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Variation in Gamma-Globin Expression before and after Induction with Hydroxyurea Associated with BCL11A, KLF1 and TAL1.

ABSTRACT: The molecular mechanisms governing γ-globin expression in a subset of fetal hemoglobin (α2γ2: HbF) expressing red blood cells (F-cells) and the mechanisms underlying the variability of response to hydroxyurea induced γ-globin expression in the treatment of sickle cell disease are not completely understood. Here we analyzed intra-person clonal populations of basophilic erythroblasts (baso-Es) derived from bone marrow common myeloid progenitors in serum free cultures and report the level of fetal hemoglobin production in F-cells negatively correlates with expression of BCL11A, KLF1 and TAL1. We then examined the effects of hydroxyurea on these three transcription factors and conclude that a successful induction of γ-globin includes a reduction in BCL11A, KLF1 and TAL1 expression. These data suggests that expression changes in this transcription factor network modulate γ-globin expression in F-cells during steady state erythropoiesis and after induction with hydroxyurea.

SUBMITTER: Grieco AJ

PROVIDER: S-EPMC4459969 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe major therapeutic benefit of hydroxyurea, the only FDA-approved pharmacologic treatment for sickle cell disease (SCD), is directly related to fetal hemoglobin (HbF) production that leads to significant reduction of morbidity and mortality. However, potential adverse effects such as infertility, susceptibility to infections, or teratogenic effect have been subject of concerns. Therefore, understanding HU molecular mechanisms of action, could lead to alternative therapeutic agents to increase HbF with less toxicity. This paper investigated whether HU-induced HbF could operate through post-transcriptional miRNAs regulation of BCL11A, KLF-1 and MYB, potent negative regulators of HbF. Both ex vivo differentiated primary erythroid cells from seven unrelated individuals, and K562 cells were treated with hydroxyurea (100 μM) and changes in BCL11A, KLF-1, GATA-1, MYB, β- and γ-globin gene expression were investigated. To explore potential mechanisms of post-transcriptional regulation, changes in expression of seven targeted miRNAs, previously associated with basal γ-globin expression were examined using miScript primer assays. In addition, K562 cells were transfected with miScript miRNA inhibitors/anti-miRNAs followed by Western Blot analysis to assess the effect on HbF protein levels. Direct interaction between miRNAs and the MYB 3'-untranslated region (UTR) was also investigated by a dual-luciferase reporter assays.ResultsDown-regulation of BCL11A and MYB was associated with a sevenfold increase in γ-globin expression in both primary and K562 cells (p < 0.003). Similarly, KLF-1 was down-regulated in both cell models, corresponding to the repressed expression of BCL11A and β-globin gene (p < 0.04). HU induced differential expression of all miRNAs in both cell models, particularly miR-15a, miR-16, miR-26b and miR-151-3p. An HU-induced miRNAs-mediated mechanism of HbF regulation was illustrated with the inhibition of miR-26b and -151-3p resulting in reduced HbF protein levels. There was direct interaction between miR-26b with the MYB 3'-untranslated region (UTR).ConclusionsThese experiments have shown the association between critical regulators of γ-globin expression (MYB, BCL11A and KLF-1) and specific miRNAs; in response to HU, and demonstrated a mechanism of HbF production through HU-induced miRNAs inhibition of MYB. The role of miRNAs-mediated post-transcriptional regulation of HbF provides potential targets for new treatments of SCD that may minimize alterations to the cellular transcriptome.

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Variation in Gamma-Globin Expression before and after Induction with Hydroxyurea Associated with BCL11A, KLF1 and TAL1.

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