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SRP162356
Homo sapiens
10 Downloadable Samples
NextSeq 500
Description
Long terminal repeat (LTR) elements are wide-spread in the human genome and have the potential to act as promoters and enhancers. Their expression is therefore under tight epigenetic control. We previously reported that a member of the THE1B class of LTR elements in classical Hodgkin Lymphoma (cHL) acted as a promoter for the growth factor receptor gene CSF1R and that expression of this gene is required for tumor survival. However, to which extent and how such elements participate in globally shaping the unique cHL gene expression program is unknown. To address this question we mapped the genome-wide activation of THE1-LTRs in cHL cells using a targeted next generation sequencing approach (RACE-Seq). Integration of these data with global gene expression data from cHL and control B cell lines showed a unique pattern of LTR activation impacting on gene expression, including genes associated with the cHL phenotype. We also show that global LTR activation is induced by strong inflammatory stimuli. Together these results demonstrate that LTR activation provides an additional layer of gene deregulation in classical Hodgkin lymphoma and highlight the potential impact of genome-wide LTR activation in other inflammatory diseases. Overall design: RNA-Seq in laser capture microdissected (LCM) tumour (TU) and non tumour cells (NTC) primary HL material from patient samples
Publication Title
Global long terminal repeat activation participates in establishing the unique gene expression programme of classical Hodgkin lymphoma.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 20 Downloadable Samples
- Illumina HiSeq 2500
Description
RUNX1 is a frequent target of translocations in acute myeloid leukemia whereby its DNA binding domain fuses to different epigenetic regulators. To assess how different RUNX1 fusion proteins interact with the epigenome we compared the global binding patterns and the chromatin landscape of t(8;21) and t(3;21) AML which express RUNX1-ETO and RUNX1-EVI-1, respectively. We found that differential prognosis for these types of AML is reflected in fundamental differences in gene expression, chromatin landscape, binding patterns of the fusion proteins and other transcription factors as identified by genome-wide digital footprinting in patients. As previously shown for RUNX1-ETO, knockdown of RUNX1-EVI-1 expression initiates differentiation of t(3;21) cells which is associated with up-regulation of genes vital for myeloid differentiation, including C/EBPa. Furthermore, by expressing either dominant-negative C/EBP or an inducible C/EBPa construct in t(3;21) cells we show that C/EBPa is necessary and sufficient for the differentiation response of these cells to RUNX1-EVI-1 knockdown. Overall design: RNA-seq expreiments have been used to study the chromatin landscape of t(8;21) and t(3;21) AML
Publication Title
RUNX1-ETO and RUNX1-EVI1 Differentially Reprogram the Chromatin Landscape in t(8;21) and t(3;21) AML.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 15 Downloadable Samples
Description
Aim: to perform a genome-wide investigation of chromatin landscape and gene expression patterns downstream of calcium and kinase signaling in Jurkat T cells. Methods: PMA and ionomycin were used to activate the calcium and kinase signalling networks involved in T cell activation. Global gene expression was measured using RNA-seq, whilst ATAC-seq was used to probe chromatin landscape following 3 hours of stimulation with PMA, ionomycin or both. All experiments were performed in triplicate. For RNA-seq all sequencing was performed using paired-end sequencing on an Illumina HiSeq2500 instrument. For ATAC-seq sequencing was performed using a HiSeq 1500. Results: we mapped approximately 60 million reads per sample for ATAC-seq, and 22 million reads per library for RNA-seq. Overall we identified 57,825 transcripts and 19,763 ATAC-seq peaks. We identifiead 1648 genes whose expression was increased by 2-fold or more by at least one treatment in comparison to untreated cells. Similarly, we identified 3972 ATAC peaks that were induced by at least 2-fold by treatment in comparison to untreated cells. Conclusions: we found that chromatin landscape was associated with gene expression downstream of calcium and kinase signaling in Jurkat cells. Further to this we found that activation of the full complement of TCR-responsive genes is dependent upon both PMA and ionomycin, and amounts to more than just the sum of both. Overall design: RNA-sequencing and ATAC-sequencing were performed after 3 hours of treatment with either PMA, ionomycin or co-treatment with PMA and ionomycin.
Publication Title
Integration of Kinase and Calcium Signaling at the Level of Chromatin Underlies Inducible Gene Activation in T Cells.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 8 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Genome-wide gene expression analysis of Reh cells following transfection with constitutively active IRF5-4D, constitutively active IKK(EE), or both in combination.
