FIGURE 3.
Calpeptin increases the levels of USF and stimulates high level globin gene expression in MEL cells. A, Western blot analysis of USF1, USF2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in MEL cells incubated with 30 μm calpeptin for 0, 3, 5, or 7 days. B, reverse transcription-PCR analysis of USF1 mRNA levels in MEL cells treated with 0.03% DMSO only or with 30 μm calpeptin, 0.03% DMSO for 0, 3, 5, and 7 days. C, reverse transcription-PCR analysis of adult βmaj-globin and α-globin mRNA levels in MEL cells incubated for 7 days with 0.04% DMSO (DMSO 7d) or with 40 μm calpeptin, 0.04% DMSO (calpeptin 7d). RNA was isolated from the cells, reverse transcribed, and analyzed by real time PCR. Globin mRNA levels in untreated control cells (MEL) were set at 1, and -fold changes were calculated with mRNA levels of the β-actin gene serving as an internal control. D, benzidine staining of hemoglobin in MEL cells grown for 7 days in the absence (MEL), in the presence of 0.04% DMSO (DMSO), or in the presence of 40 μm calpeptin, 0.04% DMSO (Calpeptin). E, relative number of benzidine-positive MEL cells in cultures incubated with DMSO only (0.03 or 0.04%) or incubated with 30 μm calpeptin, 0.03%DMSO or 40 μm calpeptin, 0.04% DMSO for 0, 3, 5, and 7 days. F, ChIP analysis of the association of USF2 and Pol II with the βmaj-globin gene promoter in MEL cells incubated for 7 days in the absence (MEL), in the presence of 0.04% DMSO (DMSO 7d), or in the presence of 40 μm calpeptin, 0.04% DMSO (calpeptin 7d). Cells were cross-linked with formaldehyde. Chromatin was isolated, fragmented, and precipitated with antibodies against USF2 and Pol II or the unspecific IgG antibody. DNA was purified from the precipitate and analyzed by quantitative real time PCR using primers specific for the adult βmaj-globin gene promoter. Bars represent the relative enrichment over the input DNA. Error bars represent S.D. from two independent experiments. d, days.