Genome-wide analysis of gene expression changes accompanied with up-regulation of GATA1 in murine hematopoietic stem/progenitors
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ABSTRACT: Analysis of hematopoietic stem/progenitors from GATA-1-GFP transgenic mouse bone marrow at gene expression level. Results provide changes in gene expression pattern accompanied with up-regulation of GATA-1 transcription factor at early stage of murine adult hematopoiesis. RNA samples obtained from isolated GATA1+LSK and other hemaopoietic stem/progenitor populations including GATA1-LSK, LMPP, CMP and LT-HSC were subjected to mRNA amplification and cDNA microarray analysis.
Project description:Analysis of hematopoietic stem/progenitors from GATA-1-GFP transgenic mouse bone marrow at gene expression level. Results provide changes in gene expression pattern accompanied with up-regulation of GATA-1 transcription factor at early stage of murine adult hematopoiesis.
Project description:Expression profiling of WT and E2A-KO LSK FLT3- and LMPP protenitor cells. Experiment Overall Design: LSK FLT3- and LMPP stem/progenitor cells from WT and E2A-KO mice were FACS sorted. Subsequently RNA was extracted, labelled and hybridized to Affymetrix microarrays. Goal of experiment was to investigate expression changes between WT and KO LMPP cells.
Project description:Haematopoietic stem cells can differentiate into all blood cell types. In this process, cells become progressively restricted to a single cell type. The order in which differentiating cells loose lineage potential, and the prospective isolation of cells with a defined potential remains a long-standing question. We performed gene expression analysis of haematopoietic cells from Gata1-EGFP reporter mice, leading to a model for hematopoiesis where the initial lineage decision consists of a separation of erythroid/megakaryocyte/mast cell/eosinophil potential from lymphopoietic/monocyte/neutrophil potential RNA was isolated from HSC EGFP-, HSC EGFP+, LMPP, CLP, preGM EGFP-, preGM EGFP+, GMP EGFP-, GMP EGFP+ and preMegE cells with the QIAGEN RNeasy Micro Kit.
Project description:The transcriptional activiy of GATA1s was compared to GATA1 through gene expression analysis in a cell line model with both erythroid and megakaryocyte differentiation. G1ME cells were derived from Gata1- mouse ES cells and have both megakaryocyte and erythrocyte differentiation potential upon reconstitution of GATA-1 expression (Stachura 2006). HA-tagged full length or short GATA-1 were expressed in G1ME cells grown in TPO via retroviral transductions. The cells were sorted for GFP positivity 68 hours post-transduction and then were allowed to recover in normal growth medium for 4h. Total RNA was then isolated using RNeasy kit from Qiagen 72 hours post-transduction.
Project description:Gata1, a member of the GATA transcription factor family, has been considered as an important factor in the hematopoietic system. As a transcriptional regulator, Gata1 has a crucial role in differentiation of blood cells such as erythrocytes, megakaryocytes, eosinophils, and dendritic cells. Recently, a novel function of Gata1 has been demonstrated in the central nervous system. Gata1, a stress-inducible gene in the brain, has been shown to increase the expression in the dorsolateral prefrontal cortex of patients with depression and function as a transcriptional repressor of synapse-related genes. In this study, we investigated the global alteration in gene expression by Gata1 using ChIPseq, mRNAseq, and smallRNAseq. From the result of ChIPseq for histone marks H3K4me3 and H3K27me3, we have identified 1127 and 4881 unique peaks respectively in the gene promoter region in the cultured cortical neurons overexpressed with Gata1 compared to the control. We also identified 113 differentially expressed mRNAs and 82 miRNAs in cultured neurons transfected with Gata1 through the mRNAseq and smallRNAseq. Through the three-sequencing data analysis, we have profiled genes that might be affected by Gata1 in the cultured cortical neurons. Gene ontology (GO) analysis revealed that Gata1 might be associated with the immune-related function. These results will provide a better understanding of the regulatory mechanisms of Gata1 that affects the pathophysiological status, such as depression.
