19 research outputs found
Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes
<div><p>Nucleosome structure and stability affect genetic accessibility by altering the local chromatin morphology. Recent FRET experiments on nucleosomes have given valuable insight into the structural transformations they can adopt. Yet, even if performed under seemingly identical conditions, experiments performed in bulk and at the single molecule level have given mixed answers due to the limitations of each technique. To compare such experiments, however, they must be performed under identical conditions. Here we develop an experimental framework that overcomes the conventional limitations of each method: single molecule FRET experiments are carried out at bulk concentrations by adding unlabeled nucleosomes, while bulk FRET experiments are performed in microplates at concentrations near those used for single molecule detection. Additionally, the microplate can probe many conditions simultaneously before expending valuable instrument time for single molecule experiments. We highlight this experimental strategy by exploring the role of selective acetylation of histone H3 on nucleosome structure and stability; in bulk, H3-acetylated nucleosomes were significantly less stable than non-acetylated nucleosomes. Single molecule FRET analysis further revealed that acetylation of histone H3 promoted the formation of an additional conformational state, which is suppressed at higher nucleosome concentrations and which could be an important structural intermediate in nucleosome regulation.</p></div
smFRET results on nucleosome stability are consistent to μpsFRET data.
<p>(<b>A</b>) Average proximity ratio calculated from all photons from double-labeled nucleosomes as a function of salt concentration. Photons from the donor and transfer channel were summed for all detected molecules, except donor-only and acceptor-only species. (<b>B</b>) Salt dependence of the fraction of intact nucleosomes in smFRET histograms from Fig. 5. For each histogram, the donor-only and acceptor-only population was excluded from the analysis. The relative fraction of FRET-active molecules (0.251/2 values, which are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057018#pone-0057018-t001" target="_blank">Table 1</a>. After fitting data were normalized to better visualize the difference between non-acetylated and H3-acetylated nucleosomes.</p
smFRET analysis reveals a conformational transition prior to nucleosome unwrapping.
<p><b>A,B</b>) smFRET histograms of non-acetylated and H3-acetylated nucleosomes at various salt concentrations and 300 pM total nucleosome concentration. Above 300 mM NaCl, a fraction of H3-acetylated nucleosomes populates a second conformation with slightly increased proximity ratio compared to non-acetylated nucleosomes, which appear to retain their initial structure. <b>C, D</b>) Overlay of histograms for salt concentrations between 150 mM and 600 mM NaCl for non-acetylated (C) and H3-acetylated nucleosomes (D). Data were smoothed once to better visualize the gradual transition of nucleosomes into the high FRET state.</p
Comparison of c<sub>1/2</sub> – values from μpsFRET and smFRET experiments.
<p>Comparison of c<sub>1/2</sub> – values from μpsFRET and smFRET experiments.</p
Operational regime of different FRET methods.
<p>Conventional smFRET and fluorometric assays are difficult to perform in the intermediate concentration regime (100 pM to 1 nM, shaded area). Quasi-bulk smFRET and microplate-scanning FRET (μpsFRET), on the contrary, allow us to accurately determine FRET efficiencies in this regime and effectively close the gap between single molecule and ensemble FRET spectroscopy. A large range of sample concentrations is now amenable to fast, high throughput estimation of bulk FRET efficiencies (μpsFRET) as well as to a detailed analysis of conformational heterogeneity within the ensemble (quasi-bulk smFRET).</p
Working range of conventional and quasi-bulk single particle FRET.
<p><b>A</b>–<b>C</b>) smFRET histograms and burst size to burst duration distributions for a binary DNA mixture (noFRET and FRET-active) at 60 pM (A), 150 pM (B), and 330 pM (C) sample concentrations. While at 60 pM both subpopulations are clearly separated, coincident detection of both species occurs at 150 pM and above. The presence of multi-particle events is evident from the burst size to burst duration distribution. While at 50 pM burst duration and burst size strongly correlate, additional populations appear outside the ellipsoidal point cloud at higher sample concentrations. <b>D, E</b>) Principle of quasi-bulk smFRET of nucleosomes. Nucleosomes were reconstituted on 5S rDNA (D) or the high affinity Widom 601 sequence (E). Histograms are shown for 5 mM or 150 mM salt concentrations and in the presence or absence of 10 nM unlabeled nucleosomes. At 5 mM NaCl (left panels) most nucleosomes were intact as expected from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057018#pone-0057018-g001" target="_blank">Figure 1A</a>. At 150 mM NaCl (right panels) and in the absence of unlabeled nucleosomes, diluted nucleosomes dissociated, whereas under quasi-bulk conditions, nucleosomes on both 5S and 601 DNA remained intact.</p
Characterization of microplate-scanning FRET spectroscopy (μpsFRET).
