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. 1997 Nov 11;94(23):12396-400.
doi: 10.1073/pnas.94.23.12396.

Hormone-regulatable neoplastic transformation induced by a Jun-estrogen receptor chimera

U Kruse  1 , J S Iacovoni, M E Goller, P K Vogt
Affiliations

Hormone-regulatable neoplastic transformation induced by a Jun-estrogen receptor chimera

U Kruse et al. Proc Natl Acad Sci U S A. .

Abstract

The v-jun oncogene encodes a nuclear DNA binding protein that functions as a transcription factor and is part of the activator protein 1 complex. Oncogenic transformation by v-jun is thought to be mediated by the aberrant expression of specific target genes. To identify such Jun-regulated genes and to explore the mechanisms by which Jun affects their expression, we have fused the full-length v-Jun and an amino-terminally truncated form of v-Jun to the hormone-binding domain of the human estrogen receptor. The two chimeric proteins function as ligand-inducible transactivators. Expression of the fusion proteins in chicken embryo fibroblasts causes estrogen-dependent transformation.

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Figures

Figure 1
Figure 1
Structure of v-Jun and v-Jun-ER fusion proteins. The basic DNA binding region (BD), the leucine zipper (LZ), and the homology boxes 1 and 2 (HOB1, HOB2) of Jun are shown as patterned boxes. Numbering of amino acid residues is according to c-Jun. The carboxyl-terminal half of the human ER HE14 (amino acids 282–595) comprising the hormone binding domain (HBD) and TAF-2 is depicted as a diagonally striped box. The v-Jun protein and the ER part are separated by a spacer consisting of the amino acids glycine-glycine-serine.
Figure 2
Figure 2
Expression of the chimeric constructs in CEFs. Secondary CEFs infected with RCAS constructs bearing the chimeric inserts were lysed in sample buffer and then separated through a 10% SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose and probed with rabbit anti-Jun antiserum (A) or rabbit anti-ER antiserum (B) followed by detection with anti-rabbit horseradish peroxidase conjugate. The position of molecular mass markers (in kDa) is shown on the left.
Figure 3
Figure 3
Transactivation by v-Jun-ER chimeras. Cells were cotransfected with a luciferase reporter plasmid containing the HTLV promoter together with RCAS expression vectors as indicated. Cells were treated with 2 μM estrogen (gray bars), 200 nM tamoxifen (black bars), or ethanol as solvent control (white bars). Luciferase activity was normalized to the protein content of the samples. The result of a typical experiment done in triplicate for each inducer is shown. The transfection was repeated three times with consistent outcomes.
Figure 4
Figure 4
Estrogen-dependent activation of the bkj gene. Quail embryo fibroblasts transfected with various RCAS expression vectors were treated with (+) or without (−) estrogen as indicated for 48 hr (Left). Ten micrograms of total RNA per lane was analyzed by Northern blotting. The blot was hybridized with the 32P-labeled bkj cDNA sequence. Equal loading was confirmed by rehybridization with a glyceraldehyde-3-phosphate dehydrogenase probe. The sizes and positions of molecular mass markers are indicated on the left. Cells were grown in the presence (+) or absence (−) of 50 μg/ml cycloheximide either with (+) or without (−) 2 μM estrogen (Right). The hormone was added to the cultures 15 min later than cycloheximide. Cells were harvested 5 hr after addition of hormone. Fifteen micrograms of total RNA per lane were analyzed by Northern blotting.
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