Gene expression profiles of murine Runx1;Runx3 double deficient cKit+Sca1+Lin- hematopoietic stem/progenitor cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE51107 | GEO | 2014/08/26
SECONDARY ACCESSION(S): PRJNA221199
REPOSITORIES: GEO
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Project description:The RUNX genes encode for transcription factors involved in development and human disease. RUNX1 and RUNX3 are frequently associated with leukemias, yet the basis for their involvement in leukemogenesis is not fully understood. Here we show that Runx1;Runx3 double knockout (DKO) mice exhibited lethal phenotypes due to bone marrow failure and myeloproliferative disorder. These contradictory clinical manifestations are reminiscent of human inherited bone marrow failure syndromes like Fanconi anemia (FA), caused by defective DNA repair. Indeed, Runx1;Runx3 DKO cells showed mitomycin C hypersensitivity, due to impairment of monoubiquitinated-FANCD2 recruitment to DNA damage foci, although FANCD2 monoubiquitination in the FA pathway was unaffected. RUNX1 and RUNX3 interact with FANCD2 independent of CBFM-NM-2, suggesting non-transcriptional role for RUNX in DNA repair. These findings suggest that RUNX dysfunction causes DNA repair defect, besides transcriptional misregulation, and promotes development of leukemias and other cancers. 6 mice were analyzed in this study. 3 Runx1;Runx3 double knockout cKit+Sca1+Lin- hematopoietic stem/progenitor cells were compared with their wild type littermate controls. RNA was isolated from 3 independent Runx1;Runx3 WT KSL samples, each pooled from 3 Runx1;Runx3 WT mice, and 3 independent Runx1;Runx3 DKO KSL samples, using the RNeasy Micro Kit (QIAgen). RNA integrity and quantity was assessed using the Agilent 2000 Bioanalyzer system. 3 M-NM-<g to 5 M-NM-<g RNA was processed using WT-Ovation Pico RNA Amplification System (NuGEN) paired with the WT-Ovation Exon Module and FL-Ovation cDNA Biotin Module (NuGEN). A detailed protocol in the userM-bM-^@M-^Ys guide kit was used without modification. cDNA were prepared for hybridization on GeneChip Mouse Gene 1.0 ST Arrays (Affymetrix) according to the instructions in GeneChip Hybridization Wash and Stain Kit for ST arrays (Affymetrix). Microarray hybridization, scanning and preliminary MAS 5.0 normalizations were completed at the A*STAR Biopolis Shared Facilities (BSF).
2014-08-26 | E-GEOD-51107 | biostudies-arrayexpress
Project description:Fanconi Anemia (FA) is a rare genetic disorder characterized by an increased susceptibility to squamous cell cancers. Fifteen FA genes are known, and the encoded proteins cooperate in a common DNA repair pathway. A critical step is the monoubiquitination of the FANCD2 protein, and cells from most FA patients are deficient in this step. How monoubiquitinated FANCD2 suppresses squamous cell cancers is unknown. Here we show that Fancd2-deficient mice are prone to Ras oncogene-driven skin carcinogenesis, while Usp1-deficient mice, expressing elevated cellular levels of Fancd2-Ub, are resistant to skin tumors. Moreover, Fancd2-Ub activates the transcription of the tumor suppressor TAp63, thereby promoting cellular senescence and blocking skin tumorigenesis. For FA patients, the reduction of FANCD2-Ub and TAp63 protein levels may account for their susceptibility to squamous cell neoplasia. Taken together, Usp1 inhibition may be a useful strategy for upregulating TAp63 and preventing or treating squamous cell cancers in the general non-FA population. Examination of FANCD2 binding after UV treatment in 293T cells
2013-06-27 | E-GEOD-46902 | biostudies-arrayexpress
Project description:Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. In this study, monoubiquitinated FANCD2-FANCI complex was characterized using crosslinking mass spectrometry in order to provide in sights into the 3D structure of the complex.
2020-03-09 | PXD017020 | Pride
Project description:Fanconi Anemia (FA) is a rare genetic disorder characterized by an increased susceptibility to squamous cell cancers. Fifteen FA genes are known, and the encoded proteins cooperate in a common DNA repair pathway. A critical step is the monoubiquitination of the FANCD2 protein, and cells from most FA patients are deficient in this step. How monoubiquitinated FANCD2 suppresses squamous cell cancers is unknown. Here we show that Fancd2-deficient mice are prone to Ras oncogene-driven skin carcinogenesis, while Usp1-deficient mice, expressing elevated cellular levels of Fancd2-Ub, are resistant to skin tumors. Moreover, Fancd2-Ub activates the transcription of the tumor suppressor TAp63, thereby promoting cellular senescence and blocking skin tumorigenesis. For FA patients, the reduction of FANCD2-Ub and TAp63 protein levels may account for their susceptibility to squamous cell neoplasia. Taken together, Usp1 inhibition may be a useful strategy for upregulating TAp63 and preventing or treating squamous cell cancers in the general non-FA population.
