Human Erythroid Day 10 ChIP using GATA, PolII, H3 and H4
ABSTRACT: A large-scale analysis of histone acetylation as well as RNA polymerase II and GATA 1 interactions on chromosome 6q in human erythroid progenitor cells. Overall design: Human erythroid progenitors from day 10 erythroid cultures were used for ChIP-chip analysis. For this purpose, the NimbleGen array system was used, following manufacturer's protocols. Samples for ChIP were taken from the same individual and processed for use with each of the following antibodies: GATA1, PolII, AcH3 and AcH4.
INSTRUMENT(S): NimbleGen Homo sapiens 384K hg18 tiling set 16
Project description:A large-scale analysis of histone acetylation as well as RNA polymerase II and GATA 1 interactions on chromosome 6q in human erythroid progenitor cells. Human erythroid progenitors from day 10 erythroid cultures were used for ChIP-chip analysis. For this purpose, the NimbleGen array system was used, following manufacturer's protocols. Samples for ChIP were taken from the same individual and processed for use with each of the following antibodies: GATA1, PolII, AcH3 and AcH4.
Project description:CRISPR/Cas9-mediated ablation of two Alas2 intronic cis-elements in G1E-ER-GATA1 cells strongly reduced GATA-1-induced Alas2 transcription, heme biosynthesis, and GATA-1 regulation of other vital constituents of the erythroid cell transcriptome. Bypassing Alas2 function in Alas2 cis-element-mutant (double mutant) cells by providing its catalytic product 5-aminolevulinic acid (5-ALA) rescued heme biosynthesis and a subset of GATA-1-dependent genetic network. Using the same system, we discovered a GATA-1- and heme-dependent circuit that regulates chromatin accessibility during erythroid maturation. Overall design: G1E-ER-GATA1 WT and double mutant cells were examined. Untreated WT, beta-estradiol-treated WT, untreated double mutant, beta-estradiol-treated double mutant and beta-estradiol/5-ALA-treated double mutant cells were subject to ATAC-seq.
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). 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:We report the occupancy of GATA1 and TAL1, two erythroid transcription factors, and H3K27ac histone modification in human erythroid precursors. Overall design: Examination of occupancy of two transcription factors and one histone mark by ChIP-seq in erythroid precursors
Project description:Regions bound by the erythroid transcription factors GATA1 and NFE2 and the general factor CTCF were identified in mouse erythroid cells using ChIP-seq. Chip-seq experiements were performed in mouse erythroid Ter119+ cells.
Project description:Missense mutations in transcription factor GATA1 underlie several distinct forms of anemia and thrombocytopenia. Clinical severity depends on the site and type of substitution, and distinct substiutions of the same residue produce disparate phenotypes. To investigate the effect of GATA1 missense mutations on erythroid differentiation we expressed conditionally activated wild type or mutant versions of GATA1 in GATA1-null G1E cells. We used gene expression microarrays to explore how GATA1 missense mutations affect erythroid transcription programs. GATA1-null G1E cells ectopically expressing conditionally activated versions of GATA1 (GATA1-ER, GATA1(R216Q)-ER, GATA1(R216W)-ER, GATA1(D218G)-ER, or GATA1(D218Y)-ER) were treated with estradiol for 24 hours to initiate erythroid differentiation. Total RNA from treated cells was extracted for Affymetrix microarray. All data were generated from three biological replicates. Transcript levels were compared in wild type vs. mutant lines.
Project description:The Affymetrix Human Gene 2.0 ST array was used to measure differential expression of RNA isolated from normal and Diamond Blackfan anemia (DBA) erythroid progenitors after ex vivo expansion of circulating, peripheral blood derived hematopoietic stem cells under erythroid growth conditions. The gene-level probe summaries reported in this series were computed using RMA as implemented in the Bioconductor package Oligo v1.36.1. Diamond Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid aplasia, usually without perturbation of other hematopoietic lineages. Approximately 65% of DBA patients with autosomal dominant inheritance have heterozygous mutations or deletions in ribosomal protein (RP) genes while <1% of patients with X-linked inheritance have been identified with mutations in the transcription factor, GATA1. Erythroid cells from patients with DBA have not been well characterized and the mechanisms underlying the erythroid specific effects of either RP or GATA1 associated DBA remain unclear. We have developed an in vitro culture system to expand peripheral blood CD34+ progenitor cells from patients with DBA and differentiate them into erythroid cells. Cells from patients with RP or GATA1 mutations showed decreased proliferation and delayed erythroid differentiation compared to controls. RNA transcript analyses of erythroid cells from controls and patients with RP or GATA1 mutations showed distinctive differences, with upregulation of heme biosynthesis genes prominently in RP-mediated DBA and failure to upregulate components of the translational apparatus in GATA1-mediated DBA. Our data show that dysregulation of translational function is a common feature of DBA caused by both RP and GATA1 mutations. Overall design: 37 samples analyzed including 10 normal CD44+/CD235+, 8 normal CD44+/235-, 5 DBA CD44+/235+, and 14 DBA CD44+/CD235- samples.
Project description:KLF1 (EKLF) regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which KLF1 operates, we performed KLF1 ChIP-seq in the mouse. We found at least 945 sites in the genome of E14.5 fetal liver erythroid cells which are occupied by endogenous KLF1. Many of these recovered sites reside in erythroid gene promoters such as β-globin, but the majority are distant to any known gene. Our data suggests KLF1 directly regulates most aspects of terminal erythroid differentiation including production of α and β-globin protein chains, heme biosynthesis, co-ordination of proliferation and anti-apoptotic pathways, and construction of the red cell membrane and cytoskeleton by functioning primarily as a transcriptional activator. Additionally, we suggest new mechanisms for KLF1 co-operation with other transcription factors, in particular the erythroid transcription factor GATA1, to maintain homeostasis in the erythroid compartment. Overall design: Examination of KLF1 occupancy in primary erythroid cells. KLF1-ChIP and input samples were run on AB SOLiD Systems 2.0 and 3.0. The genomic alignment files (*sorted.txt) and peak file (*bed) contain the combined System 2.0 and 3.0 data.
Project description:We employed a gene complementation strategy combined with microarray screening to identify miRNAs involved in the formation of erythroid (red blood) cells. To search for GATA-1-regulated erythroid miRNAs, we used the Gata-1– erythroblast line G1E. These cells proliferate in culture as immature erythroid precursors and undergo terminal maturation when GATA-1 activity is restored. G1E-ER4 is a sub-line stably expressing an estrogen-activated form of GATA-1 (GATA-1 fused to the ligand binding domain of the estrogen receptor). Treatment of G1E-ER4 cells with estradiol induces a GATA-1-regulated program of gene expression with concomitant cellular maturation. We used a microarray to evaluate the expression of 292 different miRNAs in G1E-ER4 cells at 0 versus 24 hours after GATA-1 activation. Affymetrix gene expression profiling has previously been deposited (GEO accession no. GSE628). Keywords: microRNA analysis of a cell-line model of erythroid maturation Overall design: Two condition experiment, 3 replicates each (independently grown and harvested) of untreated and estradiol-treated (24hrs) G1E-ER4 cells, which express an estrogen-responsive form of the GATA-1 transcription factor. Each sample is compared to a common reference sample, comprised of an equal mixture of all 6 experimental samples.