Publication Title
Mapping of transcription factor motifs in active chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma.
Sample Metadata Fields
Cell line
View Samples- Homo sapiens
- 7 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Genome-wide gene expression analysis of Reh cells following transfection with shRNA targeting CBFA2T3, constitutively active IKK(EE), or both in combination.
Publication Title
Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma.
Sample Metadata Fields
Cell line
View Samples- Mus musculus
- 6 Downloadable Samples
- Illumina MouseWG-6 v2.0 R2 expression beadchip
Description
Genome-wide gene expression analysis of murine splenic B-cells following retroviral transduction with a constitutively active IRF5 (IRF5-4D)
Publication Title
Mapping of transcription factor motifs in active chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma.
Sample Metadata Fields
Specimen part
View Samples- Saccharomyces cerevisiae
- 8 Downloadable Samples
- Affymetrix Yeast Genome 2.0 Array (yeast2)
Description
Cdc34 is an essential E2 ubiquitin conjugating enzyme found in nearly all eukaryotes. It contains a highly conserved motif composed of S73/S97/12 amino acid insert near the active site cysteine. This motif is unique to Cdc34/Ubc7 type E2s while other E2s contain K/D/no insert at these positions. To better understand the function of this motif we mutated Cdc34 S73/S97/insert to be K/D/no insert and observed changes in transcript levels in mid-log phase yeast cells. ABSTRACT [Cdc34 is a ubiquitin conjugating enzyme necessary for the ubiquitylation of substrates by the SCF family of ubiquitin ligases. Previous work has shown that the Cdc34 protein is phosphorylated in vivo on serine residues. Cdc34 contains two serines within its catalytic domain, S73 and S97, that together with a 12 amino acid acidic loop, constitute a highly conserved motif (serine, serine, insert) among all members of the Cdc34 family of E2 enzymes. Using phosphospecific antibodies, we show that the essential serine S97 is indeed phosphorylated in vivo. Furthermore, this phosphorylation event is regulated by treatment with pheromone in yeast. Consistently, expression of a Cdc34 mutant lacking this motif (serine, serine, insert) leads to misregulation of the SCF substrates, Sic1, Far1, Cln1 and Cln2 and suppresses the cell cycle arrest brought about by an activated mating pathway. We further explored the function of this motif by microarray analysis and show that the transcripts of nearly the entire Sic1 cluster of co-transcribed genes is altered in a strain the expresses Cdc34 lacking this motif. Our data reveals that this highly conserved motif in Cdc34 and its phosphorylation are important for modulating SCF substrate abundance both transcriptionally and post-transcriptionally.]
Publication Title
New insight into the role of the Cdc34 ubiquitin-conjugating enzyme in cell cycle regulation via Ace2 and Sic1.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 12 Downloadable Samples
- Illumina HumanHT-12 V4.0 expression beadchip
Description
We previously identified the ZTRE in genes involved in zinc homeostasis and showed that it mediates transcriptional repression in response to zinc. We now report that ZNF658 acts at the ZTRE. ZNF658 was identified by MALDI-TOF mass spectrometry of a band excised after EMSA using a ZTRE probe. The protein contains a KRAB domain and 21 zinc fingers. It has similarity with ZAP1 from Saccharomyces cerevisiae, which regulates the response to zinc restriction, including a conserved DNA binding region we show to be functional also in ZNF658. siRNA targeted to ZNF658 abrogated the zinc-induced, ZTRE-dependent reduction in SLC30A5 (ZnT5), SLC30A10 (ZnT10) and CBWD transcripts in human Caco-2 cells and the ability of zinc to repress reporter gene expression from corresponding promoter-reporter constructs. Microarray analysis of the effect of reducing ZNF658 expression by siRNA uncovered large changes in rRNA. We find that ZTREs are clustered within the 45S rRNA precursor. We also saw effects on expression of multiple ribosomal proteins. ZNF658 thus links zinc homeostasis with ribosome biogenesis, the most active transcriptional, and hence zinc-demanding, process in the cell. ZNF658 is thus a novel transcriptional regulator that plays a fundamental role in the orchestrated cellular response to zinc availability.
Publication Title
The zinc finger protein ZNF658 regulates the transcription of genes involved in zinc homeostasis and affects ribosome biogenesis through the zinc transcriptional regulatory element.
