site. Overexpression of GCNF in NT2 cells resulted in repression of CRIPTO-1 transcription, whereas expression of the transcription-activating fusion construct GCNF-VP16 led to an induction of the CRIPTO-1 gene and prevented its retinoic acid-induced down-regulation. Furthermore, we demonstrated that CRIPTO-3, a processed pseudogene of CRIPTO-1 on the X chromosome, is expressed in undifferentiated NT2 cells and is regulated by GCNF in parallel to CRIPTO-1. Thus, our study supports the hypothesis of GCNF playing a central role during differentiation of stem cells by repression of stem cell-specific genes.

The forkhead transcription factor Foxi1 directly activates the AE4 promoter.

Intercalated cells are highly specialized cells within the renal collecting duct epithelium and play an important role in systemic acid-base homoeostasis. Whereas type A intercalated cells secrete protons via an apically localized H+-ATPase, type B intercalated cells secrete HCO3-. Type B intercalated cells specifically express the HCO3-/Cl- exchanger AE4 (anion exchanger 4), encoded by Slc4a9. Mice with a targeted disruption of the gene for the forkhead transcription factor Foxi1 display renal tubular acidosis due to an intercalated cell-differentiation defect. Collecting duct cells in these mice are characterized by the absence of inter-calated cell markers including AE4. To test whether Slc4a9 is a direct target gene of Foxi1, an AE4 promoter construct was generated for a cell-based reporter gene assay. Co-transfection with the Foxi1 cDNA resulted in an approx. 100-fold activation of the AE4 promoter construct. By truncating the AE4 promoter at the 5'-end, we demonstrate that a fragment of approx. 462 bp upstream of the transcription start point is sufficient to mediate activation by Foxi1. Sequence analysis of this region revealed at least eight potential binding sites for Foxi1 in both sense and antisense orientation. Only one element was bound by recombinant Foxi1 protein in bandshift assays. Mutation of this site abolished both binding in bandshift assays and transcriptional activation by co-transfection of Foxi1 in the reporter gene assay. We thus identify the AE4 promoter as a direct target of Foxi1.

Identification of PNRC2 and TLE1 as activation function-1 cofactors of the orphan nuclear receptor ERRgamma.

Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor highly expressed in heart, skeletal muscle, kidney, and brain. To identify activation function-1 (AF-1)-dependent cofactors involved in the transcriptional function of ERRgamma, we screened for human cDNAs coding for proteins that bind to the bacterial expressed AF-1 by biopanning of a phage display library. Phages displaying fusion proteins with full-length PNRC2 (proline-rich nuclear receptor co-regulatory protein 2), already shown to be a cofactor for other nuclear receptors, and with a polypeptide of the bHLH corepressor TLE1 bound to the AF-1 containing bait. Pull-down analyses demonstrated a direct interaction of the receptor with the newly identified full-length proteins. Surprisingly, not only PNRC2 but also the corepressor TLE1 functioned as ERRgamma coactivator in a reporter gene analysis.

Structure of the murine tenascin-R gene and functional characterisation of the promoter.

The tenascin-R (TN-R) gene encodes a multidomain extracellular matrix glycoprotein belonging to the tenascin family. It is detectable mainly in oligodendrocytes and neuronal subpopulations of the central nervous system. In this report, we describe the structure of the 5'-region of the mouse TN-R gene and characterise the activity of its promoter. By in silico cloning and genome walking, we have deduced the organisation of the gene and identified the promoter sequence by 5'-RACE technology. TN-R transcripts in adult mouse brain contain non-coding exons 1 and 2 as demonstrated by the reverse transcriptase-polymerase chain reaction. The promoter displays its activity in cultured cells of neural origin, but not in a fibroblast-like cell line or an undifferentiated teratocarcimoma cell line. As for the human and rat genes, the elements required for the full and cell type-specific activity of the promoter are contained in exon 1 and 167 bp upstream of this exon. The mouse TN-R promoter sequence is similar to that of rat and human in that it displays similarly unusual features: it lacks any classical TATA-box or CAAT-box, GC-rich regions or initiator elements. The promoter contains consensus sequences for binding of a variety of transcription factors, notably p53/p73 and glucocorticoid receptors.

The germ cell nuclear factor is required for retinoic acid signaling during Xenopus development.

