UBCH7 had minimal effects on the transcriptional activity of AF-1 and AF-2 regions of hPR-B; however, it significantly enhanced the transcriptional activity of the full-length PR-B. HeLa cells were transiently transfected with either RSV.hPR-B (full-length PR-B), RSV.hPRΔA/B (AF-2), or RSV.hPRΔE (AF-1) expression plasmid along with progesterone-responsive reporter (PRE.TATA.LUC) plasmid. After 4 h, cells were treated with or without progesterone (10⁻⁷ M). Twenty-four hours later, cells were harvested and assayed for luciferase activity. Data are expressed as means and standard deviations of n-fold activation results for three different experiments. H, hormone.

UBCH7 reverses the transcriptional interference between PR and ER. HeLa cells were transfected with 0.2 μg of PR expression plasmid, 0.3 μg of ER expression plasmid, 1.0 μg of PRE.TATA.LUC, and increasing concentrations (0, 0.1, 0.15, 0.2, 0.25, and 0.3 μg) of UBCH7. Cells were then treated with progesterone (Prog.) alone or progesterone and estradiol together (each at 10⁻⁸ M). The last bar corresponds to control cells transfected with UBCH7 and PR expression plasmids only. Data are expressed as means and standard deviations of n-fold activation results from three independent transfections. The activity in the presence of hormone and absence of UBCH7 was defined as onefold, and the other bars were scaled accordingly.

UBCH7 is recruited onto ER- and PR-responsive promoters. ChIP was performed using MCF-7 and T47D/CAT0 cells in the presence or absence of estrogen or progesterone (Prog.) hormones. (A) Primers specific for the pS2 promoter were used to amplify the genomic DNA associated with ER and UBCH7 in MCF-7 cells. (B) Primers specific for the MMTV promoter were used to amplify the genomic DNA associated with PR and UBCH7 in T47D/CAT0 cells. In these experiments, 0.1% input DNA was used in the control lanes (Input). Primers specific for the GAPDH coding region were used as a loading control. The data were quantified by using the NIH image scan program version 1.62, and data are plotted as intensity versus hormone treatment.

(A) UBCH7 and E6-AP synergistically enhance PR transactivation. Cells were transiently transfected with PR-B expression plasmid and progesterone-responsive reporter plasmid (PRE.TATA.LUC) in the absence (Control) or presence of UBCH7, wild-type E6-AP, ligase-defective mutant E6-AP (Mutant E6-AP), UBCH7 plus wild-type E6-AP, or UBCH7 plus mutant E6-AP. Cells were treated with or without progesterone (P) (10⁻⁷ M). Data are expressed as means and standard deviations of n-fold activation results for three different transfections. The activation in the presence of hormone (H) using an empty vector was taken as onefold. (B) E6-AP is not required for UBCH7 to potentiate transcriptional activity of PR in vivo. E6-AP KO fibroblasts were transiently transfected with full-length PR (PR-B) expression plasmid along with the progesterone-responsive reporter plasmid (PRE.TATA.LUC). Cells were also transfected with either E6-AP or UBCH7 expression plasmid vector. After 4 h, cells were treated with progesterone (P) (10⁻⁷M) or a vehicle. Twenty-four hours later, cells were harvested and assayed for luciferase activity. Data are expressed as n-fold activation in the presence of progesterone. The activation in the presence of hormone (H) using an empty vector was taken as onefold. Data expressed are means and standard deviations of results from three different experiments.

(A) Depletion of endogenous UBCH7 levels reduces transcriptional activity of PR. HeLa cells were transiently transfected with PR and PRE.TATA.LUC expression plasmid and its response element in the presence or absence of different UBCH7 siRNAs. Transfection was also done using GAPDH siRNA as a control. Four hours after transfection, cells were treated with either progesterone (PROG) (10⁻⁷ M) or a vehicle (ethanol). Twenty-four hours after transfection, cells were harvested, and luciferase activity was assayed. The data are presented as n-fold activation. (B) Expression analysis of UBCH7 protein after siRNA treatment. Cell extracts were separated on a 4 to 20% gradient gel and transferred onto nitrocellulose paper, and protein levels were assessed by Western blotting using UBCH7 specific antibodies. Equal loading of samples was confirmed using β-actin-specific antibodies.

The ubiquitin-conjugating activity of UBCH7 is required for its coactivation function. (A) The ubiquitin conjugation-defective mutant UBCH7 (c-s) had no significant effect on the transcriptional activity of PR in the transient transfection assay. HeLa cells were transfected with PR-B expression and reporter plasmids in the presence or absence of either wild-type (w) or mutant UBCH7. Cells were treated with progesterone (P) (10⁻⁷ M), and luciferase activity was measured. The data are presented as n-fold activation. The activity of PR-B in the presence of hormone (H) and absence of UBCH7 was defined as onefold, and the other bars were scaled accordingly. (B) Wild-type and ubiquitin conjugation-defective mutant UBCH7 expressed at equal levels. Protein levels were analyzed by Western blotting using anti-UBCH7 antibody. The control lane (Ctrl) represents cells that were transfected with an empty vector. β-Actin expression was used as a loading control. (C) UBCH7 potentiates PR transcriptional activity in a cell-free transcription system on a chromatin template. Briefly, an initial reaction was carried out with Drosophila embryo extract, and core histones were assembled on a control template that lacks a PR binding site and a template that contains PR binding site PRE3-E4, purified PR-B, progesterone (Prog.) (10⁻⁷ M), no progesterone (−), or purified UBCH7 (wild-type or ubiquitin conjugation-defective mutant [C-S]). HeLa extract was added to a preassembled chromatin assembly, and the reaction was allowed to continue for 30 min at 27°C. The samples were then subjected to in vitro transcription analysis.