Sample Metadata Fields
Cell line
View Samples- Homo sapiens
- 3 Downloadable Samples
- IlluminaHiSeq2000
Description
Background: Eukaryotic cells express a complex layer of noncoding RNAs. An intriguing family of regulatory RNAs includes transcripts from the opposite strand of protein coding genes, so called natural antisense transcripts (NATs). Here, we test the hypothesis that antisense transcription triggers RNA interference and gives rise to endogenous short RNAs (endo-siRNAs). Methods/Results: We used cloned human embryonic kidney cells (HEK293) followed by short RNAseq to investigate the small genic RNA transcriptome. 378 genes gave rise to short RNA reads that mapped to exons of RefSeq genes. The length profile of short RNAs showed a broad peak of 20-24 nucleotides, indicative of endo-siRNAs. Collapsed reads mapped predominantly to the first and the last exon of genes (74%). RNAs reads were intersected with sequences occupied by RNAPolII or bound to Argonaute (AGO1 by crosslinking, ligation, and sequencing of hybrids, CLASH). In the first exon, 94% of the reads correlated with PolII occupancy with an average density of 130 (relative units); this decreased to 65%/20 in middle exons and 54%/12 in the last exon. CLASH reads mapping to multi-exon genes showed little distribution bias with an average of about 5 CLASH reads overlapping with 60% of the endo-siRNA reads. However, endo-siRNAs (21-25 nt) intersecting with CLASH reads were enriched at the 5''end and decreased towards the 3''end. We then investigated the 378 genes with particular focus on features indicative for short RNA production; however, found that endo-siRNA numbers did not correlate with gene structures that favor convergent transcription. In contrast, our gene set was found notably over-represented in the NATsDB sense/antisense group as compared to non-overlapping and non-bidirectional groups. Moreover, read counts showed no correlation with the steady-state levels of the related mRNAs and the pattern of endo-siRNAs proved reproducible after an induced mutagenic insult. Conclusions: Our results suggest that antisense transcripts contribute to low levels of endo-siRNAs in fully differentiated human cells. A characteristic endo-siRNA footprint is being produced at sites of RNAPolII transcription which is also related to AGO1. This endo-siRNA signature represents an intriguing finding and its reproducibility suggests that the production of endo-siRNAs is a regulated process with potential homoeostatic impact. Overall design: Size selected RNASeq of 3 human embryonic kidney cell (HEK293) samples. 1 control and 2 samples exposed to 100 µg/ml ethyl methanesulfonate for 24 hrs.
Publication Title
Contribution of natural antisense transcription to an endogenous siRNA signature in human cells.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 69 Downloadable Samples
- NextSeq 500
Description
The rapid improvements in single cell sequencing technologies and analyses methods afford greater scope for dissecting organoid cultures composed of multiple cell types and create an opportunity to interrogate these models to understand tissue biology, cellular behaviour and interactions. To this end, retinal organoids generated from human embryonic stem cells (hESCs) were analysed by single cell RNA-Sequencing at three time points of differentiation. Combinatorial data from all time points revealed the presence of nine clusters, five of which corresponded to key retinal cell types, namely retinal pigment epithelium (RPE), retinal ganglion cells (RGCs), cone and rod photoreceptors and Müller glia cells. The remaining four clusters expressed genes typical of mitotic cells, extracellular matrix (ECM) components and those involved in retinal homeostasis. The cell clustering analysis revealed the decreasing presence of mitotic cells and RGCs, formation of a distinct RPE cluster, the emergence of cone and rod photoreceptors from photoreceptor precursors and an increasing number of Müller Glia cells over time. The pseudotime analysis resembled the order of cell birth during retinal development, with the mitotic cluster commencing the trajectory and the large majority of Müller glia being the latest. Together, these data demonstrate the feasibility and potential of single cell RNA-Seq to dissect the inherent complexity of the organoids and the orderly birth of key retinal cell types. Overall design: A hESC (H9) cell line harbouring a CRX-GFP reporter was differentiated to retinal organoids 25. Samples were collected at 60, 90 and 200 days, dissociated, partitioned into single cells using the Fluidigm C1 Single-Cell mRNA-Seq HT IFC and processed for scRNA-Seq.
Publication Title
Deconstructing Retinal Organoids: Single Cell RNA-Seq Reveals the Cellular Components of Human Pluripotent Stem Cell-Derived Retina.
Sample Metadata Fields
Cell line, Subject, Time
View Samples