<p><b>A)</b> μpsFRET grey scale images of a nucleosome sample (nuc) and a DNA fragment (DNA) at different sample concentrations (donor channel: excitation at 488 nm, detection at 500–540 nm; transfer channel: excitation at 488 nm, detection at 595–625 nm; acceptor channel: excitation at 532 nm, detection at 595–625 nm). Due to the absence of FRET, the DNA sample has a lower signal in the transfer channel. Concentrations are (from left to right): 2.5 nM, 1.7 nM, 1.1 nM, 600 pM, 350 pM, 180 pM, 120 pM, 70 p M, 40 pM, 20 pM, The last row to the right contained pure buffer solution. <b>B</b>) A plot showing the integrated fluorescence signal (donor channel + transfer channel) as a function of sample concentration. The measured intensity is linear throughout the dilution series. Concentrations below 50 pM can still be distinguished from background. <b>C</b>) A plot showing calculated P-values of nucleosomes and DNA as a function of sample concentration. For both samples P-values were consistent at larger concentrations, while for DNA P deviated at concentrations lower than 200 pM. Nucleosomal P-values were consistent to slightly lower concentrations (100 pM). <b>D</b>) Noise analysis of P-values from a donor-only sample under sub-nanomolar concentrations. Black circles are experimental standard deviations from 25 wells, white circles show estimated shot noise values. The low signal to noise level at lowest concentrations results in a large well-to-well variation in P. Shot noise accounts for <15% of the total uncertainty only, showing that the major source of uncertainty Is of different origin. The insert figures show well-wise P-distributions for 25 pM, 100 pM and 400 pM fluorophore.</p
Analysis of the size distribution of Cdc45-containing protein complexes during the cell cycle and after UV damage.
<p>Asynchronous (Asn) UVC-treated (+UVC, 5 J/m<sup>2</sup>, 1 h post-treatment), G1/S transition or S phase synchronized HeLa S3 cells stably expressing eGFP-Cdc45 were lysed and normalized for protein content and separated by gel filtration chromatography analysed by western blotting using antibodies raised against Cdc45, Mcm5 and RPA 32. (panels a, b, c, and d, respectively). Theoretical molecular weight (kDa) and Stoke's radius (Å) of protein standards are overlayed. RPA32 acts as a marker for DNA damage response following UVC treatment. FACS analysis is provided for asynchronous (Asn), G1/S transition and S phase synchronized cells (e) and asynchronous cells treated with 5 J/m<sup>2</sup>, 1 h post-treatment (f).</p
Diffusion coefficient of eGFP-Cdc45 at G1 to S phase transition and in S phase and after UV damage.
<p>Mean and standard deviations obtained from at least 15 cells.</p
Association of Cdc45 with chromatin synchronized HeLa S3 cells and after DNA damage.
<p>Panel a, chromatin-associated lysate from 1×10<sup>6</sup> Hela S3 cells synchronized at various cell cycle stages by two consecutive thymidine block analysed by western blotting using antibodies raised against Cdc45, Mcm7, Mcm5, Lamin B1, P261 and P125 of Pol ε and <i>δ</i>, respectively. The latter serves as a loading control. Asynchronous control cells (Asn) or cells analysed at times ranging from 0 to 12 h following release from the second thymidine block (TdR 0 to TdR 12) were analysed in parallel by FACS. Corresponding FACS profiles for relevant timepoints are also shown. Panel b, western blot of chromatin-associated Cdc45 following UVC treatment. HeLa S3 cells treated with 5 J/m<sup>2</sup> UVC harvested at indicated timepoints post treatment with untreated cells (UT) acting as a control. Chromatin-associated lysates normalized for protein content were analysed by western blotting using antibodies raised against Cdc45 and Lamin B1, which serves as a loading control.</p