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Mus musculus
6 Downloadable Samples
NextSeq 500
Description
Before birth B-cells develop in the fetal liver (FL). Here we show that Gli3 activity in the FL stroma is required for B-cell development. In the Gli3-deficient FL B-cell development was reduced at multiple stages, whereas the Sonic hedgehog (Shh)-deficient FL showed increased B-cell development, and Gli3 functioned to repress Shh transcription. Use of a transgenic Hedgehog (Hh)-reporter mouse showed that Shh signals directly to developing B-cells, and that Hh pathway activation was increased in developing B-cells from Gli3-deficient fetal liver. RNAsequencing confirmed that Hh-mediated transcription is increased in B-lineage cells from Gli3-deficient FL, and showed that these cells expressed reduced levels of B-lineage transcription factors and BCR/pre-BCR-signalling genes. We showed that expression of the master regulators of B-cell development, Ebf1 and Pax5, is reduced in developing B-cells from Gli3-deficient FL and increased in Shh-deficient FL, and that in vitro Shh-treatment or neutralisation can repress or induce their expression respectively. Overall design: Wildtype and Gli3 mutant (Gli3+/- and Gli3-/-) (n=2) embryonic day 17.5 fetal livers were sorted for CD19+B220+ cells. RNA extracted from these cells was sequenced to help understand the transcriptional changes governing B cell development in the Gli3 mutants.
Publication Title
The transcription factor Gli3 promotes B cell development in fetal liver through repression of Shh.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 18 Downloadable Samples
Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Despite Hedgehogs influence on T-cell activation and proliferation, the transcriptional targets of Gli2 in lymphocytes are not known. We therefore examined the Hedgehog-dependent transcriptional response of resting and early-stage activated T-cells in order to define their transcriptional response to Hedgehog pathway activation.
Publication Title
Tissue-derived hedgehog proteins modulate Th differentiation and disease.
Sample Metadata Fields
Specimen part, Treatment
View Samples- Mus musculus
- 18 Downloadable Samples
- Affymetrix Mouse Gene 2.0 ST Array (mogene20st)
Description
We used Affymetrix microarrays to understand the genome wide differences in Wildtype and Gli3 mutant (Gli3+/- and Gli3-/-) (n=2) embryonic day 18.5 DP CD69-, DP CD69+ and SP4 thymocytes.
Publication Title
Gli3 in fetal thymic epithelial cells promotes thymocyte positive selection and differentiation by repression of <i>Shh</i>.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 24 Downloadable Samples
- Illumina HiSeq 2500
Description
In this study we used Genome Wide Transcriptional Modelling (GWTM) to investigate the temporal transcriptional changes during CD4 Th0, Th1 and Th2 differentiation in the first 24 hours after T cell activation. We measured the transcriptional response by RNA seq every four hours for a 24 hour time course. Overall design: WT CD4 T cells were isolated and purified from adult murine spleen. The purified CD4 cells were then set up in culture under three different conditions: Th0, Th1 and Th2. Cells were extracted at 4 hour timepoints during a 24hour timecourse and RNA was extracted for each timepoint under each condition. This RNA was further sequenced to analyse the genome wide transcriptional changes through time under each of the three conditions.
Publication Title
IFITM proteins drive type 2 T helper cell differentiation and exacerbate allergic airway inflammation.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 36 Downloadable Samples
- Illumina HiSeq 4000
Description
The purpose of this study was to define biomarkers of sensitivty and mechanisms of resistance to the KDM1A/LSD1 inhibtor SP-2509 (HCI-2509) in Ewing sarcoma cell lines. We report that regardless of drug sensitivity all cell lines engage the UPR and ER-stress response following treatment with SP-2509 resulting in apoptotic cytotoxicity. In addition hypersentsitive cell lines shared a common basal transcriptnomic profile, with hypersensitive cell lines signficantly inducing ETS1 which was not observed in sensitive cell lines. Overall design: 6 Ewing sarcoma cell lines were treated with either vehicle control (DMSO) or the reversible LSD1/KDM1A inhibitor SP-2509 (2uM) for 48hrs. Three SP-2509 hypersensitive (IC50< 300nM)(A673, TC32, TC252) and three SP-2509 sensitive (IC50>900nM) (EWS-502, ES-2 and TC71) cell lines were investigated. Paired RNA from three indpendent experiments per cell line was analyzed.
