Induction of erythroid gene expression by microcell fusion.
The molecular events which underlie lineage commitment and differentiation in hematopoietic cells are still incompletely understood. Microcell fusion is a versatile technique which has been utilized in characterizing and mapping genes involved in tumor suppression, cell senescence, and certain aspects of differentiation. Microcell fusion has the potential to contribute to the understanding of hematopoietic differentiation; however, application of this technique is limited by the need to use adherent cells as microcell donors, by the need to tag candidate chromosomes with a selectable marker, and by the need for prolonged selection of fused cells prior to characterization of their phenotype. We developed a modified technique of microcell fusion using square wave electroporation, which allows higher efficiency fusion than polyethylene glycol fusion. By using cross-species fusion and species-specific PCR primers, we were able to detect new gene induction events 48 h after microcell fusion. To study erythroid gene expression, we fused microcells from human erythroid K562 cells to murine B-lymphoid SP-2 cells. We found that microcell fusion induced the nonerythroid recipient cells to express alpha-globin mRNA in a dose-dependent manner. They also expressed RNA for beta-globin, GATA-1, and NF-E2. In contrast, there was no expression of heart- or liver-specific genes. We conclude that microcells from erythroid cells contain all the information necessary to induce expression of multiple erythroid genes. Analysis of the components of the microcells responsible for this new gene induction may allow the characterization of cellular factors responsible for erythroid-specific gene expression.
Kanamori
H
H
Immune Cell Biology Program, Naval Medical Research Institute, Bethesda, Maryland 20889-5607, USA.
Siegel
J N
JN
D000818:Animals; D002454:Cell Differentiation; D002459:Cell Fusion; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D018274:Electroporation; D050981:Erythroid-Specific DNA-Binding Factors; D004920:Erythropoiesis / Q000235:genetics*; D050982:GATA1 Transcription Factor; D018507:Gene Expression Regulation, Developmental; D005914:Globins / Q000096:biosynthesis / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D006822:Hybrid Cells; D051379:Mice; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C494689:Gata1 protein, mouse; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; C494693:Nfe2 protein, mouse; D014157:Transcription Factors; D005914:Globins