2013-06-27 | GSE46902 | GEO
Project description:The Fanconi Anemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks. Genetic inactivation of this pathway impairs development, prevents blood production and promotes cancer. The key molecular step in the FA pathway is the monoubiquitination of a heterodimer of FANCI-FANCD2 by the FA core complex - a megadalton multiprotein E3 ubiquitin ligase. Monoubiquitinated FANCI-FANCD2 then activates a pathway to remove the DNA crosslink. Lack of molecular insight into the FA core complex limits a detailed explanation of how this vital DNA repair pathway functions. Here we reconstituted an active, recombinant FA core complex, and used electron cryo-microscopy (cryo-EM) and mass spectrometry to determine its overall structure. The FA core complex is comprised of a central symmetric dimer of the FANCB and FAAP100 subunits, flanked by two copies of the RING finger protein, FANCL. This acts as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. The two FANCL subunits are positioned at opposite ends of the complex in an unusual asymmetric arrangement, distinct from other E3 ligases. We propose that each of the two FANCL subunits play unique roles within the complex – one is a structural component while the other monoubiquitinates FANCD2. The cryo-EM structure of the FA core complex, supported by crosslinking mass spectrometry and native mass spectrometry, therefore provides a foundation for a detailed understanding of this fundamental DNA repair pathway.
2019-10-16 | PXD014282 | Pride
Project description:Runx1 and Runx3 function redundantly in early T-development, and together drive T-lineage developmental progression by regulating distinct sets of genes in different stages. Context-specific Runx target genes are particularly enriched near Runx binding sites that dynamically shift from pre-T-commitment stages (Phase 1) to post-T-commitment stages (Phase 2). As total Runx activities (Runx1+Runx3) are maintained stably throughout early T-development stages, yet Runx factors physically interact with multiple collaborators, we hypothesized that different Runx-collaborating transcription factors compete to recruit a limited pool of Runx transcription factors. To test whether increasing Runx availability can alter Runx DNA-binding site choices, Runx1 overexpression vectors were introduced to two different systems. First, we increased Runx1 expression in a DN3-like cell line (representing a post-commitment, Phase 2 stage) in the presence or absence of exogenous Phase 1 co-factor, PU.1. Second, we increased Runx1 expression in Phase 1 primary cells which naturally express PU.1 and other Phase 1 collaborators. Then, Runx1 binding behaviors were measured using ChIP-seq or CUT&RUN (C&R). A modest increase of Runx1 levels (about 2-3 fold increase) substantially increased the number of Runx binding sites seen and the intensity of occupancy in both systems. When Runx expression was at physiological levels, PU.1 dominated Runx1 site choice before T commitment by recruiting Runx1 to PU.1 sites. The Phase 2 cell line system showed that it did this while depleting Runx1 from alternative high quality Runx motif sites. However, when Runx1 was overexpressed, Runx1 was still recruited to PU.1 sites, but this recruitment did not evacuate Runx1 occupancy from default preferred sites. Notably, Runx1 overexpression in primary Phase 1 cells caused precocious occupancy of post-commitment, Phase 2-specific sites. We found that these are often co-occupied with TCF1, E2A, and HEB, but have minimal co-binding with PU.1. In addition, Runx1 overexpression resulted in new binding sites that were not normally observed in pro-T cells, which are mostly sequestered by closed chromatin normally although they harbor more numerous Runx motifs. Thus, these data suggest that Runx DNA binding site choices are sensitive to Runx concentration and co-factors during early T-development.
