Fetal hemoglobin (HbF) induction is considered a new promising strategy for treatment of -thalassemia. Recent results suggest that BCL11A levels are inversely associated with -globin gene expression. Therefore, downregulation of BCL11A expression and/or disrupting the bindings of the BCL11A transcriptional complex to the -globin gene promoter provides a novel approach for inducing expression of the -globin genes. In the present study we investigated the effects of the HbF inducer mithramycin (MTH) on BCL11A in erythroid precursor cells (ErPC) from -thalassemia patients. The results obtained suggest that increase of -globin gene expression and HbF production is preceded by a sharp decrease of BCL11A levels in -thalassemia ErPC treated with 20 nM MTH. We demonstrated that the GC-binder MTH inhibit the binding of SP1/KLF1 activating transcription factors to BCL11A promoter causing the decrease of BCL11A transcription. Interestingly, a site recognized by MTH is present also in the -globin gene promoter at the level of the binding site of the BCL11A-complex. To investigate this aspect we have developed and characterized K562 cell clones with integrated copies of a BCL11A-XL expressing vector, producing different levels of BCL11A. EMSA experiments demonstrated that MTH interferes with the molecular interactions between the -globin gene promoter and the protein complex containing BCL11A, strongly suggesting an activity of MTH in interfering between BCL11A containing nuclear protein complexes and regulatory regions of the -globin gene. In addition, we found that a clear relationship does exist between the levels of BCL11A and the extent of differentiation. MTH was unable to induce differentiation in K562 cell clones expressing high levels of BCL11A-XL, but induced -globin mRNA in K562 clones expressing intermediate levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by MTH. In conclusion, mithramycin mediated increase of HbF and -globin gene expression is due to inhibition of molecular interactions between the -globin gene promoter and the protein complex containing BCL11A, together with downregulation of BCL11A gene transcription.
Double effects of mithramycin during induction of fetal hemoglobin: down regulation of BCL11A gene transcription and inhibition of the binding of the BCL11A transcriptional complex to the -globin gene promoter
FINOTTI, Alessia;BIANCHI, Nicoletta;ZUCCATO, Cristina;LAMPRONTI, Ilaria;BREVEGLIERI, Giulia;FABBRI, Enrica;BROGNARA, Eleonora;BORGATTI, Monica;GAMBARI, Roberto
2013
Abstract
Fetal hemoglobin (HbF) induction is considered a new promising strategy for treatment of -thalassemia. Recent results suggest that BCL11A levels are inversely associated with -globin gene expression. Therefore, downregulation of BCL11A expression and/or disrupting the bindings of the BCL11A transcriptional complex to the -globin gene promoter provides a novel approach for inducing expression of the -globin genes. In the present study we investigated the effects of the HbF inducer mithramycin (MTH) on BCL11A in erythroid precursor cells (ErPC) from -thalassemia patients. The results obtained suggest that increase of -globin gene expression and HbF production is preceded by a sharp decrease of BCL11A levels in -thalassemia ErPC treated with 20 nM MTH. We demonstrated that the GC-binder MTH inhibit the binding of SP1/KLF1 activating transcription factors to BCL11A promoter causing the decrease of BCL11A transcription. Interestingly, a site recognized by MTH is present also in the -globin gene promoter at the level of the binding site of the BCL11A-complex. To investigate this aspect we have developed and characterized K562 cell clones with integrated copies of a BCL11A-XL expressing vector, producing different levels of BCL11A. EMSA experiments demonstrated that MTH interferes with the molecular interactions between the -globin gene promoter and the protein complex containing BCL11A, strongly suggesting an activity of MTH in interfering between BCL11A containing nuclear protein complexes and regulatory regions of the -globin gene. In addition, we found that a clear relationship does exist between the levels of BCL11A and the extent of differentiation. MTH was unable to induce differentiation in K562 cell clones expressing high levels of BCL11A-XL, but induced -globin mRNA in K562 clones expressing intermediate levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by MTH. In conclusion, mithramycin mediated increase of HbF and -globin gene expression is due to inhibition of molecular interactions between the -globin gene promoter and the protein complex containing BCL11A, together with downregulation of BCL11A gene transcription.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
