A Gain-of-Function Genomic Screen Identifies the Orphan Nuclear Receptor TLX as an Enhancer of STAT1-mediated Transcription and Immunity to Toxoplasma gondii
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE55751 | GEO | 2015/05/29
SECONDARY ACCESSION(S): PRJNA240888
REPOSITORIES: GEO
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Project description:The protozoan parasite, Toxoplasma, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited this well-defined host-pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite Toxoplasma results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such "modifiers."
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Project description:The protozoan parasite Toxoplasma gondii is a highly successful intracellular pathogen, owing in part to its ability to subvert the host immune system. In particular, parasite infection suppresses STAT1 signaling in a variety of cell types, including IFN-γ activated macrophages, via a block within the nucleus. A high-throughput screen to identify genes able to overcome parasite-mediated suppression of STAT1 activity identified 9 transcription factors as enhancers of STAT1 signaling in T. gondii infected cells, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that TLX is a transcriptional regulator that drives the steady-state expression of STAT1-independent genes involved brain function and development, while enhancing the output of a subset of IFN-γ-dependent target genes. Infection of TLX deficient mice with Toxoplasma results in impaired production of interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized function for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense, and reveal that STAT1 activity can be modulated in a context-specific manner by such ‘modifiers’.
2015-05-29 | GSE55750 | GEO
Project description:The protozoan parasite Toxoplasma gondii is a highly successful intracellular pathogen, owing in part to its ability to subvert the host immune system. In particular, parasite infection suppresses STAT1 signaling in a variety of cell types, including IFN-γ activated macrophages, via a block within the nucleus. A high-throughput screen to identify genes able to overcome parasite-mediated suppression of STAT1 activity identified 9 transcription factors as enhancers of STAT1 signaling in T. gondii infected cells, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that TLX is a transcriptional regulator that drives the steady-state expression of STAT1-independent genes involved brain function and development, while enhancing the output of a subset of IFN-γ-dependent target genes. Infection of TLX deficient mice with Toxoplasma results in impaired production of interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized function for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense, and reveal that STAT1 activity can be modulated in a context-specific manner by such ‘modifiers’.
2015-05-29 | GSE55749 | GEO
Project description:Nuclear receptors (NRs) are an important group of ligand-dependent transcriptional factors. Presently, no natural or synthetic ligand has been identified for a large group of orphan NRs. Small molecules to target these orphan NRs will provide unique resources for uncovering regulatory systems that impact human health and to modulate these pathways with drugs. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and Neural Stem Cell (NSC) derived brain tumors. No chemical probes that modulate TLX activity are available, and it is not clear whether TLX is druggable. To assess TLX ligand binding capacity, we created homology models of the TLX ligand binding domain (LBD). Results suggest that TLX belongs to an emerging class of NRs that lack LBD helices ?1 and ?2 and that it has potential to form a large open ligand binding pocket (LBP). Using a medium throughput screening strategy, we investigated direct binding of 20,000 compounds to purified human TLX protein and verified interactions with a secondary (orthogonal) assay. We then assessed effects of verified binders on TLX activity using luciferase assays. As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity. We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands. While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open.
| S-EPMC4060991 | biostudies-literature
Project description:The orphan nuclear receptor tailless homologue (TLX) is expressed almost exclusively in neural stem cells acting as an essential factor for their survival and is hence considered as a promising drug target in neurodegeneration. However, few studies have characterized the roles of TLX due to the lack of ligands and limited functional understanding. Here, we identify xanthines including caffeine and istradefylline as TLX modulators that counteract the receptor's intrinsic repressor activity. Mutagenesis of residues lining a cavity within the TLX ligand binding domain altered the activity of these ligands, suggesting direct interactions with helix 5. Using xanthines as tool compounds, we observed a ligand-sensitive recruitment of the co-repressor silencing mediator for retinoid or thyroid-hormone receptors, TLX homodimerization, and heterodimerization with the retinoid X receptor. These protein-protein interactions evolve as factors that modulate the TLX function and suggest an unprecedented role of TLX in directly repressing other nuclear receptors.
