KLF1 knock-down for HbF high-expression discovered by RNA-Seq profiles under human erythropoiesis

Krueppel-like factor 1 (KLF1) is a major transcription factor, which regulates the γ-globin to β-globin gene switch during erythropoiesis. The goal of this study was to investigate genome-wide transcriptome expression pattern in the presence of KLF1 knockdown to discover targets of KLF1 regulation involved in γ-globin silencing. To achieve KLF1 gene silencing, we stably transfected human primary erythroid cells generated from CD34+ cells, with a short hairpin lentiviral vector carrying a shKLF1 (short hairpin RNA for KLF1 loop) and a puromycin resistance gene. A set of three shKLF1 were designed uusing siDesign Center (Dharmacon) with the designations: 1) shKLF1-1, located in zinc finger at 3'-terminal; 1) shKLF1-2 located in Transcription Factor II H, and 3) shKLF1-3 located in 5’-terminal without a function region. After lentiviral packaging and transduction into CD34+ cells, each expression virus with encoding GFP, puromycin resistance fragment, and a loop consists of the KLF1 knock-down domain. The three shKLF1 vectors (shKLF1-1, shKLF1-2, shKLF1-3), along with scrambled control (shScr) was used to transduce human adult CD34+ stem cells isolated from peripheral blood of healthy adults. The cells were grown in one-phase liquid culture system as previously published by our group (Li B et al. Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation. BMC Genomics. 2012; 13:153). At day-15 in the 28-day culture period, by real-time PCR and Western blot analysis we found KLF1 gene silencing mainly for shKLF-1 and shKLF-2; however, we isolated total RNA from the three stable lines for RNA-seq analysis. Twelve RNA samples including shKLF1-1, shKLF1-2, shKLF1-3 and shScr control, each in triple repeats, were harvested on day-15 with KLF1 gene silencing occurs and we previously confirmed highest γ-globin and lower β-globin expression. Total RNA was extracted with Trizol solution according to the manufacturer’s instructions. After dissolving in 30 μl of RNase free water, the RNA concentration (A260 nm) and purity (A260/280 and A260/230 ratios) were assessed with a ND-1000 spectrophotometer (Thermo Scientific, Wilmington DE); quality measurement was assayed by the Georgia Cancer Center Genomics Core facility. Total RNA was used to construct sequencing libraries using 1µg of RNA; analysis was performed by Agilent TapeStation 2200 High-Seq 2500 technology, in triple for each stable line.