ABSTRACT
BACKGROUND: Understanding the mechanism of developmental regulation of hemoglobin switching has scientific as well as clinical relevance because of the influence of fetal hemoglobin (HbF) production in adulthood on the clinical manifestation of thalassemia and sickle cell anemia. We have previously found that the normal developmental patterns of globin gene expression are recapitulated in an experimental system of primary cultures that support differentiation of erythroid progenitors. We further found that high activities of the transcriptional activators, GATA-1 and SP1, are associated with normal adult erythroid differentiation. MATERIALS AND METHODS: In the present work, we have studied, the activities of GATA-1 and SP1 during differentiation of cultured erythroid progenitors derived from cord blood and from fetal livers, as well as from beta zero-thalassemia patients. RESULTS: The results showed high GATA-1 binding activity and very low SP1 activity in the fetal liver cultures. This pattern was in contrast to cultures derived from normal adult peripheral blood, in which both GATA-1 and SP1 activities were high. Cord blood cultures showed an additive combination of "adult" and "fetal" patterns. The progenitors derived from a beta zero-thalassemia patient with high HbF production showed "fetal" pattern. On the other hand, in cultures of 2 beta zero-thalassemia patients without high HbF, "adult" pattern was observed. CONCLUSIONS: In the present work, we show that human fetal and adult erythroid progenitors are distinct in their transcription factors, and that the commitment to fetal or adult program occurs at a very early differentiation stage. Our studies also demonstrate that under anemic stress, recruitment of fetal progenitors may occur in adulthood.
Subject(s)
DNA-Binding Proteins/metabolism , Erythroid Precursor Cells/metabolism , Fetal Hemoglobin/biosynthesis , Globins/biosynthesis , Sp1 Transcription Factor/metabolism , Transcription Factors/metabolism , Adult , Base Sequence , Cell Differentiation/genetics , Cells, Cultured , DNA-Binding Proteins/blood , DNA-Binding Proteins/genetics , Erythrocytes/metabolism , Erythroid-Specific DNA-Binding Factors , Erythropoietin/pharmacology , Fetal Blood/metabolism , Fetal Hemoglobin/genetics , GATA1 Transcription Factor , Genes, Switch , Globins/genetics , Humans , Liver/cytology , Liver/embryology , Liver/metabolism , Molecular Sequence Data , RNA, Messenger/analysis , Transcription Factors/blood , Zinc Fingers , beta-Thalassemia/bloodABSTRACT
GATA-1 is a central transcription-activator of erythroid differentiation. In the present work we have studied the kinetics of its expression and activity during development of normal human erythroid progenitors, grown in primary cultures. In response to the addition of erythropoietin (Epo), the cells undergo proliferation and differentiation in a synchronized fashion. This recently developed experimental system allows biochemical dissection of erythroid differentiation in a physiological meaningful environment. No DNA-binding activity of GATA-1 could be detected before the addition of Epo, although a very low level of mRNA was observed. Following Epo addition there was a sharp parallel rise in both mRNA and DNA-binding activity, consistent with positive autoregulation of the GATA-1 gene. After reaching a peak on day 7-9, both mRNA and protein activity decreased. The binding activity of the ubiquitous factor SP1 showed a biphasic pattern; its second peak usually coincided with the GATA-1 peak, suggesting that SP1 also plays a specific role in erythroid maturation. The highest activity of GATA-1 per erythroid cell was found on day 6-8, immediately preceding the major rise in globin gene mRNA and in the number of hemoglobinized cells. The results imply that a high level of GATA-1 activity is necessary for globin gene expression and erythroid maturation, suggesting that a requirement for a threshold concentration of GATA-1 is part of the mechanism that determines the final steps of erythroid maturation.
Subject(s)
DNA-Binding Proteins/metabolism , Erythrocytes/cytology , Erythropoietin/physiology , Transcription Factors/metabolism , Adult , Base Sequence , Cells, Cultured , DNA , Erythrocytes/metabolism , Erythroid-Specific DNA-Binding Factors , Erythropoiesis , GATA1 Transcription Factor , Gene Expression Regulation , Globins/genetics , Host Cell Factor C1 , Humans , Kinetics , Molecular Sequence Data , Octamer Transcription Factor-1 , RNA, Messenger/biosynthesis , Sp1 Transcription Factor/metabolism , Transcription, GeneticABSTRACT
We have recently described a two-step liquid culture system that supports the proliferation and maturation of human erythroid progenitors. Several days after the addition of erythropoietin, the cultures undergo erythroid differentiation in a synchronized fashion. The purpose of the present study was to determine detailed kinetics of globin gene expression at the mRNA level in adult and newborn erythroid cells. Our results show that in cultures derived from normal adult peripheral blood, the mRNA levels of alpha- and beta-globin genes increased throughout most of the culture period, whereas gamma-globin mRNA remained at a low level. In contrast, high expression of all three globin genes, alpha, beta, and gamma, was observed in cultures derived from cord blood. The results demonstrate that the populations of erythroid progenitors in cord blood and in adult peripheral blood are fundamentally different, suggesting that this culture system recapitulates the normal pattern of globin gene expression, providing a valuable tool in the investigation of the regulation of the switch from fetal to adult hemoglobin.
