Nucleosomes accelerate transcription factor dissociation

Abstract

Transcription factors (TF) bind DNA-target sites within promoters to activate gene expression. TFs target their DNA-recognition sequences with high specificity by binding with resident times of up to hours in vitro. However, in vivo TFs can exchange on the order of seconds. The factors that regulate TF dynamics in vivo and increase dissociation rates by orders of magnitude are not known. We investigated TF binding and dissociation dynamics at their recognition sequence within duplex DNA, single nucleosomes and short nucleosome arrays with single molecule total internal reflection fluorescence (smTIRF) microscopy. We find that the rate of TF dissociation from its site within either nucleosomes or nucleosome arrays is increased by 1000-fold relative to duplex DNA. Our results suggest that TF binding within chromatin could be responsible for the dramatic increase in TF exchange in vivo. Furthermore, these studies demonstrate that nucleosomes regulate DNA-protein interactions not only by preventing DNA-protein binding but by dramatically increasing the dissociation rate of protein complexes from their DNA-binding sites.

Figure 1.

DNA and nucleosome constructs. Kinetic models of TF binding to (A) DNA, (B) single nucleosomes and (C) nucleosome arrays. (D) DNA constructs for single molecule TIRF measurements with Cy3 (green), Cy5 (magenta), biotin (black circle), 601 NPS (blue) and a Gal4- or LexA-target sequence (red). DNA molecules for making mononucleosome and dinucleosome array were labeled with Cy3 fluorophore as the FRET donor. DNA molecules for PIFE experiments were labeled with Cy3 as the PIFE indicator and Cy5 to help locate the molecule during single-molecule experiments. (E) Structure of the nucleosome (PDB: 1KX5) that indicates the location of the TF-target sequence (red), the Cy3 fluorophore location (green) and the Cy5 fluorophore location (magenta).

Figure 2.

Single molecule measurements of LexA binding and dissociation to DNA. (A) Single molecule PIFE traces of LexA binding to its target site in Cy3-labeled duplex DNA with 0 (top), 0.1 (middle) and 1 (bottom) nM LexA. The histogram on the right shows the distribution of Cy3 fluorescence for each trace. (B) The unbound (magenta circles) and bound (blue squares) dwell times, τ_unbound, with duplex DNA as a function of LexA concentration. Each dwell time was determined from an exponential fit to the dwell-time histogram (Supplementary Figure S2). The LexA concentration dependence of the unbound dwell times were fit to τ_unbound = A/[LexA] with A = (20 ± 6) s nM, and the bound dwell times were fit to τ_bound = constant = (290 ± 20) s, respectively. (C) The fraction of DNA bound by LexA as determined by EMSA (magenta triangles), ensemble PIFE measurements fit with a non-cooperative binding curve with a K_D = (0.13 ± 0.06) nM (blue squares) and single molecule PIFE measurements (red circles).

Figure 3.

Single molecule measurements of LexA binding and dissociation to single nucleosomes and dinucleosome arrays. (A) Single molecule FRET traces of LexA-trapping nucleosomes in partially unwrapped states with 0 (top), 5 (middle) and 50 (bottom) µM LexA. The histogram shows the distribution of the FRET for each trace. (B) The unbound (magenta circles) and bound (blue squares) dwell times with mononucleosomes, and the unbound (green triangles) and bound (red inverted triangles) dwell times with dinucleosome arrays as a function of LexA concentration. Each dwell time was determined from an exponential fit to the dwell time histogram (Supplementary Figure S5 and S6). The LexA concentration dependence of the unbound dwell times were fit to τ_unbound = A/[LexA] with A_monoNuc = (1.1 ± 0.3) × 10^–4 s nM and A_diNuc = (1.1 ± 0.3) × 10^–4 s nM, and the bound dwell times were fit to τ_bound = constant with τ_{bound(monoNuc)} = (0.31 ± 0.05) s and τ_bound(diNuc) = (0.29 ± 0.05) s. (C) The relative change in energy transfer efficiency versus the LexA concentration determined by both ensemble and single molecule measurements. The ensemble measurements relied on analysis of fluorescence spectra (Supplementary Figure S4) by the (ratio)_A method with mononucleosomes (red squares) and dinucleosome (blue squares). The fraction of time in the low- and high-FRET states were determined from single-molecule FRET time series for both mononucleosomes (red circles) and dinucleosomes (blue circles).

Figure 4.

Single molecule measurements of Gal4 binding and dissociation to DNA. (A) The fraction of DNA bound by Gal4 with a cooperative binding curve fit as determined by EMSA [magenta triangles, K_D = (240 ± 10) pM, Hill coefficient = 1.5], ensemble PIFE measurements [blue squares, K_D = (42 ± 5) pM, Hill coefficient = 1.5] and the single molecule Cy3 fluorescence-intensity histograms from panel B [red circles, K_D = (5 ± 1) pM, Hill coefficient = 2]. (B) Fluorescence distributions from Cy3-labeled DNA molecules containing the Gal4-target site with 0, 3, 10 and 100 pM Gal4 (ordered from top to bottom, respectively). The distributions were fit with the sum of two Gaussian distributions (black): the distribution without Gal4 (blue) and with 100 pM Gal4 (red). (C) Example Cy3 emission time traces of Cy3-labeled DNA containing the Gal4-recognition site without Gal4 bound (top), a dissociation and binding event (middle) and bound with Gal4 (bottom).

Figure 5.

Single molecule measurements of Gal4 binding and dissociation to nucleosomes. (A) Single FRET traces of Gal4 trapping nucleosomes in partially unwrapped states with 0 (top), 30 (middle) and 300 (bottom) pM Gal4. The histogram shows the distribution of the FRET for each trace. (B) The unbound (magenta circles) and bound (blue squares) dwell times with single nucleosomes as a function of Gal4 concentration. Each dwell time was determined from an exponential fit to the dwell-time histogram (Supplementary Figure S7). The Gal4 concentration dependence of the unbound and bound dwell times were fit to τ_unbound = A/[Gal4] with A = (2.5 ± .01) s nM, while the bound dwell times were fit to τ_bound = constant = (50 ± 2) s. (C) The relative change in energy transfer efficiency from mononucleosome versus the Gal4 concentration was determined by both ensemble and single molecule measurements. The ensemble measurements relied on analysis of fluorescence spectra (Supplementary Figure S4) by the (ratio)_A method (blue squares). The relative change in FRET was determined from single-moelcule FRET time series (red circles) by determining the fraction of time each time series is in the low- and high-FRET states.

Figure 6.

Model of competitive binding between nucleosome wrapping and TF binding. TFs could partially dissociate where part of the TF transiently releases from the DNA-target site and then rapidly fully rebinds again. However, if the site is located within the nucleosomes and the part of the TF further into the nucleosome transiently releases, than the nucleosome could rewrap preventing the TF from fully rebinding, which could increase the rate at which the TF fully dissociates.