TFII-I/GTF2I regulates globin gene expression and stress response in erythroid cells

Figure 1. Generation and cell cycle analysis of TFII-I deficient TF-1 cells.A, TF-1 cell clones harboring the control pLKO.1 vector or expressing shRNA targeted to the TFII-I mRNA (shRNA 1–5) were subjected to RT-qPCR with primers specific for TFII-I and GAPDH. TFII-I expression was normalized to GAPDH expression. B, Western blot analysis of TF-1 cell clones harboring the control pLKO.1 vector or expressing shRNA targeted to the TFII-I mRNA (shRNA 1–5). C, proliferation of TF-1 cells harboring the control pLKO.1 vector or expressing TFII-I shRNA-2 or -3. Life cells were counted every 48 h for 10 days. D, analysis of cell cycle distribution of TF-1 cells harboring the control pLKO.1 vector or expressing TFII-I shRNA-2 or -3. Cells were subjected to flow cytometry with APC-conjugated anti-BrdU and DAPI staining. Results are presented as the mean ± SD of three independent experiments (∗: p < 0.05, two-tailed Students t test).

Figure 2. TF-1 cell colony formation in TFII-I deficient cells.A, number of colonies per plate of TF-1 cells harboring the control pLKO.1 vector or vector expressing TFII-I shRNA-2 or -3. B, average cells per colony harboring the control pLKO.1 vector or vector expressing TFII-I shRNA-2 or -3. For A and B, results are represented as the mean + SD of three independent experiments (∗: p < 0.05; ∗∗: p < 0.009, two-tailed Students t test). C, representative images of colony formation of TF-1 cells harboring the control pLKO.1 vector or expressing TFII-I shRNA-2 or -3.

Figure 3. Gene expression analysis in control and TFII-I deficient TF-1 cells.A and B, expression analysis of stress-response, erythroid-specific, and cell-cycle-specific gene in TF-1 cells harboring the control pLKO.1 vector or expressing TFII-I shRNA-2 or -3. Cells were subjected to RT-qPCR using primers specific for the genes listed on the x-axis; mRNA expression was normalized to that of GAPDH, and expression in empty vector (PLKO.1) harboring cells was set to 1 (∗∗∗: p < 0.0001; ∗∗∗: p < 0.001; ∗∗: p < 0.01; ∗: p < 0.05; two-tailed Students t test). C, Western blot analysis of Elongin A, TAF15, E2F6, CTCF, and β-actin in TF-1 cells harboring the pLKO.1 vector or expressing TFII-I shRNA-2 or-3. D, expression analysis of globin genes in TF-1 cells harboring the control pLKO.1 vector or expressing TFII-I shRNA-2. Cells were subjected to RT-qPCR using primers specific for the genes listed on the x-axis; mRNA expression was normalized to that of GAPDH, and expression in cells harboring the empty vector (PLKO.1) was set to 1. The error bars reflect the SEM from three independent experiments (∗∗∗∗: p < 0.001; ∗∗: p < 0.01; ns: nonsignificant, p > 0.05; two-tailed Students t test).

Figure 4. Erythroid differentiation and globin gene expression in control and TFII-I deficient differentiating TF-1 cells.A, percentages of CD235a+ cells in control and TFII-I knockdown cells at various time points of culture with EPO. The experiment has been repeated three times (n = 3) and the error bars reflect the standard error of the mean (∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.05; two-tailed Students t test). B, hemoglobinization of TF-1 cells harboring the control pLKO.1 vector or expressing TFII-I shRNA-2. Cells were pelleted and photographed after 8 days in culture with erythropoietin. C, relative globin gene expression during erythropoietin-induced erythroid differentiation of TF-1 cells harboring the control pLKO.1 vector or expressing TFII-I shRNA-2. Cells were subjected to RT-qPCR using primers specific for the genes listed on the x-axis; mRNA expression was normalized to that of GAPDH. The error bars reflect the SEM from three independent experiments (∗: p < 0.05, ∗∗: p < 0.001, ∗∗∗: p < 0.0003, ∗∗∗∗: p < 0.0001; two-tailed Students t test).

Figure 5. Gene expression and apoptosis analysis in control and TFII-I deficient TF-1 cells during response to hypoxia as well as ER and oxidative stress.A–C, cells were treated for 8 h with 100 nM thapsigargin. Control groups were treated with DMSO. After 8 h, cells were collected for analysis of mRNA expression, western blotting, and apoptosis. A, gene expression analysis of TF-1 cells harboring the control pLKO.1 vector or expressing TFII-I shRNA-2 or -3. Cells were subjected to RT-qPCR using primers specific for the genes listed on the x-axis; mRNA expression was normalized to that of GAPDH. The experiments have been repeated three times (n = 3) and the error bars reflect the standard error of the mean (∗∗∗∗: p < 0.0001; ∗∗∗: p < 0.001; ∗∗: p < 0.01; two-tailed Students t test). B, Western blot analysis for GRP78, Elongin A and Actin in TF-1 cells harboring the pLKO.1 vector or expressing TFII-I shRNA-2 or -3 in control (C) and thapsigargin treated cells (Th). C, analysis of apoptotic frequency of TF-1 cells harboring the control pLKO.1 vector or expressing TFII-I shRNA-2 or -3 (∗∗: p < 0.01; two-tailed Students t test). D–F, cells were treated with DFO at a final concentration of 100…

Figure 6. Expression of the ATF4 and CHOP genes and interactions with TFII-I in response to hypoxia and/or ER and oxidative stress. TIF-1 cells were subjected to cellular stress with thapsigargin, DFO, or H2O2, as indicated. A, expression analysis of ATF4 and CHOP in control and stress-induced cells. The mRNA was isolated, converted to cDNA, and subjected to qPCR using primers specific for the ATF4 or CHOP genes, as indicated. The data were normalized to GAPDH and the error bars reflect the standard error of the mean of three independent experiments (∗∗∗∗: p < 0.0001; ∗∗∗: p < 0.001; two-tailed Students t test). B, interactions of TFII-I with the ATF4, CHOP, and necdin promoter, in control and stress-induced cells. Control or stress-induced TF-1 cells were crosslinked and subjected to ChIP and qPCR using primers specific for the ATF4, CHOP, or necdin promoter, as indicated. The data are shown as % input and the error bars reflect the mean of three independent experiments (∗∗: p < 0.01; ∗: p < 0.05; two-tailed Students t test).