The germ cell nuclear factor (GCNF, NR6A1) is a nuclear orphan receptor that functions as a transcriptional repressor and is transiently expressed in mammalian carcinoma cells during retinoic acid (RA) induced neuronal differentiation. During Xenopus laevis development, the spatiotemporal expression pattern of embryonic GCNF (xEmGCNF) suggests a role in anteroposterior specification of the neuroectoderm. Here, we show that RA treatment of Xenopus embryos enhances xEmGCNF expression. Moreover, we present evidence for the relevance of this finding in the context of primary neurogenesis and hindbrain development. During early development of the central nervous system, RA signals promote posterior transformation of the neuroectoderm and increase the number of cells undergoing primary neurogenesis. Our loss-of-function analyses using a xEmGCNF-specific morpholino antisense oligonucleotide indicate that xEmGCNF is required for the effect of RA on primary neurogenesis. This may be caused by transcriptional regulation of the gene encoding the RA-degrading enzyme CYP26, since this gene is derepressed after depletion of xEmGCNF and an antimorph of xEmGCNF directly activates transcription of CYP26, also in absence of protein synthesis. The effect of xEmGCNF knockdown on hindbrain patterning is similar to conditions of reduced RA signaling, which may be caused by a reduction of RAR gamma expression specifically in the presumptive hindbrain.

Characterization of calmodulin binding to the orphan nuclear receptor Errgamma.

The estrogen receptor-related receptor gamma (ERRgamma/ ERR3/NR3B3), a member of the nuclear receptor superfamily, activates transcription in the absence of ligands. In order to identify ligand-independent mechanisms of activation, we tested whether calmodulin (CaM), a key regulator of numerous cellular processes and a predominant intracellular receptor for Ca2+-signals, interacts with ERRgamma. In vitro pull-down experiments with calmodulin-Sepharose demonstrated a Ca2+-dependent interaction with cellularly expressed ERRgamma. As shown by truncation analysis, the CaM binding site is highly unusual in that it is composed of two discontinuous elements. Moreover, by surface plasmon resonance (SPR) biosensor technology, we detected a direct interaction of immobilized bacterially expressed ERR-gamma fusion protein with Ca2+-calmodulin. This is best described by a model which assumes a conformational change of the initially formed complex to a more stable form. Whereas in vitro DNA binding was calmodulin-independent, transient transfection analysis revealed a Ca2+-influx-dependent ERRgamma-mediated transcriptional activation of a luciferase reporter gene. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of ERRgamma.

PGC-1 and PERC, coactivators of the estrogen receptor-related receptor gamma.

The mouse nuclear receptor ERRgamma (estrogen receptor-related receptor gamma) is highly expressed in heart, skeletal muscle, kidney, and brain, as well as in the developing nervous system. We found that the expression of the coactivators PGC-1 (PGC-1alpha) and PERC (PGC-1beta) in mammalian cells augmented potently the transcriptional activation by ERRgamma. The constitutive activation function 2 (AF-2) of the orphan receptor was important for the synergistic enhancement. Functional receptor truncation analysis revealed an additional amino-terminal activation function, specific for the ERRgamma2 isoform and PGC-1. In vitro experiments showed a direct interaction of ERRgamma with both coactivators. Our findings suggest distinct regulatory functions for PGC-1 and PERC as tissue-specific coactivators for ERRgamma.

Domains of ERRgamma that mediate homodimerization and interaction with factors stimulating DNA binding.

The estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is an orphan member of the nuclear receptor superfamily closely related to the estrogen receptors. To explore the DNA binding characteristics, the protein-DNA interaction was studied in electrophoretic mobility shift assays (EMSAs). In vitro translated ERRgamma binds as a homodimer to direct repeats (DR) without spacing of the nuclear receptor half-site 5'-AGGTCA-3' (DR-0), to extended half-sites, and to the inverted estrogen response element. Using ERRgamma deletion constructs, binding was found to be dependent on the presence of sequences in the ligand binding domain (LBD). A far-Western analysis revealed that ERRgamma forms dimers even in the absence of DNA. Two elements, located in the hinge region and in the LBD, respectively, are necessary for DNA-independent dimerization. DNA binding of bacterial expressed ERRgamma requires additional factors present in the serum and in cellular extracts. Fusion proteins of the germ cell nuclear factor (GCNF/NR6A1) with ERRgamma showed that the characteristic feature to be stimulated by additional factors can be transferred to a heterologous protein. The stimulating activity was further characterized and its target sequence narrowed down to a small element in the hinge region.