| S-EPMC8669710 | biostudies-literature
Project description:The orphan nuclear receptor TLX regulates neural stem cell self-renewal in the adult brain and functions primarily as a transcription repressor through recruitment of Atrophin corepressors, which bind to TLX via a conserved peptide motif termed the Atro box. Here we report crystal structures of the human and insect TLX ligand-binding domain in complex with Atro box peptides. In these structures, TLX adopts an autorepressed conformation in which its helix H12 occupies the coactivator-binding groove. Unexpectedly, H12 in this autorepressed conformation forms a novel binding pocket with residues from helix H3 that accommodates a short helix formed by the conserved ALXXLXXY motif of the Atro box. Mutations that weaken the TLX-Atrophin interaction compromise the repressive activity of TLX, demonstrating that this interaction is required for Atrophin to confer repressor activity to TLX. Moreover, the autorepressed conformation is conserved in the repressor class of orphan nuclear receptors, and mutations of corresponding residues in other members of this class of receptors diminish their repressor activities. Together, our results establish the functional conservation of the autorepressed conformation and define a key sequence motif in the Atro box that is essential for TLX-mediated repression.
| S-EPMC4335298 | biostudies-literature
Project description:Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.
| S-EPMC3366998 | biostudies-literature
Project description:Using Illumina’s digital gene expression (DGE) system, we investigated the transcriptional response to TLX overexpression in beta cells. Finally, an average of 13,791,918 and 12,476,623 reads of high quality clean tags were obtained from control and TLX-overexpressing beta cells, respectively. Results reveals that overexpression of TLX resulted in suppression of 176 genes by ?50%, and also results in ?2-fold upregulation of 49 genes. Expression profiling of stable TLX overexpressing MIN6 cells and control cells by high throughput sequencing
2015-03-06 | E-GEOD-66551 | biostudies-arrayexpress
Project description:The chemokine receptor CCR7 is a well-established homing receptor for dendritic cells and T cells. Interactions with its ligands, CCL19 and CCL21, facilitate priming of immune responses in lymphoid tissue, yet CCR7-independent immune responses can be generated in the presence of sufficient antigen. In these studies, we investigated the role of CCR7 signaling in the generation of protective immune responses to the intracellular protozoan parasite Toxoplasma gondii. The results demonstrated a significant increase in the expression of CCL19, CCL21, and CCR7 in peripheral and central nervous system (CNS) tissues over the course of infection. Unexpectedly, despite the presence of abundant antigen, CCR7 was an absolute requirement for protective immunity to T. gondii, as CCR7(-/-) mice succumbed to the parasite early in the acute phase of infection. Although serum levels of interleukin 12 (IL-12), IL-6, tumor necrosis factor alpha (TNF-alpha), and IL-10 remained unchanged, there was a significant decrease in CCL2/monocyte chemoattractant protein 1 (MCP-1) and inflammatory monocyte recruitment to the site of infection. In addition, CCR7(-/-) mice failed to produce sufficient gamma interferon (IFN-gamma), a critical Th1-associated effector cytokine required to control parasite replication. As a result, there was increased parasite dissemination and a significant increase in parasite burden in the lungs, livers, and brains of infected mice. Adoptive-transfer experiments revealed that expression of CCR7 on the T-cell compartment alone is sufficient to enable T-cell priming, increase IFN-gamma production, and allow the survival of CCR7(-/-) mice. These data demonstrate an absolute requirement for T-cell expression of CCR7 for the generation of protective immune responses to Toxoplasma infection.
| S-EPMC2863519 | biostudies-literature
Project description:Using Illumina’s digital gene expression (DGE) system, we investigated the transcriptional response to TLX overexpression in beta cells. Finally, an average of 13,791,918 and 12,476,623 reads of high quality clean tags were obtained from control and TLX-overexpressing beta cells, respectively. Results reveals that overexpression of TLX resulted in suppression of 176 genes by ≥50%, and also results in ≥2-fold upregulation of 49 genes.
2015-03-06 | GSE66551 | GEO