2023-07-09 | GSE218147 | GEO
Project description:Runx transcription factors play pivotal roles in the development of hematopoietic cells, including T cells. However, the Runx-target genes in early stages of thymic T cell development have been only partially elucidated. Moreover, the relationships of different Runx paralogs, whether they compete or collaborate with each other, are unknown. We have performed CRISPR-Cas9 mediated acute deletion of Runx1 and Runx3, alone and in combination, to define the functions of Runx factors and their target genes. We focused on two distinct stages of early T cell development; before lineage commitment (Phase1) and after commitment (Phase2). Our data suggest that Runx1 and Runx3 are functionally redundant in Phase1, hence the absence of one factor was compensated by the other factor. In Phase2, Runx1 becomes a major contributor, while low levels of Runx3 still strengthened the Runx1-mediated target gene regulation and showed an additive effect. Of importance, we found that Runx1 and Runx3 upregulate T cell programs and inhibit alternative lineage genes. In order to capture the developmental status of Runx-perturbed cells, we performed supervised principal component analysis (PCA) using a fixed frame of PC loadings from single cell RNA-seq analysis of normal in vivo-derived thymocytes. Unlike the reference in vitro and in vivo pro-T cell population or sgControl introduced cells, Runx knockout cells either failed to progress fully (Phase1) or deflected from the normal developmental trajectory (Phase2), suggesting essential roles of Runx factors in early stages of T development. In addition, we analyzed the correlation between stage-specific genomic occupancy of Runx1 and Runx3 (using previously published data for Runx1, GSE103953) and Runx-mediated gene regulation responses at each stage. Interestingly, genes with dynamic, stage-sensitive expression showed a very significant association with phase-specific genomic interactions of Runx factors, and an especially strong correlation with Runx activated target genes. In summary, our data suggest a pivotal role for Runx transcription factors in thymic T cell development by imposing T lineage specification in Phase1 and instructing the lineage-committed progenitor cells to progress through a normal T developmental trajectory.
2020-07-14 | GSE154304 | GEO
Project description:In B cells infected by the cancer-associated Epstein-Barr virus (EBV), RUNX3 and RUNX1 transcription is manipulated to control cell growth. The EBV-encoded EBNA2 transcription factor (TF) activates RUNX3 transcription leading to RUNX3-mediated repression of the RUNX1 promoter and the relief of RUNX1-directed growth repression. We show that EBNA2 activates RUNX3 through a specific element within a -97 kb super-enhancer in a manner dependent on the expression of the Notch DNA-binding partner RBP-J. We also reveal that the EBV TFs EBNA3B and EBNA3C contribute to RUNX3 activation in EBV-infected cells by targeting the same element. Uncovering a counter-regulatory feed-forward step, we demonstrate EBNA2 activation of a RUNX1 super-enhancer (-139 to -250 kb) that results in low-level RUNX1 expression in cells refractory to RUNX1-mediated growth inhibition. EBNA2 activation of the RUNX1 super-enhancer is also dependent on RBP-J. Consistent with the context-dependent roles of EBNA3B and EBNA3C as activators or repressors, we find that these proteins negatively regulate the RUNX1 super-enhancer, curbing EBNA2 activation. Taken together our results reveal cell-type specific exploitation of RUNX gene super-enhancers by multiple EBV TFs via the Notch pathway to fine tune RUNX3 and RUNX1 expression and manipulate B-cell growth.
2016-03-03 | GSE76869 | GEO
Project description:Runx transcription factors are essential for generating functional T cells starting from the earliest stages in thymic T-development. To evaluate how Runx factors instruct T cell development, we perturbed Runx activities using 1) CRISPR-Cas9, simultaneously deleting two predominant, redundant Runx factors, Runx1 and Runx3, and 2) Runx1-overepxression at a stage before pro-T cells commit to a T cell fate. Then single-cell transcriptome analysis was performed. Runx1/Runx3 knockout (KO) vs. Runx1 overexpression (OE) resulted in contrasting deviations from the normal developmental trajectory. The separation of both perturbation conditions from the controls implied that Runx factors regulate gene networks in individual cells to control separate pathways instead of changing distributions of cells among different stages along the control pathway. Notably, a common core set of genes responded to both KO and OE, in opposite directions. Runx activation target genes were selectively enriched for T-identity program and innate lymphoid cell program genes, whereas inhibition targets were significantly associated with stem/progenitor program and myeloid lineage pathways. Pseudotime inference analysis suggested that Runx perturbations also substantially regulate developmental speed, as Runx KO significantly slowed down developmental progression, whereas Runx1 OE resulted in pronounced acceleration. This interpretation was largely supported by Runx activities regulating key transcription factors involved in establishing distinct gene network modules during T cell development. Indeed, further analysis showed that Runx target genes get combinatorial inputs from these Runx-regulated transcription factors as well as from Runx factors themselves. Together, our data suggest that Runx transcription factors drive early T developmental progression by launching gene expression modules associated with T-identity and innate lymphoid cells through mediating other transcription factor cooperativities.
2023-07-09 | GSE218149 | GEO
Project description:The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.
2022-02-22 | PXD031168 | Pride