Bulk RNAseq of megakaryocytes and Lin- cells of mice with Srsf2-P95H mutation, Jak2-V617F mutation, both, or none
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina NovaSeq 6000
ORGANISM(S): Mus musculus
SUBMITTER: Lucie Laplane
PROVIDER: E-MTAB-11960 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:SRSF2 is an RNA binding protein that plays important roles in splicing of mRNA precursors. Mutations in SRSF2 are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how they affect SRSF2 function has only begun to be examined. Here we used CRISPR/Cas9 to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these, 374 involve the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more included exons and a distinct motif (UGGA/UG) in the more excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG, but less tightly to UGGAG sites. The pattern of exon inclusion or exclusion thus correlated in most cases with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings not only shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation, but also reveal a group of mis-spliced mRNA isoforms for potential therapeutic targeting. Examination of differentially spliced events in K562 CRISPR cell clones (with wild-type or mutant SRSF2) by RNA sequencing
2015-08-10 | E-GEOD-71299 | biostudies-arrayexpress
Project description:Bulk RNAseq of megakaryocytes and Lin- cells of mice with Srsf2-P95H mutation, Jak2-V617F mutation, both, or none
| PRJEB54749 | ENA
Project description:We showed that the over-expression of the SR protein SRSF2 led to the regulation of transcript abundance of many genes. H358 cell line with 6 samples with SRSF2 over-expression and 6 samples as control.
2013-08-30 | E-GEOD-50467 | biostudies-arrayexpress
Project description:SRSF2 is an RNA binding protein that plays important roles in splicing of mRNA precursors. Mutations in SRSF2 are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how they affect SRSF2 function has only begun to be examined. Here we used CRISPR/Cas9 to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these, 374 involve the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more included exons and a distinct motif (UGGA/UG) in the more excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG, but less tightly to UGGAG sites. The pattern of exon inclusion or exclusion thus correlated in most cases with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings not only shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation, but also reveal a group of mis-spliced mRNA isoforms for potential therapeutic targeting.
2015-08-10 | GSE71299 | GEO
Project description:Purpose: the goal of this study is to investigate the consequences of USP3 deletion on gene expression in mouse LSK hematopoietic progenitors and in splenic B cells Methods: mRNA profiles of 8 weeks-old wild-type (WT) and ubiquitin specific protease 3 knockout (Usp3−/−) mice were generated by deep sequencing, in duplicate, using Illumina Hiseq2000. The sequence reads that passed quality filters were mapped with TopHat and the gene expressions were calculated using HTSeq-count. qRT–PCR validation was performed using SYBR Green assays Results: We assigned about 8-16 million reads per sample uniquely to a gene of the mouse reference genome (mm9). We identified 23,429 genes in the LSKs, naive B cells and activate B cells of WT and USP3−/− mice using TopHat in combination with HTSeq-count. Comparison of the RNAseq data from LSK with naive or activated B cells show that both the wt and the Usp3-/- LSKs largely exibited a gene expression profile that is specific for wt LSK and distinct from B cells (as supported by statistical significant difference between the transciptional profile of LSK versus naive or activated B cells, p value<0.0001 by Student t test). Comparison of normalized gene expression data for Wt LSKs versus naive B cells of one representative experiment shows Pearson coefficient of r=0.874, and R2=0.763. Distinct LSK-specific expressed genes (such as the MlI receptor and the Kit receptor) and B cells specific genes (such as the MS4A1/CD20 and Spi-B transcription factors) are identified. Expression of a set of 19 genes was assessed by RT-qPCR in three independent LSK mRNA per each genotype. qRT-PCR and the RNA-seq normalized expression data for these genes had a good linear relationship, validating the RNAseq analysis. Comparison of normalized gene expression data for Usp3-/- versus Wt LSK show Pearson coefficient r=0.986; R2=0.9738), naive B cells (Pearson coefficient r=0.987, R2=0.974) and LPS activated B cells (Pearson coefficient r=0.991, R2=0.983). RT-qPCR of a subset of hematopoietic stem cell genes, including Mlp2, ENg, Tek and Fdzl3, show no significant difference beteewn wt and Usp3-/- LSK cells. Less than 100 genes showed differential expression (up or down regulated) between the Wt and Usp3-/- LSK, with a fold change ≥1.5 and p value <0.05. Conclusions: Our results represent the first detailed analyis of the consequences of USP3 deletion on gene expression in hematopoietic populations such as LSKs progenitors and B cells by genome wide expression profiling in wt and Usp3-/- mice. RNAseq of two freshly isolated biological replicas of sorted LSKs from 8 weeks old Usp3-/- animals showed a very limited number of genes either slighly up or down regulated (<100 out of about 25.000) in Usp3-/- LSKs, none of which are reported to be directly involved in hematopoietic stem cell maintenance or to be linked to premature differentiation. We confirmed that Usp3-/- and wt LSKs express hematopoietic stem cell-specific genes to a similar extent. We conclude that young adult hematopoietic stem and progenitor cells (LSKs) perpetuated a stable gene expression program regardless of the homozygous deletion of USP3. mRNA profiles of 8 weeks-old wild type (WT) and Usp3-/- mice were generated by deep sequencing, in duplicate, using Illumina Hiseq2000. For each experiment wt n=4, Usp3-/- n=4 mice were analized. FACS sorted cells from from individual animals were pooled and subjected to deep sequencing. Cells were: LSK (Lin- Sca1+ cKit+) from bone marrow, sorted naive B cells from spleens (CD19+) and activated B cells harvested and FACS sorted after 4 days stimulation with lipopolysaccharide (LPS) in culture.
