Genome-wide mapping of Runx1-bound sites in early B-cell progenitors
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ABSTRACT: The t(12;21) chromosomal translocation, targeting the gene encoding the RUNX1 transcription factor, is observed in 25% of pediatric acute lymphoblastic leukemia (ALL) and is an initiating event in the disease. To elucidate the mechanism by which RUNX1 disruption initiates leukemogenesis, we investigated its normal role in murine B-cell development. Gene expression analysis and genome-wide Runx1-occupancy studies support the hypothesis that Runx1 reinforces the transcription factor network in B-cell progenitors governing early B-cell survival and development . ChIP-seq experiments were performed in the proB-cell line BMiFLT3(15-3), stably transduced with the transcription factor Runx1, to identify Runx1-bound sites in early B-cell progenitors.
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.
Project description:This SuperSeries is composed of the following subset Series: GSE24777: Regulation of Megakaryocytic differentiation in Cell Line Models by Dynamic Combinatorial Interactions of RUNX1 with Its Cooperating Partners GSE24778: Expresssion data in K562 cells, before and after TPA induction and including a RUNX1 knockout construct or a control structure Refer to individual Series
Project description:The sequential activation of distinct developmental gene networks governs the ultimate identity of a cell, but the mechanisms by which downstream programs are activated are incompletely understood. The preB-cell receptor (preBCR) is an important checkpoint of B-cell development and essential for a preB-cell to traverse into an immature B-cell. Here, we show that activation of Mef2 transcription factors by preBCR is necessary for initiating the subsequent genetic network. We demonstrate that B-cell development is blocked at the preB-cell stage in mice deficient for Mef2c and Mef2d transcription factors and that preBCR signaling enhances the transcriptional activity of Mef2c/d through phosphorylation by the ERK5 mitogen activating kinase. This activation is instrumental in inducing Krüppel-like factor 2 and several immediate early genes of the AP1 and Egr family. Finally, we show that Mef2 proteins cooperate with the products of their target genes (Irf4 and Egr2) to induce secondary waves of transcriptional regulation. Our findings uncover a novel role for Mef2c/d in coordinating the transcriptional network that promotes early B-cell development. ChIP-seq experiments were performed in the proB-cell line BMiFLT3(15-3) to identify Mef2c-bound sites in early B-cell progenitors.
Project description:Analysis of patient-specific nucleotide variants is a cornerstone of personalised medicine. Although only 2% of the genomic sequence is protein-coding, mutations occurring in these regions have the potential to influence protein structure and may have severe impact on disease aetiology. Of special importance are variants that affect modifiable amino acid residues, as protein modifications involved in signal transduction networks cannot be analysed by genomics. Proteogenomics enables analysis of proteomes in context of patient- or tissue-specific non-synonymous nucleotide variants. Here, we developed a proteogenomics workflow and applied it to study resistance to serine/threonine-protein kinase B-raf (BRAF) inhibitor (BRAFi) vemurafenib in malignant melanoma cell line A375. This approach resulted in high identification and quantification of non-synonymous nucleotide variants and (phospho)proteins. We integrated multi-omic datasets to reconstruct the perturbed signalling networks associated with BRAFi resistance and to predict drug therapies with the potential to disrupt BRAFi resistance mechanism in A375 cells. Notably, we showed that aurora kinase A (AURKA) inhibition is effective and specific against BRAFi resistant A375 cells. Furthermore, we investigated nucleotide variants that interfere with protein post-translational modification (PTM) status and potentially influence cell signalling. Mass spectrometry (MS) measurements confirmed variant-driven PTM changes in 12 proteins; among them was the runt-related transcription factor 1 (RUNX1) displaying a variant on a known phosphorylation site S(Ph)276L. We confirmed the loss of phosphorylation site by MS and demonstrated the impact of this variant on RUNX1 interactome.