Project description:17b-Estradiol added to MEL cells expressing Gata1-ER or PU.1-ER transgenes to stimulate either erythropoietic Gata-1 dependent or myeloid PU.1 dependent gene espression in different time points
Project description:GATA1 is repressed in hematopoietic stem cells. To understand the significance of Gata1 gene silencing in HSC we generated GATA1-HSC overexpressing mice (MG-G1). RNA-seq was performed to analyze the gene expression profiles between MG-G1 and WT adult bone marrow LSK cells to examine the consequence of GATA1 overexpression upon HSC.
Project description:Analysis of erythroid differentiation using Gata1 gene-disrupted G1E ER4 clone cells. Estradiol addition activates an ectopically expressed Gata-1-estrogen receptor fusion protein, triggering synchronous differentiation. 30 hour time course corresponds roughly to late burst-forming unit-erythroid stage (t=0 hrs) through orthochromatic erythroblast stage (t=30 hrs). Experiment Overall Design: G1E ER4 cells cultured in G1E medium were treated at 6 time points with estradiol to initiate erythroid differentiation by activating Gata1 transcription factor and total RNAs from treated cells were extracted for microarray experiment. The erythroid differentiation status was confirmed by cell pellet color and expression of microRNA miR451. The design was similar to an earlier studies (Welch, J. J., Watts, J. A., Vakoc, C. R., Yao, Y., Wang, H., Hardison, R. C., Blobel, G. A., Chodosh, L. A., and Weiss, M. J. (2004)). Global regulation of erythroid gene expression by transcription factor GATA-1. Blood 104, 3136-3147), except that a more recent version of Affymetric chip was used to acheive greater transcriptome coverage.
Project description:The 3' untranslated region (3'UTR) of mRNA plays several important roles in post-transcriptional gene regulation. Some of its functions include regulating mRNA stability by polyadenylation and microRNAs. However, the overall function of the Gata1 3’UTR in mammals has not been defined. In this study, we used CRISPR/CAS9 technology to knock out the sequence of the mouse Gata1 3’UTR. We found a defect in erythropoiesis in mutant mice, evidenced by macrocytic anemia at the baseline. Ablation of Gata1 3’UTR also resulted in a reduced number of erythroid precursors that might be associated with the cell cycle, especially G2/M disruption in fetal livers. Mechanistically, deletion of the Gata1 3’UTR destabilizes the Gata1 mRNA and ultimately reduces Gata1 protein levels. The low stability of the Gata1 mRNA is unlikely to be caused by the loss of binding of microRNAs or lack of polyadenylation; rather, in part, by the dissociation of AU-rich elements in the 3’UTR with a trans-activating factor Elavl1. Specifically, we transcribed Gata1 3’UTR in vitro and performed an RNA pulldown assay followed by mass spectrometry to profile the proteins that bind the 3’UTR. Gene Ontology analysis demonstrated that several proteins specifically targeting the 3’UTR were found to potentially bind Gata1 3’UTR, among which Elavl1 was in almost all categories related to mRNA stabilization. Western blotting and RNA immunoprecipitation confirmed the direct interaction of Gata1 3’UTR with Elavl1. Manipulation of Elavl1 activity and protein levels by the small molecule inhibitor Dihydro-tanshinone-I and Elavl1 overexpression in fetal liver erythroblasts confirmed Elavl1 as a stabilizing factor for Gata1 mRNA. Our findings shed light on the functional significance of the 3’UTR of Gata1 mRNA in the context of erythroid development. More importantly, our findings highlight the complexity and diversity of regulatory mechanisms that govern Gata1 mRNA stability and precise expression at the post-transcriptional levels. In addition, our findings prove that miRNAs are not always essential in vivo for controlling transcription factor levels to maintain body homeostasis.