Page 65 - Haematologica - Vol. 105 n. 6 - June 2020

DKC1 drives upregulation of telomerase in normal human erythroblasts
in transfected HEL 92.1.7 erythroleukemia cells. The results demonstrated that both GATA sites contributed to promoter activity, and that concurrent ablation of the two GATA sites substantially diminished promoter activity (Figure 5D). Collectively the results provide strong evi- dence that the erythroid-restricted transcription factor GATA1 contributes to the regulation of DKC1.
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Discussion
This report is the first to implicate GATA1 in the regu- lation of telomerase and to describe a biological context in which telomerase activity is upregulated by induction of DKC1. Here it is shown that GATA1 binds the DKC1 pro- motor and contributes to the upregulation of DKC1,
CD
EF
G
Figure 4. Downregulation of DKC1 expression suppresses telomerase activity and inhibits erythroid cell proliferation but not differentiation. Cord blood-derived CD34+ hematopoietic stem and progenitor cells were transduced with retroviral vectors encoding DKC1-targeted shRNA (D2 and D3) or non-silencing RNA (NS) and green fluorescent protein (GFP). GFP+ cells were sprted by fluorescent activated cell sorting (FACS) (week 1), then cultured with stem cell factor plus erythropoietin (SE) for a further 2 weeks. Expression of telomerase genes and telomerase enzyme activity were measured by quantitative real-time polymerase chain reaction and the qTRAP assay, respectively. (A) DKC1 mRNA, (B) telomerase enzyme activity, (C) TERT mRNA and (D) TERC abundance. (E) Expansion of FACS-sorted GFP+ cells determined from cell counts using typan blue exclusion of dead cells. (F) Percentage of glycophorin A-positive cells in cultures determined by weekly FACS analysis. (G) Erythroid progenitors in the GFP+ sorted fraction were quantified as burst-forming units-erythroid in methylcellulose assays. Values are means ± standard error of mean from six independent experiments. *P<0.05, **P<0.01, ****P<0.0001 from the Dunnett multiple comparison of D2 and D3 to NS. AU: arbitrary units; BFU-E: burst-forming unit-erythroid; GLYA+: glycophorin A-positive cells.
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