Project description:The t(12;21) chromosomal translocation, targeting the gene encoding the RUNX1 transcription factor, is observed in 25% of pediatric acute lymphoblastic leukemia (ALL) and is an initiating event in the disease. To elucidate the mechanism by which RUNX1 disruption initiates leukemogenesis, we investigated its normal role in murine B-cell development. Gene expression analysis and genome-wide Runx1-occupancy studies support the hypothesis that Runx1 reinforces the transcription factor network in B-cell progenitors governing early B-cell survival and development . 1) To identify Runx1 target genes in early B-cell progenitors, we used a whole genome microarray for analyzing the gene expression profile in a proB-cell line [BMiFLT3(15-3)] engineered to overexpresses the Runx1 transcription factor fused to the ligand binding domain of the estrogen receptor. The use of an inducible Runx1 fusion protein allowed specific translocation of Runx1 into the nucleus activation of Runx1 target genes. 2) To identify Runx1 target genes in early B-cell progenitors, we used a whole genome microarray for analyzing the gene expression profiles of proB-cells (CD19+/B220med/CD93+) isolated from Runx1+/+Cd79ahCre/+ and Runx1fl/flCd79ahCre/+ mice. Two independent experiments were peformed.
Project description:The t(12;21) chromosomal translocation, targeting the gene encoding the RUNX1 transcription factor, is observed in 25% of pediatric acute lymphoblastic leukemia (ALL) and is an initiating event in the disease. To elucidate the mechanism by which RUNX1 disruption initiates leukemogenesis, we investigated its normal role in murine B-cell development. Gene expression analysis and genome-wide Runx1-occupancy studies support the hypothesis that Runx1 reinforces the transcription factor network in B-cell progenitors governing early B-cell survival and development . Refer to individual Series
Project description:Regulation of Megakaryocytic differentiation in Cell Line Models by Dynamic Combinatorial Interactions of RUNX1 with Its Cooperating Partners Examination of RUNX1 binding in K562 cells, before and following TPA induction and CMK cells. Examination of GATA1 and FOS binding and H3K4me1 and H3K27me3 modification levels following TPA induction in K562 cells.
Project description:RUNX1 transcription factor (TF) is a key regulator of megakaryocytic development and when mutated is associated with familial platelet disorder and predisposition to acute myeloid leukemia (FPD-AML). We used mice lacking Runx1 specifically in megakaryocytes (MKs) to characterize the Runx1-mediated transcriptional program during advanced stages of MK differentiation. Gene expression and chromatin-immunoprecipitation-sequencing (ChIP-seq) of Runx1 and p300 identified functional Runx1-bound MK enhancers. Runx1/p300 co-bound regions showed significant enrichment in genes important for MK and platelet homeostasis. Runx1-bound regions were highly enriched in RUNX and ETS motifs and to a lesser extent in GATA motif. The data provides the first example of genome-wide Runx1/p300 occupancy in maturating FL-MK, unravels the Runx1-regulated program controlling MK maturation in vivo and identifies its bona fide regulated genes. It advances our understanding of the molecular events that upon mutations in RUNX1 lead to the predisposition to familial platelet disorders and FPD-AML. Examination of RUNX1 and P300 binding in WT mouse megakaryoctye cells using ChIP-Seq. The supplementary 'GSE45372_PeakList.txt' file includes a list of regions identified as binding for P300 or RUNX1 or both.
Project description:Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study, we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFbeta transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate considerable chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFbeta and functional regulation of a significant fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFbeta deficiency generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via Polycomb Repressive Complex 2 (PRC2) independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells. ChIP-seq against Runx1, CBFb and Ring1b in L8057 cells (induced & uninduced with biological replicates) and thymocytes from control and Runx1 KO mice
Project description:We identified directly and indirectly regulated target genes utilizing an inducible TEL-AML1 system derived from the murine pro B-cell line BA/F3 and a monoclonal antibody directed against TEL-AML1. By integration of promoter binding identified with ChIP-on-chip, gene expression and protein output through microarray technology and stable labelling of amino acids in cell culture (SILAC), we identified directly and indirectly regulated targets of the TEL-AML1 fusion protein. Bound promoter regions of immunoprecipitated TEL-AML1 associated genes were first compared to input material and enrichment was calculated. The same was performed for empty vector control cell lines, also treated with the induction reagent mifepriston. Enriched promoter regions were then compared of the both sets. 2 independent replicates each (4 arrays) were performed.