Compaction of Single-Molecule Megabase-Long Chromatin under the Influence of Macromolecular Crowding

Abstract

The megabase-sized length of chromatin is highly relevant to the state of chromatin in vivo, where it is subject to a highly crowded environment and is organized in topologically associating domains of similar dimension. We developed an in vitro experimental chromatin model system reconstituted from T4 DNA (approximately 166 kbp) and histone octamers and studied the monomolecular compaction of this megabase-sized chromatin fiber under the influence of macromolecular crowding. We used single-molecule fluorescence microscopy and observed compaction in aqueous solutions containing poly(ethylene glycol) in the presence of monovalent (Na⁺ and K⁺) and divalent (Mg²⁺) cations. Both DNA and chromatin demonstrated compaction under comparable conditions in the presence of poly(ethylene glycol) and Na⁺ or Mg²⁺ salt. However, the mechanism of the compaction changed from a first-order phase transition for DNA to a continuous folding for megabase-sized chromatin fibers. A more efficient and pronounced chromatin compaction was observed in the presence of Na⁺ compared to K⁺. A flow-stretching technique to unfold DNA and chromatin coils was used to gain further insight into the morphology of partially folded chromatin fibers. The results revealed a distribution of partially folded chromatin fibers. This variability is likely the result of the heterogeneous distribution of nucleosomes on the DNA chain. The packaging of DNA in the form of chromatin in the crowded nuclear environment appears essential to ensure gradual conformational changes of DNA.

Figure 1

Single-molecule FM observations of T4 DNA conformational changes upon reconstitution with HOs. (A) Fluorescence micrographs show a YOYO-labeled single T4 DNA molecule and T4 DNA reconstituted with HO at loading degrees 45 and 113%, observed in bulk solution of TE buffer with 0.1 M NaCl. (B) Quasi-three-dimensional fluorescence intensity profiles of DNA and chromatin images presented in (A) are shown. (C) Long-axis length distributions of T4 DNA molecules and chromatin at loading degrees 45 and 113% under conditions of (A) are shown. (D) The average long-axis length of T4 DNA and DNA reconstituted with HOs is shown as a function of a loading degree. The error bars indicate the SDs of the average values measured over 100 individual DNA or chromatin molecules. To see this figure in color, go online.

Figure 2

Compaction of T4 DNA and reconstituted chromatin in solution of PEG 10,000 and NaCl. (A and B) Typical fluorescence micrographs of a YOYO-labeled T4 DNA (A) and chromatin at 45% HO loading (B) are shown, observed at different concentrations of PEG in a bulk solution of TE buffer with 0.1 M NaCl. The concentrations of PEG are indicated for the corresponding rows of images. The time interval between snapshots is approximately 0.5 s. (C–E) Changes in the average long-axis length of YOYO-labeled T4 DNA (C) and chromatin at 45% (D) and 113% (E) HO loading are shown at various concentrations of PEG in a bulk solution of TE buffer with 0.1 M NaCl. Blue areas correspond to PEG concentrations at which T4 DNA or chromatin molecules are completely compacted, i.e., in a globule state. The error bars indicate the SDs of the average values measured over at least 100 individual T4 DNA or chromatin coils. The percentage values above rgw experimental points in the coexistence region indicate the percentage of T4 DNA in the unfolded and compact states, respectively. To see this figure in color, go online.

Figure 3

Long-axis length distributions of T4 DNA molecules and T4 DNA reconstituted with HOs at loading degrees of 45% and 113% in TE buffer solution containing 10% (w/v) PEG and 0.15 M KCl (top panel) or NaCl (bottom panel). At least 100 randomly selected molecules of T4 DNA and 50 molecules of chromatin were measured to build each distribution. To see this figure in color, go online.

Figure 4

Compaction of T4 DNA and reconstituted chromatin in solutions of PEG 10,000 and MgCl₂. Changes in the average long-axis length of YOYO-labeled T4 DNA (0.2 μM) (A) and chromatin with HO loading (0.2 μM) at 50% (B) and 100% (C) are shown at various concentrations of PEG in a bulk solution of TE buffer containing 4 mM MgCl₂. The blue area corresponds to PEG concentrations at which DNA or chromatin molecules are completely compacted (globule state). The error bars indicate the SDs of the average values measured over at least 100 individual DNA or chromatin molecules. The percentage values above the experimental points in the coexistence region indicate the percentage of T4 DNA or reconstituted chromatin in the unfolded and compact states, respectively. To see this figure in color, go online.

Figure 5

Intrachain segregation and formation of compacted structures on chromatin. (A) A fluorescence micrograph shows a YOYO-labeled T4 DNA molecule in the coil-globule coexistence region at 4 mM Mg²⁺ and 10% PEG concentration. The imaged DNA molecule was stretched by hydrodynamic flow on a glass surface (36). (B) Fluorescence micrographs show stretched chromatin at 50% loading in solution with 4 mM Mg²⁺ and 10% PEG. (C) Fluorescence micrographs show stretched chromatin at 100% loading in solution with 2 mM Mg²⁺ and 10% PEG. The corresponding length distributions of the stretched DNA and chromatin molecules are shown on the right. At least 50 randomly selected stretched molecules of T4 DNA or chromatin were measured to build each distribution. The compact globular objects without the unfolded DNA part were not measured for the distributions. To see this figure in color, go online.