Publication Title
Therapeutic Targeting of KDM1A/LSD1 in Ewing Sarcoma with SP-2509 Engages the Endoplasmic Reticulum Stress Response.
Sample Metadata Fields
Treatment, Subject
View Samples- Mus musculus
- 12 Downloadable Samples
- Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
To better elucidate epigenetic mechanisms that correlate with the dynamic gene expression program observed after T cell activation, we investigated the genomic landscape of histone modifications in antigen-experienced CD8+ T cells. Using a ChIP-Seq approach coupled with global gene expression profiling, we generated genome-wide histone H3 lysine 4 (H3K4me3) and H3 lysine 27 (H3K27me3) trimethylation maps in distinct subsets of CD8+ T cells-nave, stem cell memory, central memory, and effector memory-to gain insight into how histone architecture is remodeled during the differentiation of activated T cells. We show that H3K4me3 histone modifications are associated with activation of genes, while H3K27me3 is negatively correlated with gene expression at canonical loci and enhancers regions associated with T cell metabolism, effector function, and memory. Our results also reveal histone modifications and gene expression signatures that distinguish the recently identified stem cell memory T cell from other antigen-experienced CD8+ T cell subsets. Taken together, our results suggest that antigen-experienced T cells may undergo chromatin remodeling in a progressive fashion that may have implications for our understanding of peripheral T cell ontogeny and the formation of immunological memory.
Publication Title
Lineage relationship of CD8(+) T cell subsets is revealed by progressive changes in the epigenetic landscape.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 33 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Adoptive immunotherapies using genetically-redirected T cells expressing a chimeric antigen receptor (CAR) or T cell receptor (TCR) are poised to enter mainstream clinical practice. Despite encouraging results, some patients fail to respond to current therapies. In part, this phenomenon has been associated with infusion of a reduced number of early memory T cells. Herein, we report that pharmacologic disruption of AKT-signaling (AKTi) is compatible with the transduction of both CARs and TCRs into human T cells and promotes a minimally differentiated CD62L-expressing phenotype. Critically, this intervention did not compromise cell yield. Mechanistically, disruption of AKT-signaling preserved MAPK activation and promoted the intra-nuclear accumulation of FOXO1, a key transcriptional regulator of T-cell memory. Consequently, AKTi synchronized the T-cell transcriptional profile for FOXO1-dependent target genes across multiple donors. Expression of an AKT-resistant FOXO1 mutant phenocopied the influence of AKTi while addition of AKTi to T cells expressing mutant FOXO1 failed to further augment the frequency of CD62L-expressing cells. Finally, CD19 CAR-modified T cells transduced and expanded in AKTi treated established B-cell acute lymphoblastic leukemia superiorly to conventionally grown T cells in a murine xenograft model. Thus, inhibition of AKT-signaling represents a generalizable strategy to generate large numbers of receptor-modified T cells with an early memory phenotype.
Publication Title
Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy.
Sample Metadata Fields
Treatment, Subject, Time
View Samples- Homo sapiens
- 15 Downloadable Samples
- Illumina HiSeq 2000
Description
RNA from A673 cells with shRNA-mediated knockdown of GFP (4 libraries), EWS-FLI1 (4 libraries), or lnc277 (7 libraries) was isolated with TRIzol (Invitrogen). Each sample was DNase treated and further purified on an RNeasy Mini column (Qiagen) before quality analysis on an Agilent 2100 Bioanalyzer. For each sample, 100-150ng of RNA was synthesized into cDNA, sheared on a Covaris ultrasonicator, and amplified using the NuGen Encore Complete kit (NuGen) to produce strand-specific and rRNA-depleted libraries. Samples were multiplexed (4/lane) for 2x100bp paired-end sequencing on an Illumina HiSeq 2000 Overall design: RNA from A673 cells with shRNA-mediated knockdown of GFP (4 libraries), EWS-FLI1 (4 libraries), or lnc277 (7 libraries) was isolated with TRIzol (Invitrogen).
