School of Biosciences, University of Kent, Canterbury, United Kingdom.
Department of Biology & Biochemistry, University of Bath, Bath, United Kingdom.
J Biol Chem. 2022 Aug;298(8):102229. doi: 10.1016/j.jbc.2022.102229. Epub 2022 Jul 1.
The AP-1 transcription factor family crucially regulates progression of the cell cycle, as well as playing roles in proliferation, differentiation, and the stress response. The two best described AP-1 family members, cFos and cJun, are known to dimerize to form a functional AP-1 heterodimer that binds to a consensus response element sequence. Although cJun can also homodimerize and bind to DNA, the canonical view is that cFos cannot bind DNA without heterodimerizing with cJun. Here, we show that cFos can actually bind to DNA in the absence of cJun in vitro. Using dual color single molecule imaging of cFos alone, we directly visualize binding to and movement on DNA. Of all these DNA-bound proteins, detailed analysis suggested 30 to 46% were homodimers. Furthermore, we constructed fluorescent protein fusions of cFos and cJun for Förster resonance energy transfer experiments. These constructs indicated complete dimerization of cJun, but although cFos could dimerize, its extent was reduced. Finally, to provide orthogonal confirmation of cFos binding to DNA, we performed bulk-phase circular dichroism experiments that showed clear structural changes in DNA; these were found to be specific to the AP-1 consensus sequence. Taken together, our results clearly show cFos can interact with DNA both as monomers and dimers independently of its archetypal partner, cJun.
AP-1 转录因子家族对细胞周期的进展起着至关重要的调节作用，同时在增殖、分化和应激反应中也发挥作用。两个最著名的 AP-1 家族成员 cFos 和 cJun 被认为可以二聚化形成功能性的 AP-1 异源二聚体，结合到一个共有反应元件序列上。尽管 cJun 也可以自身二聚化并与 DNA 结合，但传统观点认为 cFos 不能与 cJun 异源二聚化而与 DNA 结合。在这里，我们表明 cFos 实际上可以在没有 cJun 的情况下在体外与 DNA 结合。我们单独使用双色单分子成像技术研究 cFos，直接观察其与 DNA 的结合和在 DNA 上的移动。在所有这些与 DNA 结合的蛋白质中，详细分析表明有 30%到 46%是同源二聚体。此外，我们构建了 cFos 和 cJun 的荧光蛋白融合体，用于Förster 共振能量转移实验。这些构建体表明 cJun 完全二聚化，尽管 cFos 可以二聚化，但程度降低。最后，为了提供 cFos 与 DNA 结合的正交确认，我们进行了批量圆二色性实验，结果表明 DNA 发生了明显的结构变化；这些变化被发现是 AP-1 共有序列特异性的。总之，我们的研究结果清楚地表明，cFos 可以与 DNA 相互作用，无论是作为单体还是二聚体，都独立于其典型的伴侣 cJun。
