Alterations of globin-gene cluster expression mediated through cytokine signal transduction have been previously established. In adult erythroblasts, ex vivo combinatorial signaling from cytokines such as erythropoietin (EPO), stem cell factor (SCF), and transforming growth factor-beta (TGF-B) causes a robust reactivation of fetal hemoglobin. To determine if cytokine-activated expression of fetal hemoglobin significantly alters the transcript and protein abundance of nuclear transcription factors, profiling studies of transcription factor expression were performed. Primary CD34+ cells were cultured as donor-matched pairs using cytokine combinations that produced low vs. high levels of gamma-globin mRNA and fetal hemoglobin (Bhanu et al. Blood, 2005). To identify candidate genes, total RNA from 15 separate healthy volunteer donors was collected and combined into 5 pools. The pooled RNA was labeled and hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips. Informatics strategies were aimed toward identifying differential expression of those transcription factors with binding motif on any of the 10 human globin gene promoter regions (553 transcription factors; 115 binding motif families), versus all transcription factors demonstrating robust expression with changes of at least 3-fold. Combining both strategies, 11 candidate transcription factors were identified (BCL11A, CBFB, EGR-1, ELK-1, HHEX, ID2, MAFF, MNDA, SOX-6, TCF3, and THRB). RNA-based descriptions of gene activity do not necessarily reflect changes in protein levels; therefore, Western analyses were performed. GATA-1 was utilized as a control, since no significant change in GATA-1 mRNA was detected by expression array profiling. Nuclear protein extracts were obtained from 3 additional donors’ CD34+ cells cultured under conditions of low vs. high gamma-globin mRNA synthesis (1.6E+06 ± 3.8E+05 and 1.5E+07 ± 6.3E+06 copies/ng total RNA, respectively). Western blot analysis revealed reproducible and robust differences in protein expression levels for 7 of the 11 candidate transcription factors (EGR-1, ELK-1, HHEX, ID2, MAFF, MNDA, and SOX-6). Two other candidates were expressed below the protein detection limit. GATA-1 and the remaining 2 candidates demonstrated no change in the nuclear protein levels. Among the group with confirmed changes in gene expression, ELK-1 and EGR-1 are downstream targets of the MAP kinase signal transduction cascade. MAFF, ID2, and SOX-6 are known regulators of globin gene expression. The differential expression of HHEX and MNDA warrants further investigation. These data demonstrate that cytokine signal transduction causes changes in the intranuclear levels of at least 7 transcription factors concurrently with activation of gamma-globin gene expression.

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