During Terminal Differentiation, HbF-Inducing Cytokines Cause Decreased Expression and Reduced Globin Locus Occupancy of BCL11A in Human Erythroblasts.

Abstract 457

Genetic association studies and gene regulation studies demonstrate that the transcription factor BCL11A is a regulator of fetal hemoglobin (HbF) expression in humans. Cytokine signal transduction also regulates fetal hemoglobin expression in cultured adult human erythroblasts. To further explore the potential for BCL11A in the cytokine-mediated induction of HbF during adult erythropoiesis, transcript and protein expression levels of BCL11A were measured during erythroblast differentiation. BCL11A expression was detected at all stages of erythroid differentiation with the highest level expression in proerythroblasts during the first week in culture under both low-HbF (%HbF ≤3) and high-HbF (%HbF ≥30) culture conditions. Despite a reduction in BCL11A mRNA expression, Western analyses failed to demonstrate reduced levels of BCL11A nuclear protein expression at the proerythroblast stage of differentiation. However, BCL11A protein expression in the high-HbF producing cells was reduced relative to the low-HbF cells during the later period of culture as the cells underwent terminal differentiation. During this later culture period, hemoglobinization occurred, and cells grown in the high-HbF condition revealed a pancellular distribution of HbF compared with a heterocellular distribution in the low-HbF culture condition. Chromatin immunoprecipitation further demonstrated that the addition of HbF-inducing cytokines caused a nearly complete loss of BCL11A chromatin occupancy within the beta-globin locus under the high-HbF culture condition. Specifically, the loss of chromatin occupancy was detected in a region approximately 3 kb downstream of the (A)gamma-globin gene. Further examination of this genomic region demonstrated several BCL11A binding domains located on a cluster of non-coding, intronless RNAs previously named “BGL3” that possess an expression pattern in vivo that is largely restricted to the fetal-liver. In addition to increased and pancellular expression of fetal hemoglobin in the high-HbF erythroblasts, the loss of BCL11A chromatin occupancy in that region of the beta-globin locus was associated with increased expression of BGL3 mRNA (GenBank: AY034471) measured by RT-PCR. These findings demonstrate that defined combinations of cytokines regulate the expression level and chromatin occupancy of BCL11A in adult human erythroblasts as they undergo terminal differentiation. In addition to inheritance and ontogeny, the data also support a role for BCL11A in the regulation of HbF by cytokine signal transduction.