CRISPR/Cas9 screening of RNA binding proteins (RBPs) that regulate RUNX1 isoform production
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ABSTRACT:
PROVIDER: PRJNA608901 | ENA |
REPOSITORIES: ENA
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Project description:The proper balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for normal hematopoiesis and disrupted in hematologic malignancy. Among regulators of HSC fate, transcription factors have a well-defined, central role and mutations promote malignant transformation. More recently, studies have illuminated the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) in hematopoiesis and leukemia development. However, the RBPs involved and breadth of regulation are only beginning to be elucidated. Furthermore, the intersection between post-transcriptional regulation and hematopoietic transcription factor function is poorly understood. Here, we studied the post-transcriptional regulation of RUNX1, a key hematopoietic transcription factor. Alternative polyadenylation (APA) of RUNX1 produces functionally antagonistic protein isoforms (RUNX1a versus RUNX1b/c) that mediate HSC self-renewal versus differentiation, an RNA-processing event that is dysregulated in malignancy. Consequently, RBPs that regulate this event directly contribute to healthy and aberrant hematopoiesis. We modeled RUNX1 APA using a split GFP minigene reporter and confirmed the sensitivity of our model to detecting changes in RNA-processing. We utilized this reporter in a CRISPR screen consisting of single guide-RNAs exclusively targeting RBPs and uncovered HNRNPA1 and KHDRBS1 as antagonistic regulators of RUNX1a isoform generation. Overall, our study provides mechanistic insight into the post-transcriptional regulation of a key hematopoietic transcription factor and identifies RBPs which may have a widespread, important function in hematopoiesis.
2021-03-16 | GSE145968 | GEO
Project description:Aneuploidy and structural aberrations affecting chromosome 21 (Hsa21) are the most frequent in cytogentic events in acute myeloid leukemia. However, it remains unclear why leukemic blasts select for amplifications of Hsa21 or parts of it and why children with Down syndrome (i.e. trisomy 21) are at a high risk of developing leukemia. Here, we propose that disequilibrium of the RUNX1 isoforms and resultant RUNX1A dominance are key to trisomy 21-associated leukemogenesis. Using a Hsa21-focussed CRISPR-Cas9 screen, we uncovered a strong and specific RUNX1 dependency in myeloid leukemia associated with Down syndrome (ML-DS). High levels of RUNX1A – as seen in ML-DS – synergized with the pathognomonic Gata1s mutation in ML-DS pathogenesis, an effect that was reversed upon restoration of the normal RUNX1A:RUNX1C equilibrium. Mechanistically, RUNX1A displaces RUNX1C from its endogenous binding sites and recruits the MYC cofactor MAX to induce oncogenic programs and perturb normal differentiation. This presents a therapeutic vulnerability that can be exploited by interfering with MYC:MAX dimerization. Our study highlights the importance of alternative splicing in leukemogenesis, and paves the way for developing specific and targeted therapies for ML-DS as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium.
2023-05-10 | PXD030616 | Pride
Project description:RUNX1 and ETV6-RUNX1 possess the same DNA-binding runt domain and are therefore expected to bind to canonical RUNX motifs. As the ETV6-RUNX1 fusion arises in the context of native RUNX1 expression, and since RUNX1 is retained or amplified in B-ALL, the two proteins are likely to compete for the same target sites. To assess this, we performed RUNX1 ChIP-seq in the presence of exogenous ETV6-RUNX1 (or non DNA binding ETV6-RUNX1-R139G) and the reciprocal experiment: ETV6-RUNX1 ChIP (using a V5 tag) in the presence of exogenous RUNX1 or vector control.
2022-10-31 | E-MTAB-12209 | biostudies-arrayexpress
Project description:RNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs recognized by RBPs are typically a poor predictor of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory potential. To probe the potential for RBP–RBP complex formation, we assembled a library of 978 mammalian RBPs and used rec-Y2H screening to detect direct interactions between RBPs, sampling >1M possible interactions. We discovered 1994 new interactions and demonstrate that our interaction screening discovers RBP pairs that bind RNAs adjacently. We further find that the mRNA binding region preferences of an RBP can deviate, depending on its adjacently binding interaction partner. Finally, we reveal novel RBP–RBP interaction networks among major RNA processing steps and show that RBP mutations observed in cancer rewire spliceosomal interaction networks.
2021-09-01 | E-MTAB-9612 | biostudies-arrayexpress
Project description:Human histone deacetylase 3 (HDAC3) plays an important role in gene transcription in diseased human cells, such as leukemia. The t(8;21) chromosomal translocation is one of the most commonly observed genetic abnormalities associated with acute myeloid leukemia. This translocation generates the AML1-ETO fusion protein between the wild-type RUNX1 transcription factor and wild-type ETO transcriptional corepressor. To better understand the role of HDAC3 in t(8;21) leukemogenesis, the human HDAC3-containing complexes were isolated from stably-transfected HeLa cells by using anti-FLAG immunoprecipitation. The resulting complexes were resolved in SDS-PAGE. The components of the complexes were identified using LC-MS/MS. We report here that the human RUNX1 transcription is a component of the HDAC3 complexes. We demonstrate that HDAC3 and RUNX1 collaboratively repress AML1-ETO-mediated transcription. These results reveal new insight into how AML1-ETO, RUNX1, and HDAC3 crosstalk to deregulate gene transcription in t(8;21) leukemia cells.
2020-02-17 | PXD017230 | Pride
Project description:Transcriptome-wide profiling of RNA-binding by four RBPs in v-Abl transformed 220-8 pro-B mouse cell line. Binding sites are characterized by T-to-C cross-linking mutations, depth, location on transcript, and overlap/exclusivity across different RBPs.