(A) UBCH7 interacts with SRC-1 protein. Interaction of UBCH7 with SRC-1, SRC-2, and SRC-3 was determined in a GST pull-down assay. SRC-1, SRC-2, and SRC-3 were labeled by in vitro translation and incubated overnight at 4°C with E. coli-expressed GST alone (control), GST-UBCH7 bound to beads, or GST-UBCH5B in NETN buffer (50 mM NaCl, 1 mM EDTA, 20 mM Tris [pH 8.0], 0.1% NP-40). Bound proteins were analyzed by 7.5% SDS-PAGE and autoradiographed. (B) In vivo UBCH7 interacts with SRC-1 protein. After cells were grown for 24 h, the cell lysates were prepared in RIPA buffer. Then lysates were immunoprecipitated (IP) with either control serum, UBCH7-specific antibody (α-UBCH7), or UBCH5 specific antibody (α-UBCH5), and precipitated proteins were immunoblotted with either anti-SRC-1 antibody (top panel, WB: α-SRC-1), anti-UBCH7 antibody (middle panel, WB: α-UBCH7), or anti-UBCH5 antibody (bottom panel, WB: α-UBCH5). (C) Overexpression of UBCH7 has no effect on the expression levels of SRC-1 protein. HeLa cells were transfected with either control or UBCH7 expression plasmid. Cells were harvested 48 h after transfection, and SRC-1 levels were assessed by Western blotting using SRC-1 specific antibodies. Equal loading of samples was confirmed using β-actin-specific antibodies. (D) SRC-1 is required for UBCH7 to potentiate transcriptional activity of PR in vivo. SRC-1 KO fibroblasts were transiently trans-fected with full-length PR (PR-B) expression plasmid along with the progesterone-responsive reporter plasmid (PRE.TATA.LUC). Cells were also transfected with either SRC-1 or UBCH7 expression plasmid vectors or both. After 4 h, cells were treated with progesterone (P) (10⁻⁷ M) or a vehicle. Twenty-four hours later, cells were harvested and assayed for luciferase activity. Data are expressed as n-fold activation in the presence of progesterone. The activation in the presence of hormone (H) using an empty vector was taken as onefold. Data expressed are means and standard deviations of results from three different experiments.

(A) UBCH7 modulates the hormone-dependent transcriptional activity of various nuclear hormone receptors. HeLa cells were transiently transfected with receptor expression plasmid for PR-B, GR, AR, RAR, and their cognate hormone response elements in the presence or absence of UBCH7. Later, the cells were treated with hormones (H) as follows: PR, progesterone (10⁻⁷ M); AR, R1881 (2.5 × 10⁻¹⁰ M); GR, dexamethasone (10⁻⁷ M); and RAR, retinoic acid (10⁻⁷ M), respectively. Cells were harvested after 24 h and assayed for luciferase activity. Bars indicate means and standard deviations of three different determinations. The data are presented as n-fold activation. The activity of receptor in the presence of hormone and the absence of UBCH7 was defined as onefold, and the other bars were scaled accordingly. (B) UBCH7 modulates the hormone-dependent transcriptional activity of endogenous PR in T47D cells. T47D cells were transiently transfected with a reporter plasmid that contained the progesterone response element in the presence and absence of UBCH7. Cells were treated with progesterone (P) (10⁻⁷ M), and after 24 h cells were harvested and assayed for luciferase activity. The data are presented as n-fold activation. The activity of receptor in the presence of hormone (H) and in the absence of UBCH7 was defined as onefold, and the other bars were scaled accordingly. (C) Overexpression of UBCH7 in T47D cells had no significant effect on the expression levels of PRs. T47D cells were transfected with either control vector (pBKRSV) or UBCH7 vector (pBKRSV-UBCH7). Cells were treated with progesterone (PROG) (10⁻⁷ M), and after 48 h cells were harvested. The expression levels of PR protein were assessed by Western blot analysis using PR-specific antibodies. β-Actin expression was used as a loading control. (D) Coexpression of UBCH7 had no significant effect on the transcriptional activity of nonnuclear hormone receptor transcription factors p53 and VP-16 activation domain. HeLa cells were transiently transfected with expression plasmid and either p53 or VP-16 activation domain along with their respective reporter plasmids, p21 promoter-LUC and 17mer-LUC, in the presence or absence of UBCH7. Data are expressed as means and standard deviations of n-fold activation results from three different transfections. (E) Other ubiquitin-conjugating enzymes are not involved in the steroid receptor transactivation pathway. Cells were transiently transfected with PR-B expression and progesterone-responsive reporter plasmids in the presence of either control vector, UBCH5B expression plasmid, UBCH7 expression plasmid, UBCH8 expression plasmid, or UBC12 expression plasmid. Cells were treated with progesterone (P) (10⁻⁷ M), and after 24 h cells were harvested and assayed for luciferase activity. The data are presented as n-fold activation. The activity of receptor in the presence of hormone (H) and in the absence of ubiquitin conjugation enzyme was defined as onefold, and the other bars were scaled accordingly.