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 (Mg) cations. Both DNA and chromatin demonstrated compaction under comparable conditions in the presence of poly(ethylene glycol) and Na or Mg 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.

Organization of fast and slow chromatin revealed by single-nucleosome dynamics

“…S9, we show the dependence of the simulated radius of gyration of the ring on the interaction strength of ϵnormalI=ϵnormalInormalI=ϵ. This ring is open extended when ϵ/knormalBT<0.6 and compact condensed when ϵ/knormalBT>1.2, showing a continuous coil–globule transition as in the microscopic observation of chromatin (55). We examine ϵnormalI and ϵnormalInormalI around this transition regime.…”

Internal Motion of Chromatin Fibers Is Governed by Dynamics of Uncompressed Linker Strands

“…Although many studies have addressed the structure and dynamics of smaller segments of chromatin (12,13), only a few have focused on larger chromatins that provide a good model system for TADs. Here, we will use reconstituted chromatin by complexation of over-expressed, refolded, and purified histone octamer on bacteriophage T4-DNA (165.6 kbp) (14)(15)(16). This system can serve as a model of (smaller) TADs because it approaches their molecular weight, there are no preferential histone binding sites, and the flexibility in nucleosome position is generally conserved in vivo human chromatin (17).…”

Crowding-Induced Elongated Conformation of Urea-Unfolded Apoazurin: Investigating the Role of Crowder Shape in Silico

“…Under the assumption that the dominant interaction between a crowding agent and a biopolymer is steric repulsion, the macromolecular crowding is shown to stabilize the conformation of compact biopolymers as shown in proteins, 6–8 nucleic acids, 9 and chromatins. 10…”