Publication Title
Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis.
Sample Metadata Fields
No sample metadata fields
View Samples
SRP045869- Homo sapiens
- 6 Downloadable Samples
- IlluminaGenomeAnalyzerII
Description
Ewing sarcoma is a highly aggressive tumor characterized by a translocation between members of the FET family of RNA binding proteins and one of several ETS transcription factors, with the most common translocation being EWS-FLI1. EWS-FLI1 leads to changes in gene expression through mechanisms that are not completely understood. We performed RNA sequencing analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify novel target genes. This analysis identified lnc277 as a previously uncharacterized long non-coding RNA upregulated by EWS-FLI1 in pMPCs. Inhibiting the expression of lnc277 diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar whereas inhibiting lnc277 had no effect on other cell types tested. By analyzing gene expression after shRNA knockdown, we found that both EWS-FLI1 and lnc277 repressed many more genes that they induced and that a significant fraction of EWS-FLI1 repressed targets were also repressed by lnc277. Analysis of primary human Ewing sarcoma RNA sequencing data further supports a role for lnc277 in mediating gene repression. We identified hnRNPK as an RNA binding protein that interacts directly with lnc277. We found a significant overlap in the genes repressed by hnRNPK and those repressed by both EWS-FLI1 and lnc277, suggesting that hnRNPK participates in lnc277 mediated gene repression. Thus, lnc277 is a previously uncharacterized long non-coding RNA downstream of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes. Our studies identify a novel mechanism of oncogenesis downstream of a chromosomal translocation and underscore the importance of lncRNA-mediated gene repression as a mechanism of EWS-FLI1 transcriptional regulation. Overall design: RNA from primary human bone marrow derived mesenchymal cells either control or with inducible expression of EWS-FLI1 for 13 days was used to prepare PolyA selected cDNA libraries.
Publication Title
Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis.
Sample Metadata Fields
No sample metadata fields
View Samples SRP045870- Homo sapiens
- 4 Downloadable Samples
- IlluminaHiSeq2000
Description
Ewing sarcoma is a highly aggressive tumor characterized by a translocation between members of the FET family of RNA binding proteins and one of several ETS transcription factors, with the most common translocation being EWS-FLI1. EWS-FLI1 leads to changes in gene expression through mechanisms that are not completely understood. We performed RNA sequencing analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify novel target genes. This analysis identified lnc277 as a previously uncharacterized long non-coding RNA upregulated by EWS-FLI1 in pMPCs. Inhibiting the expression of lnc277 diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar whereas inhibiting lnc277 had no effect on other cell types tested. By analyzing gene expression after shRNA knockdown, we found that both EWS-FLI1 and lnc277 repressed many more genes that they induced and that a significant fraction of EWS-FLI1 repressed targets were also repressed by lnc277. Analysis of primary human Ewing sarcoma RNA sequencing data further supports a role for lnc277 in mediating gene repression. We identified hnRNPK as an RNA binding protein that interacts directly with lnc277. We found a significant overlap in the genes repressed by hnRNPK and those repressed by both EWS-FLI1 and lnc277, suggesting that hnRNPK participates in lnc277 mediated gene repression. Thus, lnc277 is a previously uncharacterized long non-coding RNA downstream of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes. Our studies identify a novel mechanism of oncogenesis downstream of a chromosomal translocation and underscore the importance of lncRNA-mediated gene repression as a mechanism of EWS-FLI1 transcriptional regulation. Overall design: A673 Ewing cells expressing an shRNA targeting hnRNPK or control were subjected to paired end RNA sequencing and compared to shGFP control.
Publication Title
Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis.
Sample Metadata Fields
No sample metadata fields
View Samples