FIG. 2.
Repression of β-globin gene expression by TFII-I. (A) Diagram outlining the experimental system for expressing wild-type or dominant-negative proteins. Coding sequences for TFII-I, p70, USF1, and A-USF are under the control of the CMV enhancer/promoter. A Tet operator sequence interacts with the Tet repressor, for which an expression construct is cotransfected into these cells. Doxycycline interacts with the Tet repressor and relieves repression. TRE, tetracycline-responsive element. (B) Analysis of TFII-I function in K562 cells. The expression of dominant-negative mutant p70 was induced in stably transfected K562 cells by using 1 μg doxycycline per ml culture medium for 12 h. The expression of p70 was analyzed by Western blotting (left panel). RNA was isolated from transfected cell lines, reverse transcribed, and analyzed by quantitative PCR for expression of the β-globin gene. β-Globin gene expression is presented as the ratio of expression in pTO/p70 cells using GAPDH as the internal reference, relative to expression in cells transfected with the pTO vector, which was set at 1. +, doxycycline-treated cells; +/−, pTO+Dox cells served as control for doxycycline-treated cells, and pTO-Dox served as control for untreated cells. (C and D) Analysis of TFII-I function in MEL cells. The expression of wild-type TFII-I (C) or the dominant-negative mutant p70 (D) was induced in stably transfected MEL cells lines by using 1 μg doxycycline per ml culture medium for 36 h. The expression of TFII-I or p70 was analyzed by Western blotting and/or RT-PCR analysis as indicated. For RT-PCR analysis of TFII-I expression, RNA was isolated from induced MEL cells harboring expression constructs for TFII-I or the empty vector pTO and converted to cDNA. The cDNA was analyzed by PCR with forward primers corresponding to the 3′ region of the p70 and TFII-I-coding region and the reverse bovine growth hormone primer hybridizing to the pTO vector. For gene expression analysis, RNA was isolated from transfected cell lines, reverse transcribed, and analyzed by quantitative PCR for the expression of the βmaj-globin gene. βmaj-Globin expression is presented as the ratio of expression in pTO/p70 or pTO/TFII-I cells and expression in pTO cells using GAPDH as the internal reference. Doxycycline-treated pTO MEL cells were used as controls for all studies in which protein expression was induced by doxycycline. Data marked with an asterisk have a P value above 0.05. Error bars indicate standard deviations.