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MicroRNA expression dynamics during murine and human erythroid differentiation.

ABSTRACT: MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that regulate diverse cellular functions by sequence-specific inhibition of gene expression. We determined miRNA expression profile during erythroid differentiation and putative roles in erythroid differentiation.The expression profile of 295 miRNAs before and after their erythroid differentiation induction was analyzed using microarray. Fluorescein-activated cell sorting analysis was used to isolate mouse spleen erythroblasts at different differentiation stages. Human cord blood CD34+ progenitors were differentiated in vitro. Real-time reverse transcriptase polymerase chain reaction was used to confirm the results of miRNA microarray. Synthetic oligonucleotides for miR-451 overexpression or knockdown were transfected into MEL cells.More than 100 miRNAs were found to be expressed in erythroid cells. The majority of them showed changes in their expression levels with progression of erythroid differentiation. Further analysis revealed that overall miRNA expression levels are increased upon erythroid differentiation. Of the miRNAs analyzed, miR-451 was most significantly upregulated during erythroid maturation. Functional studies using gain of function and loss of function approaches showed that miR-451 is associated with erythroid maturation.Dynamic changes in miRNA expression occurred during erythroid differentiation, with an overall increase in the levels of miRNAs upon terminal differentiation of erythroid cells. MiR-451 may play a role in promoting erythroid differentiation.

SUBMITTER: Zhan M

PROVIDER: S-EPMC1955220 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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MicroRNA expression dynamics during murine and human erythroid differentiation.

Zhan Mei M Miller Chris P CP Papayannopoulou Thalia T Stamatoyannopoulos George G Song Chao-Zhong CZ

Experimental hematology 20070701 7

<h4>Objective</h4>MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that regulate diverse cellular functions by sequence-specific inhibition of gene expression. We determined miRNA expression profile during erythroid differentiation and putative roles in erythroid differentiation.<h4>Methods</h4>The expression profile of 295 miRNAs before and after their erythroid differentiation induction was analyzed using microarray. Fluorescein-activated cell sorting analysis was used to isola ...[more]

PMID: 17588470

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Project description:The aim of the present study has been to establish serum-free culture conditions for ex vivo expansion and differentiation of human CD34(+) cells into erythroid lineage and to study the chromatin structure, gene expression, and transcription factor recruitment at the alpha-globin locus in the developing erythron.A basal Iscove's modified Dulbecco's medium cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for expansion and differentiation of the CD34(+) cells. Expression patterns of the alpha- and beta-like genes at various stages of erythropoiesis was studied by reverse transcriptase quantitative polymerase chain reaction analysis, profile of key erythroid transcription factors was investigated by Western blotting, and the chromatin structure and transcription factor recruitment at the alpha-globin locus was investigated by chromatin immunoprecipitation quantitative polymerase chain reaction analysis.Human CD34(+) cells in the serum-free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the alpha-globin locus during erythroid differentiation of CD34(+) cells. Nuclear factor erythroid-derived 2 (NF-E2) was present at upstream activator sites even before addition of erythropoietin (EPO), while bound GATA-1 was only detectable after EPO treatment. After 7 days of EPO treatment, H3K4Me2 modification uniformly increases throughout the alpha-globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2, and GATA-1 were restricted to certain hypersensitive sites of the enhancer and theta gene, and were conspicuously low at the alpha-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the alpha-globin locus as CD34(+) cells differentiated into erythroid pathway.Our results indicate that remodeling of the upstream elements may be the primary event in activation of alpha-globin gene expression. Activation of alpha-globin genes upon EPO treatment involves initial binding of Pol II, downregulation of pre-existing factors like NF-E2, removal of CTCF from the locus, then rebinding of CTCF in an altered pattern, and concurrent or subsequent binding of transcription factors like GATA-1.

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MicroRNA expression dynamics during murine and human erythroid differentiation.

Publications

MicroRNA expression dynamics during murine and human erythroid differentiation.

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