Torque modulates nucleosome stability and facilitates H2A/H2B dimer loss

Abstract

The nucleosome, the fundamental packing unit of chromatin, has a distinct chirality: 147 bp of DNA are wrapped around the core histones in a left-handed, negative superhelix. It has been suggested that this chirality has functional significance, particularly in the context of the cellular processes that generate DNA supercoiling, such as transcription and replication. However, the impact of torsion on nucleosome structure and stability is largely unknown. Here we perform a detailed investigation of single nucleosome behavior on the high affinity 601 positioning sequence under tension and torque using the angular optical trapping technique. We find that torque has only a moderate effect on nucleosome unwrapping. In contrast, we observe a dramatic loss of H2A/H2B dimers upon nucleosome disruption under positive torque, while (H3/H4)2 tetramers are efficiently retained irrespective of torsion. These data indicate that torque could regulate histone exchange during transcription and replication.

DNA Looping Mediates Nucleosome Transfer

“…For the first such transfer event on each unzipped template (Supplementary Table 2), 67% of traces showed a force signature consistent with that of a nucleosome [10][11][12][13][14][15][16] and 32% were consistent with that of a tetrasome 10,13 , though it is possible that some of these may have been hexasomes 17,18 . These results are in agreement with previous findings that parental H3/H4 tetrasomes generally remain intact after replication fork passage, while H2A/H2B dimers are more labile [19][20][21] .…”

“…Histones were purified using hydroxyapatite precipitation from HeLa-S3 cells purchased from the National Cell Culture Center [10][11][12]16,20 IGEPAL CA-630 (NP-40) nonionic detergent, 1 tablet per 50 ml Complete protease inhibitor cocktail (Roche) and 3 mM 2-mercaptoethanol) 47 . The nuclei pellets were frozen in liquid nitrogen and stored at À 80°C.…”

“…Aliquots of purified histones were stored in À 80°C at a final concentration of 2.7 mM. Nucleosomes were assembled on the Widom 601 nucleosome-positioning element 49 by salt dialysis [10][11][12][13]15,16,20,[50][51][52][53] . For the mechanical displacement assay, nucleosomes were assembled on a 764 bp template at a molar ratio of 1.25:1.00 of histone octamer to DNA.…”

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

“…The chromatin fiber containing histone octamers, with or without the linker histone, has this property (Recouvreux et al 2011). However, positive superhelicity may also promote the dissociation of H2A/H2B dimers, thus promoting transcription elongation by loosening DNA wrapping (Sheinin et al 2013). In bacteria, the octameric form of a mutant HUα protein has also been shown to constrain positive supercoils (Kar et al 2006), although there is no direct evidence that the wild-type protein has this capability.…”

Optical tweezers studies of transcription by eukaryotic RNA polymerases

“…In combination with fluorescence microscopy, optical tweezers were used in DNA overstretching experiments and for determining DNA-protein interactions (88)(89)(90). Angular optical trapping (AOT) was developed for simultaneous detection of force and torque (91,92), which enabled studying transcription dynamics and nucleosome stability under conditions of applied torque on the DNA (93). Recent advancements in combining optical tweezers with confocal microscopy, as well as development of advanced microfluidics systems will likely increase the range of applications and enable investigation of conditions more similar to the in vivo situation.…”