Gene Expression Profiles during the γ- to β-Globin Switch in Erythroid Progenitors.

The design and evaluation of therapies for sickle cell disease rely on our understanding of the sequential expression of the β-like globin genes during development. Drug inducers that reactivate γ-globin transcription after birth ameliorate symptoms in sickle cell patients. Therefore, the objective of our study is to identify trans-factors involved in γ-gene activation during the γ- to β-globin switch recapitulated in erythropoiesis. We previously established the one-phase liquid culture system using buffy coat mononuclear cells grown in erythropoietin (Epo; 4U/mL), stem cell factor (50ng/ml) and Interleukin-3 (IL-3; 10ng/ml). We observed high γ-globin transcription on day 7, equal γ- and β-globin mRNA levels by day 20 followed by high β-globin expression on day 28. Cell surface markers were measured by quantitative PCR (qPCR) and correlated with globin gene expression. During lineage commitment and erythroid maturation CD34⁺ cells decreased while the erythroid markers CD71, glycophorin A and Epo receptor increased 12 to 144-fold, followed by a decrease on day 28. We concluded that the γ- to β-globin switch was recapitulated in our system. Subsequent studies were performed with microarray and gene profiling by principle component analysis (PCA) to characterize proteins involved in switching. The Discover Chip from ArrayIt (Sunnyvale, CA) containing 380 human genes was used. cDNA from day 0/7, 7/21 and 21/28 were labeled with Cy3 and Cy5 respectively and used for standard chip hybridization. Fluorescence data was acquired using the Axon scanner and GenePixPro 6.0 software. By day 7, 88 genes were silenced and 68 activated; at day 28 an additional 38 genes were silenced and 47 activated. PCA defined the four major gene profiles consistent with developmental globin gene regulation. Profile 1 genes including IL-3, IL-9, heat shock protein 1a and neurofibromin were activated greater than 2-fold on day 7 suggesting positive regulators of γ-globin; trans-activators were not identified. Profile 2 genes were highly expressed around the switch on day 20. GATA-1, guanine nucleotide binding protein, ZF protein 132 and ERK1 expression peaked during this time. This data is consistent with a γ-globin silencer role previously demonstrated for ERK. Profile 3 genes also peaked on day 20 but continued to be highly expressed during β-globin transcription; EKLF, IL-8, Ras, Fos, and histone H3 were identified in this group. Experimental data support EKLF binding in the locus control region to compete with γ-globin expression. Finally Profile 4 genes increased steadily and peaked on day 28 consistent with positive β-globin regulators including NF-E2, ATF-2, IL-12, and thymosin β10. The microarray data were confirmed by qPCR in 90% of the genes tested. Our findings support the one-phase erythroid liquid culture system as a model for globin switching. We will expand gene profiling efforts using the Agilent 44K chip and PCA to confirm this model.