TY - JOUR
T1 - Spontaneous switch from Aγ- to β-globin promoter activity in a stable transfected dual reporter vector
AU - Migliaccio, Giovanni
AU - Di Baldassarre, Angela
AU - Di Rico, Cristina
AU - Di Noia, Antonella
AU - Nakamoto, Betty
AU - Cao, Hua
AU - Skarpidi, Eva
AU - Migliaccio, Anna Rita
N1 - Funding Information:
The authors gratefully acknowledge Prof. Francesco Antonio Manzoli for continuous support. This study was supported by, Progetti di ricerca di Interesse Nazionale 2001 and 2002 from the Ministry of Health, MIUR 60% Grant 2002, Grant E.1172 from Telethon Foundation, Progetti FIRB 2002 and 2003, National Project on Stem Cells, Institutional Funds from Istituto Superiore di Sanità, and grant NIH-HL20899.
PY - 2005
Y1 - 2005
N2 - Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for Aγ- and β-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 Aγ- and β-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 β-promoter increased 10-fold more than that driven by the Aγ one, the ratio between Aγ-driven/ (Aγ-driven + β-driven) reporter activity in the cells decreased after 1 month and became similar to the γ/(γ + β) 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 Aγ-promoter, only cells from late bulk culture decreased, as normal primary erythroblasts do, the activity of the reporter driven by the β-promoter. These results suggest that the rapid changes in activity driven by the Aγ- and β-globin promoters occurring during the first month after transfection may represent a novel in vitro model to study epigenetic regulation of the Aγ- and β-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.
AB - Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for Aγ- and β-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 Aγ- and β-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 β-promoter increased 10-fold more than that driven by the Aγ one, the ratio between Aγ-driven/ (Aγ-driven + β-driven) reporter activity in the cells decreased after 1 month and became similar to the γ/(γ + β) 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 Aγ-promoter, only cells from late bulk culture decreased, as normal primary erythroblasts do, the activity of the reporter driven by the β-promoter. These results suggest that the rapid changes in activity driven by the Aγ- and β-globin promoters occurring during the first month after transfection may represent a novel in vitro model to study epigenetic regulation of the Aγ- and β-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.
KW - Dual Luciferase reporter assay
KW - Epigenetic regulation
KW - GM979 cell line
KW - Hb switch
UR - http://www.scopus.com/inward/record.url?scp=13844270534&partnerID=8YFLogxK
U2 - 10.1016/j.bcmd.2004.11.005
DO - 10.1016/j.bcmd.2004.11.005
M3 - Article
C2 - 15727902
AN - SCOPUS:13844270534
SN - 1079-9796
VL - 34
SP - 174
EP - 180
JO - Blood Cells, Molecules, and Diseases
JF - Blood Cells, Molecules, and Diseases
IS - 2
ER -