Global gene expression reveals a set of new genes involved in the modification of cells during erythroid differentiation

Abstract

Objectives: Erythroid differentiation is a dynamic process in which a pluripotent stem cell undergoes a series of developmental changes that commit it to a specific lineage. These alterations involve changes in gene expression profiles. In this study, gene expression profiles during differentiation of human erythroid cells of a normal blood donor were evaluated using SAGE.

Materials and methods: Global gene expression was evaluated in cells collected immediately before addition of erythropoietin (0 h) and 192 and 336 h after addition of this hormone. Real-time PCR was used to evaluate activation of differentially expressed genes.

Results: The data indicate that global aspects of the transcriptome were similar during differentiation of the majority of the genes and that a relatively small set of genes is probably involved in modification of erythroid cells during differentiation. We have identified 93 differentially expressed genes during erythroid development, and expression of some of these was confirmed by qPCR. Various genes including EYA3, ERH, HES6, TIMELESS and TRIB3 were found to be homologous to those of Drosophila melanogaster and here are described for the first time during erythroid development. An important and unique carboxypeptidase inhibitor described in mammalians, LXN, was also identified.

Conclusions: The results of this study amplify previously published data and may contribute to comprehension of erythroid differentiation and identification of new target genes involved in some erythroid concerning diseases.

Figure 1

Validation of SAGE technique – eighteen genes arbitrarily selected for study by qRT‐PCR in the same samples used to generate libraries. Results showed a 95% concordance (17 of 18).

Figure 2

 Cluster analysis of differentially expressed genes associated with erythroid differentiation. Three clusters were found according to up‐regulation of each stage of development. Colour code: blue, low expression; red, high expression. Intensity of colour reflects reliability of expression data.

Figure 3

 Cluster analysis of 15 differentially expressed genes homologous to D. melanogaster found during erythroid differentiation. These genes have been identified in humans; however, most of them do not have any described function. Genes HES6, EYA3, ERH and TRIB3 were found with high expression at the end of differentiation, while TIMELESS showed high expression in the intermediate phase. With the exception of ERH, expression of these genes were hardly observed at the beginning of differentiation. Intensity of colour reflects reliability of expression data.

Figure 4

 Gene expression of selected genes during erythroid differentiation. Gene expressions of six selected genes were evaluated by qPCR in three different two‐phase liquid cultures (a) and in a CD34+ culture (b). Expressions observed in both cultures are the same as those identified by SAGE analysis. The pattern observed in SAGE libraries is displayed together with two‐phase culture.

Figure 5

 Differential expression of selected genes in several tissues using a cDNA tissue library (Clontech Laboratories Inc).