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Evidence for Homodimerization of the c-Fos Transcription Factor in Live Cells Revealed by Fluorescence Microscopy and Computer Modeling - PubMed

Nikoletta Szalóki  1 ,

Evidence for Homodimerization of the c-Fos Transcription Factor in Live Cells Revealed by Fluorescence Microscopy and Computer Modeling

Nikoletta Szalóki et al. Mol Cell Biol. 2015 Nov.

Erratum in

Abstract

The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 μM) and c-Fos-c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development.

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Figures

FIG 1

Schematic drawing of c-Fos, its mutant forms, and c-Jun. From the top: full-length Fos with fluorescent protein tag at the C terminus, C-terminally truncated Fos215 and FosΔΔ where the DNA-binding and dimerization domains were deleted, and Jun. Pink denotes the DNA-binding domain, yellow denotes the leucine zipper, and the dotted line denotes the linker between Fos/Jun and the fluorescent-protein tag (ECFP, EYFP, EGFP, or mRFP1).

FIG 2

Possible combinations of fluorescently tagged and endogenous Fos and Jun. (A) In the monomer-heterodimer equilibrium, fluorescently tagged and endogenous, unlabeled Fos and Jun molecules participate. The three species containing a donor tag contribute to the measured value of FRET efficiency, Emeas, i.e., the doubly labeled heterodimer having a FRET efficiency of E0 and donor-labeled Fos in complex with endogenous Jun or present as a monomer; the latter two species are characterized by zero FRET efficiency. The fractions of the different heterodimers follow a multinomial distribution. Emeas is a weighted average of the species-specific E values (see equation S12 in the supplemental material). (B) In the monomer-homodimer equilibrium, donor-tagged, acceptor-tagged, and endogenous Fos and endogenous Jun participate. Four heterodimeric species and the donor-tagged monomer contribute to Emeas (see equation S14 in the supplemental material).

FIG 3

Subcellular pixel-by-pixel analysis of dimerization by confocal microscopic FRET on HeLa cells. ECFP (donor channel) was excited at 458 nm and detected at 475 to 525 nm. In the transfer channel, excitation was at 458 nm and detection was at 530 to 600 nm. EYFP (acceptor channel) was excited at 514 nm and detected at 530 to 600 nm. Full-length Fos-ECFP–Fos-EYFP (top row), Fos215-ECFP–Fos215-EYFP (second row), Fos-ECFP–Jun-EYFP (third row), and Fos215-ECFP–Jun-EYFP (fourth row) showed nuclear localization. The negative control, ECFP and EYFP expressed independently, and the positive control, the ECFP-EYFP fusion protein (fifth and sixth rows), were evenly distributed in the whole cell. FRET efficiency (E) was calculated in each pixel. Histograms show the statistics of the subcellular distribution of E.

FIG 4

Cell-by-cell analysis of dimerization by confocal microscopic FRET. (A, B) FRET efficiencies of donor (ECFP)- and acceptor (EYFP)-tagged Fos215 or full-length Fos molecules as a function of the acceptor-to-donor molecular ratio (NA/ND). Data from 300 cells were grouped into three subsets as a function of donor intensity (low, <800; medium, 800 to 1,200; high, >1,200 [arbitrary units]). Cellular data were binned in 0.25-wide intervals of the NA/ND values to reduce data scatter. FRET efficiencies increased with increasing NA/ND ratios. (C) Saturation values of FRET efficiencies at high NA/ND ratios (>0.95). ECFP-EYFP fusion protein served as a positive control, and independently expressed ECFP and EYFP served as a negative control. The FRET data of the Fos-Jun and Fos215-Jun pairs were previously published in reference .