2019-06-27 | GSE98465 | GEO
Project description:RUNX1 is frequently mutated in myeloid and lymphoid malignancies. It has been shown to negatively regulate Toll-like receptor 4 (TLR4) signaling through nuclear factor kappa-B (NF-κB) in lung epithelial cells. Here we show that RUNX1 regulates TLR1/2 and TLR4 signaling and inflammatory cytokine production by neutrophils. Hematopoietic-specific RUNX1 loss increased the production of pro-inflammatory mediators, including tumor necrosis factor α (TNF-α), by bone marrow (BM) neutrophils in response to TLR1/2 and TLR4 agonists. Hematopoietic RUNX1 loss also resulted in profound damage to the lung parenchyma following inhalation of the TLR4 ligand lipopolysaccharide (LPS). However, neutrophils with neutrophil-specific RUNX1 loss lacked the inflammatory phenotype caused by pan-hematopoietic RUNX1 loss, indicating that dysregulated TLR4 signaling is not due to loss of RUNX1 in neutrophils per se, and single cell RNA sequencing indicates the dysregulation originates in a neutrophil precursor. Enhanced inflammatory cytokine production by neutrophils following pan-hematopoietic RUNX1 loss correlated with increased degradation of the inhibitor of NF-κB signaling (IκBα), and RUNX1-deficient neutrophils displayed broad transcriptional upregulation of many of the core components of the TLR4 signaling pathway. Hence early, pan-hematopoietic RUNX1 loss de-represses an innate immune signaling transcriptional program that is maintained in terminally differentiated neutrophils, resulting in their hyper-inflammatory state. We hypothesize that inflammatory cytokine production by neutrophils may contribute to leukemia associated with inherited RUNX1 mutations.
2020-02-17 | GSE144482 | GEO
Project description:ETV6-RUNX1 is a first-hit mutation in childhood B cell precursor acute lymphoblastic leukaemia. ETV6-RUNX1 is a fusion protein which inherits the DNA-binding runt domain from RUNX1. Here we performed chromatin precipitation for native RUNX1 and ETV6-RUNX1 using RUNX1 antibodies and specifically for the ETV6-RUNX1 fusion using a V5-tag pull down.
2022-10-31 | E-MTAB-12208 | biostudies-arrayexpress
Project description:Purpose: The goal of this study was to identify the RBPs that regulate the inclusion of the exons in the muscular isoform of the DMD gene (Dp427m). We compared the splicing pattern of the Dp427m from C25Cl48 treated with a control siRNA or siRNA targeting 16 different RBPs. Method: The C25Cl48 were treated by RNAi in duplicates (or n=4 for the control condition) and submitted to 3 days of myogenic differentiation. The RBPs tested were CELF1, DAZAP1, DDX5/DDX17, hnRNPA1, hnRNPA2, MBNL1, PTBP1, QKI, RBFOX1, RBFOX2, RBM4, SRSF1, SRSF2, TDP-43 and Tra2b. The silencing efficiency was controlled by RT-qPCR and Western blotting analysis. RNA was reverse transcribed using oligodT and the Dp427m (11.3kb) coding sequence was amplified by Long-Range PCR and sequenced on a 454 GS junior platform. Reads that passed quality filters were mapped to the human X chromosome with STAR. The Percent-Spliced-In (PSI) was calculated for each splicing event using intron-centric metrics (Pervouchine et al., 2013), with the SJPIPE pipeline, from the ipsa package. Junctions were filtered to keep only those covered by a minimum of 5 reads in at least one out of the two biological replicates or 2/4 in the control condition. Results: Using this DMD-targeted RNA-seq approach, we obtained an average read depth of 1638X, allowing reliable detection of alternative splicing events. Based on a cutoff of |[delta]PSI|≥0.05, we identified 8 exons that were deregulated in at least one of the RBPs tested, and 15 RNAi conditions induced splicing changes. Conclusion: The 8 deregulated exons identified here are mainly exons that were previously found alternatively spliced in other dystrophin isoforms.
2020-10-22 | GSE153803 | GEO
Project description:RUNX1 is frequently mutated in myeloid and lymphoid malignancies. It has been shown to negatively regulate Toll-like receptor 4 (TLR4) signaling through nuclear factor kappa-B (NF-κB) in lung epithelial cells. Here we show that RUNX1 regulates TLR1/2 and TLR4 signaling and inflammatory cytokine production by neutrophils. Hematopoietic-specific RUNX1 loss increased the production of pro-inflammatory mediators, including tumor necrosis factor α (TNF-α), by bone marrow (BM) neutrophils in response to TLR1/2 and TLR4 agonists. Hematopoietic RUNX1 loss also resulted in profound damage to the lung parenchyma following inhalation of the TLR4 ligand lipopolysaccharide (LPS). However, neutrophils with neutrophil-specific RUNX1 loss lacked the inflammatory phenotype caused by pan-hematopoietic RUNX1 loss, indicating that dysregulated TLR4 signaling is not due to loss of RUNX1 in neutrophils per se, and single cell RNA sequencing indicates the dysregulation originates in a neutrophil precursor. Enhanced inflammatory cytokine production by neutrophils following pan-hematopoietic RUNX1 loss correlated with increased degradation of the inhibitor of NF-κB signaling (IκBα), and RUNX1-deficient neutrophils displayed broad transcriptional upregulation of many of the core components of the TLR4 signaling pathway. Hence early, pan-hematopoietic RUNX1 loss de-represses an innate immune signaling transcriptional program that is maintained in terminally differentiated neutrophils, resulting in their hyper-inflammatory state. We hypothesize that inflammatory cytokine production by neutrophils may contribute to leukemia associated with inherited RUNX1 mutations.
2020-02-17 | GSE144481 | GEO