Mi2β regulates the expression of the γ-globin gene in human primary erythroid cells. CD34⁺ human hematopoietic progenitor cells were infected with lentivirus vectors harboring shRNA either for scramble control, 2 different Mi2β constructs, or MBD2. (A) Knockdown with shMi2β 1 leads to a 7.2-fold induction of γ-globin gene expression determined by qPCR. Knockdown with shMi2β 2 leads to a ∼20-fold increase in γ-globin expression and a slight decrease in β-globin gene expression. Shown below the graph are RNA levels in cells infected with scramble control or knockdown shRNA vectors. (B) Partial knockdown of Mi2β (construct 2) leads to a 20-fold increase in γ/γ+β-globin gene expression. (C) Knockdown of MBD2 leads to a ninefold increase in expression of γ/γ+β-globin gene expression. (D) Fluorescence activated cell sorting analysis showing erythroid differentiation of 81.1% of CD34⁺ progenitor cells in which Mi2β is knocked down compared with 93.8% of scramble shRNA control cells and 92.9% of cells in which MBD2 is knocked down. Values signify standard deviations for ≥3 independent experiments. (E) qPCR analysis showing a ∼40% knockdown level of Mi2β RNA in double-positive cells taken at the end of differentiation (quadrant 2). (F) qPCR analysis showing a 90% knockdown of Mi2β RNA in double-negative cells taken at the end of differentiation (quadrant 3). (G-I) Wright-Giemsa stain of scramble control, Mi2β knockdown, and MBD2 knockdown cell populations. Photomicrographs were generated using an Olympus (Center Valley, PA) BX41 compound microscope and Olympus DP71 digital camera at ×100 magnification. Images were acquired with Olympus DP Controller software. Error bars represent the standard deviation of ≥3 independent experiments. *P < .05 and **P < .02 according to the Student t test.