Fig. 1.
Fig. 1. Estrogen-dependent forms of E2a/Pbx1 are produced by replacement of Pbx1 sequences with the ER HBD. / (A) EPΔ578ER and EPΔ623ER were created by internal fusion of the HBD (amino acids 282-595) of the Gly400Val mutant human ER. The HBD replaces Pbx1 sequences, upstream of the DNA-binding homeodomain, that are dispensable for the biochemical and transforming properties of E2a/Pbx1. (B) EPΔ578ER and EPΔ623ER demonstrate conditional transformation of NIH3T3 fibroblasts. NIH3T3 fibroblasts infected with empty vector (MSCV) or stably expressing E2a/Pbx1, EPΔ578ER, or EPΔ623ER, were assayed for density-dependent growth by culture in the presence or absence of 1 μM β-estradiol. (B, inset) Immunoblot (α-E2a antibody) analysis of E2a/Pbx1 and EPΔ623ER (and EPΔ578ER, data not shown) in NIH3T3 fibroblasts demonstrates equivalent protein expression in the presence and absence of estrogen. (C) Combined Hox-independent and Hox-dependent transcriptional activation was assayed in NIH3T3 fibroblasts by cotransfection of constructs encoding wild-type E2a/Pbx1, EPΔ578ER, EPΔ623ER (denoted Δ578ER, and Δ623ER, respectively) and the 6xTGATTGAT_luciferase reporter in the presence (solid bars) or absence (empty bars) of 1 μM β-estradiol. (C, inset) The concentration of β-estradiol required for maximal transactivation of EPΔ623ER in NIH3T3 fibroblasts was determined by measuring luciferase activity in transfected cells cultured over a range of β-estradiol concentrations. (D) Hox-independent transactivation was assayed on the 6xTGATTGAT_luciferase reporter in human pre-B Nalm-6 cells, which lack significant expression of endogenous Hox protein partners. (E) Hox partner-dependent cooperative transactivation was assayed in Nalm-6 cells on the 6xTGATTTAT_luciferase reporter by cotransfection of E2a/Pbx1 constructs with constructs encoding HoxC8 (E) or HoxA9 (F). Lanes are described in the legends accompanying each graph. In panels C-F, luciferase activity was first normalized to the internal Renilla luciferase transfection control, and the fold transactivation was determined in comparison to cells cotransfected with empty MSCV vector DNA and reporter plasmid. The results represent the average of duplicate or triplicate transfections performed 2 times on separate days.

Estrogen-dependent forms of E2a/Pbx1 are produced by replacement of Pbx1 sequences with the ER HBD.

(A) EPΔ578ER and EPΔ623ER were created by internal fusion of the HBD (amino acids 282-595) of the Gly400Val mutant human ER. The HBD replaces Pbx1 sequences, upstream of the DNA-binding homeodomain, that are dispensable for the biochemical and transforming properties of E2a/Pbx1. (B) EPΔ578ER and EPΔ623ER demonstrate conditional transformation of NIH3T3 fibroblasts. NIH3T3 fibroblasts infected with empty vector (MSCV) or stably expressing E2a/Pbx1, EPΔ578ER, or EPΔ623ER, were assayed for density-dependent growth by culture in the presence or absence of 1 μM β-estradiol. (B, inset) Immunoblot (α-E2a antibody) analysis of E2a/Pbx1 and EPΔ623ER (and EPΔ578ER, data not shown) in NIH3T3 fibroblasts demonstrates equivalent protein expression in the presence and absence of estrogen. (C) Combined Hox-independent and Hox-dependent transcriptional activation was assayed in NIH3T3 fibroblasts by cotransfection of constructs encoding wild-type E2a/Pbx1, EPΔ578ER, EPΔ623ER (denoted Δ578ER, and Δ623ER, respectively) and the 6xTGATTGAT_luciferase reporter in the presence (solid bars) or absence (empty bars) of 1 μM β-estradiol. (C, inset) The concentration of β-estradiol required for maximal transactivation of EPΔ623ER in NIH3T3 fibroblasts was determined by measuring luciferase activity in transfected cells cultured over a range of β-estradiol concentrations. (D) Hox-independent transactivation was assayed on the 6xTGATTGAT_luciferase reporter in human pre-B Nalm-6 cells, which lack significant expression of endogenous Hox protein partners. (E) Hox partner-dependent cooperative transactivation was assayed in Nalm-6 cells on the 6xTGATTTAT_luciferase reporter by cotransfection of E2a/Pbx1 constructs with constructs encoding HoxC8 (E) or HoxA9 (F). Lanes are described in the legends accompanying each graph. In panels C-F, luciferase activity was first normalized to the internal Renilla luciferase transfection control, and the fold transactivation was determined in comparison to cells cotransfected with empty MSCV vector DNA and reporter plasmid. The results represent the average of duplicate or triplicate transfections performed 2 times on separate days.

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