In vitro single molecule and bulk phase studies reveal the AP-1 transcription factor cFos binds to DNA without its partner cJun

James Leech, Andrew Brennan, Nicola Don, Jody Mason, Neil Kad

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Abstract

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.

Original languageEnglish
Article number102229
JournalJournal of Biological Chemistry
Volume298
Issue number8
Early online date1 Jul 2022
DOIs
Publication statusPublished - 31 Aug 2022

Bibliographical note

Funding Information:
This work was supported by the Biotechnology and Biological Sciences Research Council BB/R017921/1 , BB/P00847X/1 to N. M. K. and BB/R017956/1 and BB/T018275/1 to J. M. M. J. M. M. is grateful to Cancer Research UK ( A26941 ) and to the MRC (MR/T028254/1).

Funding Information:
We thank Dr Joseph Muretta (University of Minnesota, USA) for insightful discussions on interpreting FRET data. J. T. L. A. B. and N. A. D. investigation; J. T. L. A. B. and N. A. D. methodology; J. T. L. and N. M. K. writing–original draft.: J. T. L. A. B. J. M. M. and N. M. K. writing–review & editing; J. T. L. A. B. J. M. M. and N. M. K. formal analysis; J. M. M. and N. M. K. conceptualization, J. M. M. and N. M. K. supervision; J. M. M. and N. M. K. project administration; J. M. M. and N. M. K. funding acquisition. This work was supported by the Biotechnology and Biological Sciences Research Council BB/R017921/1, BB/P00847X/1 to N. M. K. and BB/R017956/1 and BB/T018275/1 to J. M. M. J. M. M. is grateful to Cancer Research UK (A26941) and to the MRC (MR/T028254/1).

Keywords

  • AP-1
  • CD
  • DNA tightropes
  • fluorescence
  • single-molecule
  • transcription factors

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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