FIG 5

Determination of the dissociation coefficients of Fos-Jun heterodimers and Fos-Fos homodimers from flow cytometric FRET titrations. (A, C) FRET efficiency measured in cells cotransfected with Fos215-EGFP–Jun-mRFP1 and Jun-EGFP–Fos215-mRFP1 and plotted as a function of the donor-tagged Fos215 or Jun concentration. Data were grouped according to acceptor-to-donor molecular ratios (NA/ND) and fitted as described elsewhere (see equation S12 in the supplemental material) (solid lines), yielding the Kd value of the heterodimers and the FRET efficiency (E0) of individual donor-acceptor pairs. Endogenous Fos and Jun were also taken into account. (B, D) The solid lines represent the maximal theoretically attainable E values at different NA/ND ratios (assuming E0 values of 15 and 14.1% based on the fits) when all available Jun-mRFP1 molecules are engaged in heterodimers with Fos; the marked points correspond to the experimental NA/ND ratios (see equation S13 in the supplemental material). (E) Dependence of the Kd values from the fits on the Fos/Jun ratio. (F) FRET efficiency of Fos215-EGFP–Fos215-mRFP1 homodimers as a function of the donor-tagged Fos215 concentration with Kd and E0 yielded from a linked fit (see equation S14 in the supplemental material). (G) Maximal attainable FRET efficiencies at different NA/ND ratios (assuming an E0 value of 9.47% based on the fit) when all Fos molecules form homodimers (see equation S15 in the supplemental material).

FIG 6

FCS-based concentration calibration and brightness analysis. (A) The EGFP concentration in HeLa cells was determined from the amplitude of the ACF. The curve was fitted to a two-component free-diffusion model with triplet correction. (B) Diffusing particle concentration (1/G0) as a function of the fluorescence intensity per pixel of EGFP, FosΔΔ-EGFP, Fos-EGFP, and Fos-EGFP coexpressed with Jun-mRFP1. Data were fitted with straight lines by Deming regression. (C) Fluorescence per particle or molecular brightness values characterizing the aggregation state plotted as a function of the concentration of the EGFP tag. Symbols are the same as in panel B. (D) Normalized ACFs fitted to a two-component free-diffusion model. (E) Diffusion constants and fractions of the second, slow component derived from the fits (n, number of cells).

FIG 7

SPIM-FCCS data analysis showing codiffusion and DNA binding of Fos homodimers. (A) ACFs and CCFs from SPIM-FCCS measurements. Green, EGFP ACF; red, mRFP1 ACF. Solid lines indicate the experimental data, whereas dashed lines are fits assuming two diffusing components (ACF curves) or one component (CCFs). The red horizontal line is the cross talk-corrected red ACF amplitude, and the blue horizontal line is the level of cross-correlation due to cross talk. Cross-correlation above this value is due to the codiffusion of green and red molecules. (B) The first two columns are fluorescence intensity maps of EGFP or mRFP1 from a selected cell. The third column is a map of the fraction of GR dimers among all of the molecules detected [cGR/(cG-only + cR-only + cGR)], determined from the fits, and the histograms show their distributions. (C and D) Average fractions of GR dimers (C) and diffusion coefficients, Dcross (D), from the cross-correlation fits (mean ± SD; n > 20 for each sample). Fits were carried out on a pixel-by-pixel basis, and the median of the respective parameter from each cell was then averaged. ***, P < 0.0001 (t test).

FIG 8

Both Fos-Jun and Fos-Fos complexes form stable dimers and bind to DNA. (A and B) MD simulations were carried out on small Fos-Jun (A) and Fos-Fos (B) fragments bound to the DNA fragment. Ribbon representation (colored cyan) was applied for the helical secondary structure of the Fos protein fragment (A, B). The atomic details of constituent residues are shown by stick representation with the C, H, N, O, and S atoms in gray, white, blue, red, and yellow, respectively. For the Jun fragment (A) or the second Fos fragment (B), a solvent-excluded surface representation was applied by using the above-described color codes. (C to F) Visual representation of trajectories from MD simulations of the Leu zipper region of the Jun-Fos (C, E) and Fos-Fos (D, F) dimeric structures. Wild-type protein fragments (C, D) and virtually mutated (Leu280Asp and Leu294Asp in c-Jun and Leu165Asp and Leu179Asp in c-Fos) fragments (E, F) were considered. Mutant residues are shown by stick representation with the color scheme of the atoms as above. Jun is represented by the orange helix, and Fos is represented by the green and yellow helixes. From each 500-ns dynamic trajectory, 100 frames were saved equidistantly and superimposed (after removal of rotation and translation). Wild-type protein fragments (C, D) demonstrate stable coiled-coil motifs with relatively small fluctuations. The mutations in the Fos-Jun fragment (E) resulted in a somewhat distorted structure and larger fluctuations, indicating weakening of the interaction between the monomers. This is even more strongly expressed in the mutant dimeric Fos-Fos fragment (F), where the hydrophobic interaction between regions affected by the mutations is completely destroyed.

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