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In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells
Published on: July 23, 2010
In vivo viral and cellular Jun complexes exhibit differential interaction with a number of in vitro generated 'AP-1-
1Department of Microbiology and Immunology, Eastern Virginia Medical School, Norfolk 23501.
Abstract:
A direct comparison of the relative DNA-binding capabilities of in vivo Jun-containing complexes derived from overexpression of the highly transforming viral Jun (VJ-1 CEF), the weakly transforming chicken cellular Jun (CJ-3 CEF) or background endogenous Jun (RCAS CEF) was assessed by gel mobility-shift assays using a synthetic oligonucleotide containing the consensus sequence TGACTCA (consensus AP-1). Chicken embryo fibroblasts (CEFs) expressing background c-Jun levels (RCAS CEF) contain almost undetectable levels of c-Jun but retain significant DNA-binding activity with two distinct complexes capable of binding specifically to the consensus AP-1 site. CEFs overexpressing either v-Jun or c-Jun contain these same two complexes and, while showing marked increases in Jun protein levels, do not exhibit any increase in DNA binding or transcriptional activation activity, suggesting that much of the overexpressed protein is inactive. Gel-shift assays performed in the presence of a Jun-specific antibody revealed a reduction in binding by both complexes, suggesting that each contains Jun or a Jun cross-reactive protein. Antibodies specific for Jun B, c-Fos, Fos B and CREB failed to interact with either complex. However, antibody specific for Fra-2 caused a slight supershift, suggesting that one or both complexes may contain Fra-2. Gel-shift competition assays with 16 'AP-1- and CREB-like' target sequences revealed that, within each cell type, the two protein complexes varied in their ability to recognize the mutant target sequences. These results clearly indicate differences in potential target recognition by each specific in vivo complex, and suggest that each may preferentially bind its own subset of target DNAs. In addition, a comparison of binding by individual complexes derived from CEFs overexpressing v-Jun and c-Jun also revealed differences in target recognition. Thus, in vivo complexes formed by overexpression of v-Jun and c-Jun vary in their ability to recognize and bind to a number of 'AP-1- and CREB-like' target sequences. This has important implications with regard to the mechanisms involved in cell transformation by v-Jun.
Insights
Overexpressed Jun proteins in chicken cells bind DNA similarly to endogenous Jun, but with altered target recognition. This suggests specific Jun complexes may influence cell transformation mechanisms.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Jun proteins are transcription factors involved in cell proliferation and transformation.
- Viral Jun (v-Jun) is known to be highly transforming, while cellular Jun (c-Jun) has weaker transforming potential.
- Understanding the DNA-binding properties of Jun complexes is crucial for deciphering their role in oncogenesis.
Purpose of the Study:
- To directly compare the DNA-binding capabilities of in vivo Jun-containing complexes from overexpressed viral Jun (v-Jun) and cellular Jun (c-Jun) versus endogenous Jun.
- To investigate the composition and target recognition specificity of these Jun complexes.
- To elucidate the implications for cell transformation mechanisms mediated by v-Jun.
Main Methods:
- Gel mobility-shift assays were used to assess DNA-binding activity.
- Synthetic oligonucleotides with the consensus AP-1 binding site (TGACTCA) were employed.
- Jun-specific and other relevant antibodies were used to identify complex components and assess binding.
- Gel-shift competition assays with various target sequences were performed to determine binding specificity.
Main Results:
- Chicken embryo fibroblasts (CEFs) with endogenous Jun levels showed significant DNA-binding activity with two distinct complexes, despite low c-Jun protein levels.
- Overexpression of v-Jun or c-Jun in CEFs resulted in the same two complexes, with increased protein but no enhanced DNA binding or transcriptional activity, suggesting protein inactivation.
- Jun-specific antibodies reduced binding, indicating Jun or a cross-reactive protein in both complexes. Fra-2 antibody caused a slight supershift, suggesting its potential presence.
- Competition assays revealed differential target recognition among the complexes within each cell type and between v-Jun and c-Jun overexpressing cells.
Conclusions:
- In vivo Jun complexes exhibit distinct DNA-binding capabilities and target recognition profiles.
- Overexpressed Jun proteins may exist in inactive forms, and their complexes display altered DNA-binding specificity.
- These findings have significant implications for understanding how v-Jun-mediated transformation occurs through specific DNA target interactions.
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