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Objective To study the function of ten-eleven translocation 2 (Tet2) in γglobin gene expression in patients with β-thalassemia.Methods Gamma globin expression was induced by 5-azacytidine and Tet2 gene expression was knocked down by short hairpin RNA (shRNA) in a human immortalized myelogenous leukemia K562 cell line.The global 5-hydroxymethylcytosine (5hmC) level was measured by an ELISA kit.5hmC level of γglobin gene was quantified by sulfite sequencing.The mRNA level of Tet2,γglobin,and related transcription factors Nfe4 and Klfl were quantified by real-time PCR.Results Tet2 knockdown resulted in a decreased global 5hmC level from 0.14％ to 0.03％ as of the control group in K562 cells.The expression of γ globin was enhanced after 5-azacytidine treatment in vitro.However,γglobin mRNA level in Tet2 knockdown cells was only 55％ as that in control group.The CG sites on γ globin gene were unmethylated.As Tet2 was down-regulated,the expression levels of Nfe4 and Klf1 decreased by about 80％ and increased to 3.5 folds,respectively.Conclusions Tet2 appears to maintain 5hmC level and facilitates γ globin gene activation.Moreover,Tet2 more likely regulates γglobin expression via affecting transcription factors rather than the gene itself.Thus,Tet2 could be a potential therapeutic target for β thalassemias.
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MicroRNAs (miRs) play an important role in regulating diverse cellular processes.It has been reported that miRs are associated with the formation and maturation of erythrocytes, and the expression of globin genes at post-transcriptional level.Compared with normal human enrythrocytes, various miRs are altered in the patients with thalassemia.These changes also happen in the patients with diverse clinical manifestations.In this paper, we systematically summarized the recent progress about the expression dysregulation of miRs in β-thalassemia and their roles in regulating the levels of γ-globin and fetal hemoglobin.During β-like globin gene expression, miRs directly or indirectly regulate the levels of erythroid-specific transcription factors through post-transcriptional action, such as B-cell lymphoma 11A (BCL11A), myeloblastosis oncogene (MYB), specificity protein 1 (Sp1), Kruppel-like factor 3 (KLF3) and GATA1.These effects subsequently regulate the switch between γ-and β-globin gene expression and affect fetal hemoglobin production.Targeting miRs might be a novel therapeutic strategy for β-thalassmeia.
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OBJECTIVE: To investigate the mechanisms of sirolimus inducing γ-globin gene expression in K562 cells. METHODS: K562 cells were cultured in the presence of 10 nmol · L-1 sirolimus, butylate, or DMSO for 3 d. Western blot and real time PCR-based chromatin immunoprecipitation was employed to measure the levels of p38MAPK and acetylated histone H3 (acH3) at γ-globin gene promoter regions, respectively. RESULTS: In K562 cells with 10 nmol · L-1 sirolimus treatment, phospholylated p38MAPK (p-p38MAPK) was 2.8-fold greater and acH3 was 9.8-fold greater than that in untreated K562 cells, and there was a 2.9-fold in p-p38MAPK and a 9.1-fold in acH3 increase comparing with K562 cells treated with DMSO, no significant difference in p-p38MAPK and acH3 level was found between cells treated with sirolimus and with butylate. SB203580 completely abolished induction of p38MAPK activation and acH3 increase by sirolimus. CONCLUSION: Our results indicate that sirolimus actives p38MAPK signal and increases acetylation of H3 at γ-globin gene promoter regions, which may be the mechanisms of induced expression of γ-globin genes by sirolimus in K562 cells.