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Transcriptome of the maturing erythroblast

ABSTRACT: Understanding the pattern of gene expression and identifying the specific genes expressed during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here we have isolated four distinct populations of erythroblasts at successive erythropoietin-dependent stages of erythropoiesis including the terminal, pyknotic stage. The transcriptome has been determined using Affymetrix arrays. First, we show that cells sorted by surface expression profile express not only significantly fewer genes than unsorted cells, but also significantly more differences in the expression levels of particular genes between stages than unsorted cells, demonstrating the importance of working with defined cell populations to identify lineage and temporally-specific patterns of gene expression. Second, using standard software and matched filtering we identify eleven differentially regulated genes and one continuously expressed gene previously undetected in erythroid expression studies with unknown roles in erythropoiesis (CA3, CALB1, CTSL2, FKBP1B, GSDMB, ITLN1, LIN7B, RRAD, RUNDC3A, UNQ1887, ZNF805, MYL12B). Finally, using transcription factor binding site analysis we identify potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts are a resource for functional studies of erythropoietic protein function and gene regulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE22552 | GEO | 2011/03/02

SECONDARY ACCESSION(S): PRJNA128183

REPOSITORIES: GEO

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Project description:Understanding the pattern of gene expression and identifying the specific genes expressed during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here we have isolated four distinct populations of erythroblasts at successive erythropoietin-dependent stages of erythropoiesis including the terminal, pyknotic stage. The transcriptome has been determined using Affymetrix arrays. First, we show that cells sorted by surface expression profile express not only significantly fewer genes than unsorted cells, but also significantly more differences in the expression levels of particular genes between stages than unsorted cells, demonstrating the importance of working with defined cell populations to identify lineage and temporally-specific patterns of gene expression. Second, using standard software and matched filtering we identify eleven differentially regulated genes and one continuously expressed gene previously undetected in erythroid expression studies with unknown roles in erythropoiesis (CA3, CALB1, CTSL2, FKBP1B, GSDMB, ITLN1, LIN7B, RRAD, RUNDC3A, UNQ1887, ZNF805, MYL12B). Finally, using transcription factor binding site analysis we identify potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts are a resource for functional studies of erythropoietic protein function and gene regulation. PBMC-derived erythroblasts grown in vitro were harvested at the CFU-E, Pro-E, Int-E and Late-E stages and FACS sorted based on expression of cell surface markers. RNA was extracted from those populations which met rigorous specifications of time cultured in the presence of erythropoietin, morphology and CD36, CD71 and CD235a expression. For each stage, 3 chips were hybridised, each representing a pool of 4 different samples. In addition to these 12 chips, one chip representing a pool of RNA extracted from unsorted cells at each stages was also hybridised.

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.

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Transcriptome of the maturing erythroblast

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