Unknown,Transcriptomics,Genomics,Proteomics

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Expression profiling of erythroid developmental subsets isolated from mouse fetal liver.

ABSTRACT: The S1 and S3 erythroid developmental subsets were isolated using flow cytometry and the cell surface markers CD71 and Ter119 as described by Pop et. al. 2010 (PMID: 20877475) Expression profiles for S1 and S3 subsets were generated using Affymetrix GeneChips. Results were used to identify genes that are differentially expressed during erythropoiesis. Single cell suspensions were prepared by mechanically dissociating whole fetal livers obtained from E12.5 to E13.5 Balb/C mouse embryos. Cells were stained for CD71, Ter119, and a cocktail containing lineage-specific antibodies. S1 and S3 erythroid developmental subsets were identified and isolated using flow cytometric sorting as described by Pop et. al. 2010 (PMID: 20877475). S1 and S3 subsets were isolated on 3 seperate days to generate total RNA (biological replicates). 20 ng of total RNA from each biological replicate was converted to cDNA, linearly amplified and biotinylated using Ovation reagents (Nugen, San Carlos, CA). Samples were hybridized to Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA). Microarray suite 5 (MAS5) processed sample data were normalized to the average of 18SRNA (AFFX-18SRNAMur/X00686_M_at), GAPDH (AFFX-GapdhMur/M32599_3_at) and M-NM-2-actin (1419734_at) expression values. These gene expression profiles were performed as part of the manuscript by Shearstone et. al. Global DNA Demethylation During Erythropoiesis In Vivo

ORGANISM(S): Mus musculus

SUBMITTER: Merav Socolovsky

PROVIDER: E-GEOD-32214 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Global DNA demethylation during mouse erythropoiesis in vivo.

Shearstone Jeffrey R JR Pop Ramona R Bock Christoph C Boyle Patrick P Meissner Alexander A Socolovsky Merav M

Science (New York, N.Y.) 20111101 6057

In the mammalian genome, 5'-CpG-3' dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide demethylation is thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. These demethylation events are followed by de novo methylation, setting up a pattern inherited throughout development and modified only at tissue-specific loci. We studied DNA methylation in differentiating mouse erythroblasts in vivo by usin ...[more]

PMID: 22076376

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Project description:It is unclear how epigenetic changes regulate the induction of erythroid-specific genes during terminal erythropoiesis. Here we use global mRNA sequencing (mRNA-seq) and chromatin immunoprecipitation coupled to high-throughput sequencing (CHIP-seq) to investigate the changes that occur in mRNA levels, RNA Polymerase II (Pol II) occupancy and multiple post-translational histone modifications when erythroid progenitors differentiate into late erythroblasts. Among genes induced during this developmental transition, there was an increase in the occupancy of Pol II, the activation marks H3K4me2, H3K4me3, H3K9Ac and H4K16Ac, and the elongation methylation mark H3K79me2. In contrast, genes that were repressed during differentiation showed relative decreases in H3K79me2 levels yet had levels of Pol II binding and active histone marks similar to those in erythroid progenitors. We also found that relative changes in histone modification levels-in particular, H3K79me2 and H4K16ac-were most predictive of gene expression patterns. Our results suggest that in terminal erythropoiesis both promoter and elongation-associated marks contribute to the induction of erythroid genes, while gene repression is marked by changes in histone modifications mediating Pol II elongation. Our data maps the epigenetic landscape of terminal erythropoiesis and suggests that control of transcription elongation regulates gene expression during terminal erythroid differentiation. Mouse fetal liver cells are double-labeled for erythroid-specific TER119 and non erythroid-specific transferrin receptor (CD71) and then sorted by flow-cytometry. E14.5 fetal livers contain at least five distinct populations of cells (R1 through R5); as they progressively differentiate they gain TER119 and then gain and subsequently lose CD71. CFU-E cells and proerythroblasts make up the R1 population; R2 consists of proerythroblasts and early basophilic erythroblasts; R3 includes early and late basophilic erythroblasts; R4 is mostly polychromatophilic and orthochromatophilic erythroblasts; and R5 is comprised of late orthochromatophilic erythroblasts and reticulocytes. We have sorted for R2-R5 cells for RNA-seq experiment.

Dataset Information

Expression profiling of erythroid developmental subsets isolated from mouse fetal liver.

Publications

Global DNA demethylation during mouse erythropoiesis in vivo.

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