2014-07-24 | E-GEOD-58495 | biostudies-arrayexpress
Project description:We present cell line SET-2 as potential model system for DNA methylation analysis. This cell line was established from a patient with essential thrombocythemia at megakaryoblastic transformation and carries the JAK2 V617F mutation. Cell line SET-2 carries the DNMT3A R882H mutation, recurrent in cytogenetically normal AML.
2019-11-13 | E-MTAB-8323 | biostudies-arrayexpress
Project description:Mutations within genes encoding spliceosomal proteins are the most common class of mutations in patients with myelodysplastic syndromes, yet it is currently not well understood how these mutations impact hematopoiesis or RNA splicing. Here we report that mutations affecting the splicing factor SRSF2 alter its normal RNA recognition activity, resulting in impaired hematopoietic differentiation and myelodysplasia. Commonly occurring SRSF2 mutations impaired wildtype SRSF2’s normal RNA-binding avidity and preference for specific exonic splicing enhancer RNA motifs. Integration of murine and human transcriptome data identified recurrent mis-splicing of key transcriptional regulators in the presence of mutant SRSF2, including promotion of a highly conserved “poison” exon of EZH2 that results in nonsense-mediated decay and contributes to impaired hematopoiesis. These data provide a mechanistic basis for the enrichment of specific mutations in spliceosomal proteins in myelodysplasia, and suggest that altered RNA recognition activity is a novel mechanism of leukemogenesis. mRNA profiles of murine model and K562 cells expressing SRSF2 WT, mutants and knockdown of SRSF2 in TF-1 cells generated by deep sequencing.
2015-05-12 | E-GEOD-65349 | biostudies-arrayexpress
Project description:Pegylated interferon alpha (pegIFNα) can induce molecular remissions in JAK2-V617F-positive myeloproliferative neoplasms (MPN) patients by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFNα. We investigated if DNMT3A loss leads to alterations in JAK2-V617F LT-HSCs functions conferring resistance to pegIFNα treatment in a mouse model of MPN and in hematopoietic progenitors from MPN patients. Long-term treatment with pegIFNα normalized blood parameters, reduced splenomegaly and JAK2-V617F-chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFNα in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F-chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared to VF were less prone to accumulate DNA damage and exit dormancy upon pegIFNα treatment. RNA-sequencing showed that IFNα induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ compared to VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFNα signaling. Transplantations of bone marrow from pegIFNα treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from MPN patients with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFNα exposure, whereas in patients with JAK2-V617F alone the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFNα combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.
2024-03-29 | GSE255253 | GEO
Project description:The activation of PD-1 (Programmed Death receptor-1) on T cells can cause T cell exhaustion and immune tolerance. Some tumors up-regulate the expression of the ligand of PD-1, namely PD-L1 (Programmed Death Receptor-Ligand 1), thus preventing anti-tumor immune response and promoting immune-escape. Previous studies have shown that JAK2 (Janus Kinase 2) signaling can promote PD-L1 expression in Hodgkin Lymphoma. In Myeloproliferative Neoplasms (MPN), JAK2 is frequently characterized by the the presence of the point-mutation V617F, which leads to its constitutive activation and to uncontrolled cell proliferation and survival. Accordingly, tumor cell lines expressing JAK2 V617F express higher levels of PD-L1 as compared to tumor cell lines negative for such mutations. In this experiment, we transfected BaF3 cells with a vector (plasmid for Murine Stem Cell Virus) containing the gene for JAK2 with the point-mutation V617F. As control, we used BaF3 cells transfected with the same vector, but without the gene for JAK2 V617F (empty vector). Both the cell lines (with/without JAK2 V617F) were co-cultured with primary murine T cells. When co-cultured with BaF3 cells expressing JAK2 V617F, T cells upregulated genes connected to senescence pathways, showed increased apoptosis, less cytokine production, and displayed other forms of dysfunction which can be associated with the activation of PD-1.
2018-12-01 | E-MTAB-5028 | biostudies-arrayexpress
Project description:Causal mutations in multiple classes of genes have been identified in MDS patients with some patients harboring more than one mutation. Interestingly, double mutations tend to occur in different classes, rather than the same classes of genes, as exemplified by frequent co-occurring mutations in the transcription factor RUNX1 and the splicing factor SRSF2. Here we report a mouse model in which Runx1 knockout was combined with the Srsf2 P95H mutation to cause multi-lineage hematopoietic defects. Besides their additive and synergistic effects, we also unexpectedly noted a degree of antagonizing activity of single mutations in specific hematopoietic progenitors. To uncover the mechanism, we further developed a cellular model using human K562 cells and performed parallel gene expression and splicing analyses in both human and murine contexts. We performed RNA-seq on sorted Lineage- c-Kit+ (LK) hematopoietic progenitors from bone marrow of mice from WT, runx1 KO , Srsf2 P95H knock in and runx1 Srsf2 P95H double mutants. We also performed RNA-seq on set of isogenic K562 cell lines: wild type (WT), SRSF2 P95H knock-in (SRSF2P95H/+/+), RUNX1 knockdown (RUNX1 KD), and SRSF2 P95H knock-in with RUNX1 knockdown (Double). Strikingly, while RUNX1 deficiency was responsible for altered transcription in both single and double mutants, it also induced dramatic changes in global splicing, as seen with mutant SRSF2, and only their combination induced mis-splicing of genes selectively enriched in the DNA damage response and cell cycle checkpoint pathways. Collectively, these data reveal the convergent impact of a prototypic MDS-associated double mutant on RNA processing and suggest that aberrant DNA damage repair and cell cycle regulation critically contribute to MDS development.
2022-09-27 | GSE200446 | GEO