Induction of fetal hemoglobin (HbF) is considered a new promising strategy to treat -thalassemia, where the production of adult hemoglobin (HbA) is impaired by mutations affecting the -globin gene. Recent results suggest that BCL11A-XL levels are associated with -globin gene expression. Therefore, disrupting the bindings of the BCL11A-XL transcriptional complex to the -globin gene promoter provides a novel approach for inducing expression of the -globin genes. However, few information is available on the roles, if any, of BCL11A-XL on other erythroid genes. In order to verify this we have developed K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We have characterized twelve K562 clones expressing different levels of BCL11A-XL and found that a clear relationship does exist between the levels of BCL11A-XL and the extent of differentiation. Using mithramycin as inducer, we found that MTH was unable to induce differentiation in K562 cell clones expressing high levels of BCL11A-XL. By sharp contrast BCL11A-XL was able to induce the increase of -globin mRNA in K562 clones expressing intermediate levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by MTH. Interestingly, a site recognized by MTH is present in the -globin gene promoter and is specific for the binding of the BCL11A-complex. In addition, 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
Development and characterization of K562 cell clones expressing at high levels BCL11A: induction of erythroid differentiation by mithramycin
FINOTTI, Alessia;BREVEGLIERI, Giulia;COSENZA, Lucia Carmela;BIANCHI, Nicoletta;BORGATTI, Monica;GAMBARI, Roberto
2013
Abstract
Induction of fetal hemoglobin (HbF) is considered a new promising strategy to treat -thalassemia, where the production of adult hemoglobin (HbA) is impaired by mutations affecting the -globin gene. Recent results suggest that BCL11A-XL levels are associated with -globin gene expression. Therefore, disrupting the bindings of the BCL11A-XL transcriptional complex to the -globin gene promoter provides a novel approach for inducing expression of the -globin genes. However, few information is available on the roles, if any, of BCL11A-XL on other erythroid genes. In order to verify this we have developed K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We have characterized twelve K562 clones expressing different levels of BCL11A-XL and found that a clear relationship does exist between the levels of BCL11A-XL and the extent of differentiation. Using mithramycin as inducer, we found that MTH was unable to induce differentiation in K562 cell clones expressing high levels of BCL11A-XL. By sharp contrast BCL11A-XL was able to induce the increase of -globin mRNA in K562 clones expressing intermediate levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by MTH. Interestingly, a site recognized by MTH is present in the -globin gene promoter and is specific for the binding of the BCL11A-complex. In addition, 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 geneI documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
