A single molecule approach to investigate how AP1 transcriptional regulators find their target sites on DNA

Transcriptional regulator protein family members Activator Protein-1 (AP1) bind to their target site TGAC/GTCA during the normal cell cycle. Their over-expression is linked to the initiation of cancer. Regulating cFos and cJun interactions with AP1 binding sites is a potential cancer therapy strategy. How the proteins find their target sites and whether non-specific DNA binding occurs will be investigated. The Protein Fragment Complementation Assay (PCA) derived inhibitor FosW is also capable of interfering with its target cJun. To study these proteins, DNA tightropes were created where single strands of λ, pUC19, pUCap1 and target-free λ (TFλ) DNA were suspended above the surface of a glass coverslip on 5 μm high pedestals. Oblique Angle Fluorescence (OAF) microscopy was used to image Quantum dot (Qdot) conjugated proteins in vitro. The protein combinations cFos:cFos, cJun:cJun, cFos:cJun, FosW and FosW+cJun (Mason et al. 2006, Worrall and Mason 2011) were studied interacting with the different DNA substrates and within the AP1 family. 71 ± 3.1% cJun:cJun, 53 ± 6.1% cFos:cJun heterodimers diffused 3-Dimensionally and 1-Dimensionally along λ DNA, indicating this is a crucial part of their search mechanism. Surprisingly, cFos is capable of dimerising, a previously unseen observation. 45 ± 3.7% of these cFos:cFos homodimers also diffused 3-Dimensionally and 1-Dimensionally. Diffusion decreased when the proteins interacted with pUC19 and pUCap1 and cJun only showed 3 ± 1.5% movement on TFλ, an unexpected observation. The interaction between FosW and cJun:cJun indicated clear interference with cJun dimerization. 55 ± 11.0% FosW and 39 ± 11.0% FosW+cJun diffused 3-Dimensionally and 1-Dimensionally. This was observed to occur directly on DNA and clarifies the mechanism of competitive inhibition and partner exchange in the AP1 family. This insight may significantly impact our understanding on how these proteins regulate transcription and help define new mechanisms of inhibition.