ABSTRACT: TLX (NR2E1), as an orphan nuclear receptor, plays a critical role in growth, self-renewal, and tumorigenesis of glioblastoma stem cells. We used microarrays to investigate the global gene expression regulated by TLX and identified downstream targets of TLX in glioblastoma stem cells. We further investigated the global gene expression regulated by TET3, a downstream target of TLX, to identified downstream targets of TLX-TET3 axis in glioblastoma stem cells.
Project description:Tlx (nr2e1) is an orphan nuclear receptor that is highly expressed in proliferating neural stem cells (NSCs) in the adult mouse forebrain. The goal was to identify Tlx-regulated genes in this specific cell population. Two populations of Tlx-positive neural stem cells were isolated from 2-month-old male mice based on a LacZ marker that was knocked into the Tlx locus. The first population, Tlx(f/Z;CreER), contains a floxed allele of Tlx (f), the LacZ marker (Z), and a CreER fusion transgene. Addition of tamoxifen (4OH-tamoxifen) into this NSC population leads to Cre-mediated deletion of the floxed allele of Tlx. The second NSC population, Tlx(f/Z), does not contain a CreER transgene; thus it does not respond to tamoxifen treatment and was used as a control. Experiment Overall Design: Total RNA samples from two biological replicates were collected at 36 hr or at 60 hr after tamoxifen or vehicle treatment. Total of 16 samples were used to analyze the global gene expression changes using Affymetrix chips.
Project description:Tlx (nr2e1) is an orphan nuclear receptor that is highly expressed in proliferating neural stem cells (NSCs) in the adult mouse forebrain. The goal was to identify Tlx-regulated genes in this specific cell population. Two populations of Tlx-positive neural stem cells were isolated from 2-month-old male mice based on a LacZ marker that was knocked into the Tlx locus. The first population, Tlx(f/Z;CreER), contains a floxed allele of Tlx (f), the LacZ marker (Z), and a CreER fusion transgene. Addition of tamoxifen (4OH-tamoxifen) into this NSC population leads to Cre-mediated deletion of the floxed allele of Tlx. The second NSC population, Tlx(f/Z), does not contain a CreER transgene; thus it does not respond to tamoxifen treatment and was used as a control. Keywords: Nuclear receptor-dependent gene expression
Project description:We previously showed that the orphan nuclear hormone receptor, TLX (also known as NR2E1), selectively modifies the output of IFN-gamma-driven, STAT1-dependent gene expression. Since IFN-gamma and STAT1 are also known to regulate myeloid cell development and function, we tested whether ectopic expression of TLX or COUPTF2 (a closely related orphan nuclear receptor) might influence hematopoietic differentiation. Indeed, TLX promotes lineage commitment toward myeloid development and suppresses erythropoiesis, phenocopying IFN-gamma treatment. Moreover, TLX expression in our assay is sufficient to activate a proinflammatory transcriptional program with two hallmarks: differential regulation of STAT1-dependent genes, and suppression of an anti-inflammatory program in favor of a fatty acid signature – a process known to be essential for cellular remodeling during macrophage maturation and phagocytosis. These results demonstrate an important role for a new nuclear hormone receptor in STAT1 signaling, and link TLX to myeloid lineage commitment and function.
Project description:Development of targeted therapies will be a critical step towards reducing the mortality associated with triple-negative breast cancer (TNBC). To achieve this, we searched for targets that met three criteria: (1) pharmacologically targetable, (2) expressed in TNBC, and (3) expression is prognostic in TNBC patients. Since nuclear receptors have a well-defined ligand-binding domain and are thus highly amenable to small-molecule intervention, we focused on this class of protein. Our analysis identified TLX (NR2E1) as a candidate. RNA-Seq analysis revealed that TLX reduced the expression of genes implicated in the pro-proliferative KRAS signaling pathway as well as epithelial-mesenchymal transition (EMT), a cellular program known to drive metastatic progression. Indeed, TLX overexpression significantly decreased cell proliferation, migration and invasion, and robustly decreased the metastatic capacity of TNBC cells in murine models. Taken together, our work indicates that TLX retards the progression of TNBC. Several ligands have been shown to regulate the transcriptional activity of TLX, providing a framework for the future development of this receptor for therapeutic intervention.
Project description:PUS7, a pseudouridine synthase, plays a critical role in growth, self-renewal, and tumorigenesis of glioblastoma stem cells. We used RNA-seq to investigate the global gene expression regulated by PUS7 and identified downstream pathway of PUS7 in glioblastoma stem cells.
Project description:Glioblastoma (GBM) is a fatal disease with a poor prognosis, whose aetiology involves profound molecular alterations. Given the limited progress made in recent years, research into new therapeutic avenues may improve the treatment of GBM patients. In this work, we have characterised the epigenomic landscape of patient-derived glioblastoma stem cells in the context of a proneural GBM subtype (U31117). We performed a systematic knockdown of each of the TET proteins (TET1, TET2 and TET3) and explored the consequences of their deletion at the level of DNA methylation (5mC) and hydroxymethylation (5hmC). Global 5hmC levels were then restored using a low dose of ascorbic acid, and the epigenetic landscape of all these conditions was examined using high-content DNA methylation microarrays (Illumina MethylationEPIC Beadchip platform).
Project description:Analysis of the function of Tlx in regulating brain tumor stem cells (BTSCs) at the gene expression level. Based on our knowledge, we know that Tlx is specifically expressed in the neural stem cells in mouse brain. It also functionally regulates neurogenesis during the postnatal stages. The hypothesis tested in the present study is that Tlx influences the maintenance and progression of mouse brain gliomas through regulating the self-renewal of BTSCs and it is also a specific marker of brain tumor stem cells (BTSCs). Results provide important information about the function of Tlx in brain gliomas. Brain gliomas were initiated from the Nestin-CreERT2;Tlx flox/flox;Nestin-Tva transgenic mouse line. After 2.5 weeks, when the brain gliomas were fully developed, tamoxifen was administered through intraperitoneal injection. Tlx was knocked-out from the Cre-expressing littermates, but the littermates without Cre expression maintained intact Tlx function. Then, total RNA was obtained from mouse brain gliomas with severe neurological symptoms from the Tlx knockout and Tlx intact groups.
Project description:DNA hydroxymethylation is frequently lost in glioblastoma. We hypothesized that reduced 5hmC levels might be related to the impaired expression of TET proteins in brain tumors. In this study we performed a genome-wide methylation analysis of LN229 cells stably transfected with scramble or TET3 overexpressing vectors. TET3 overexpression partially restored the genome-wide patterns of 5hmC characteristic of control brain samples in glioblastoma cell lines.
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’.