Spontaneous switch from Agamma- to beta-globin promoter activity in a stable transfected dual reporter vector.

Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for (A)gamma- and beta-globin gene, each one linked to a different Luciferase, in stably transfected GM979 cells for as long as 1-2 years from transfection. The transfected GM979 cells rapidly (within 1 month) evolved into a stable population which expresses constant levels of reporters for more than a year of continuous bulk culture. No silencing of the inserted construct was observed over time. In contrast, after 1 month, the reporter activity (both from (A)gamma- and beta-promoter) expressed per cell increased over time. The analysis of the Luciferase contained in single cell clones indicated that the higher reporter activity was due to increased gene expression per cell rather than to clonal selection of the most expressing clones. Since the activity driven by the beta-promoter increased 10-fold more than that driven by the (A)gamma one, the ratio between (A)gamma-driven/((A)gamma-driven + beta-driven) reporter activity in the cells decreased after 1 month and became similar to the gamma/(gamma + beta) globin mRNA ratio expressed by adult erythroid cells. Moreover, although both cells from early and late bulk culture responded to incubation with butyric acid, a known inducer of fetal globin gene expression, by increasing the reporter activity driven by the (A)gamma-promoter, only cells from late bulk culture decreased, as normal primary erythroblasts do, the activity of the reporter driven by the beta-promoter. These results suggest that the rapid changes in activity driven by the (A)gamma- and beta-globin promoters occurring during the first month after transfection may represent a novel in vitro model to study epigenetic regulation of the (A)gamma- and beta-promoter during the fetal to adult hemoglobin switch and confirm GM979 cells stably transfected with the dual reporter construct as a reliable assay for automated screening of chemical inducers of fetal globin gene activation.

5-azacytidine reactivates the erythroid differentiation potential of the myeloid-restricted murine cell line 32D Ro.

32D cells grown for 1 year in interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) generated the 32D Ro cell line which retained the parental mast cell phenotype but lost ability to generate erythroid cells in response to erythropoietin (EPO). In order to clarify the mechanisms underlying such restriction, we compared 32D and 32D Ro cells for their capacity to express erythroid-specific transcription factors (Gata1, Gata2, Scl, Nef2, Eklf, and Id) and the capacity of short exposure to 5-azacytidine (5-AzaC) to reactivate erythroid differentiation potential in 32D Ro cells. By Northern analysis, the two cell lines expressed similar levels of all these genes. However, after being treated with 5-AzaC, 32D Ro cells acquired the ability to generate EPO-dependent clones (1 clone/10(4) cells) which gave rise to EPO-dependent cell lines. All the 10 EPO-responsive cell lines independently isolated from 5-AzaC-treated 32D Ro cells had erythroid morphology and expressed high levels of alpha- and beta-globin. In contrast, none of the IL-3-dependent and granulocyte/macrophage colony-stimulating factor-dependent clones concurrently isolated, as a control, showed erythroid properties. Therefore, 5-AzaC treatment reactivates the potential of the myeloid-restricted 32D Ro cells to generate EPO-responsive erythroid clones suggesting that gene methylation played an important role in the G-CSF-mediated restriction/activation of the differentiation potential of these cells.