Nanoscale decompaction of nucleosomal DNA revealed through multi-color super-resolution microscopy

Abstract

Super-resolution imaging of histones and DNA reveals that DNA is compacted by groups of nucleosomes -clutches -at the nanoscale level and clutch compaction of DNA is affected by histone tail acetylation especially in highly folded regions containing several nearby clutches. AbstractChromatin organization plays an important role in regulating gene expression. Previously, we showed that chromatin is organized in the form of nucleosome groups or clutches. The size and nucleosome packing density of clutches decreased in hyperacetylated cells having more open chromatin. While hyperacetylation is thought to disrupt histone-DNA and inter-nucleosome interactions, its impact on higher order DNA compaction by groups of nucleosomes in vivo is not clear. To elucidate this question, we carried out two-color super-resolution imaging of histones and DNA in cells treated with the Histone Deacetylase (HDAC) inhibitor Trichostatin A (TSA). We showed that a lower percentage of DNA was associated to clutches in hyperacetylated cells, suggesting a decrease in nucleosome occupancy. We further identified the presence of "clutch" DNA within a nanoscale distance around the clutches. Upon histone hyperacetylation, the radius of the clutch DNA decreased leading to DNA release from the clutches, consistent with disruption of DNA-histone interactions. Finally, the most dramatic decompaction was observed for groups of clutches in close spatial proximity, suggesting that neighboring clutches influence each other´s DNA compaction.

Nuclear softening expedites interstitial cell migration in fibrous networks and dense connective tissues

“…These results are in line with a more folded chromatin confirmation in (−)TSA cells, which opens and decondenses after TSA treatment. These results are also consistent with recent super-resolution analysis, which showed that TSA-treated fibroblasts have small nucleosome nanodomains that are more uniformly distributed in the nuclear space compared to control fibroblasts (29,30). This decondensation was also confirmed in TSA-treated bovine mesenchymal stem cells (MSCs), where TSA treatment decreased the number and area of H2B nanodomains ( fig.…”

“…S3C) and the infiltration depth measured on cross-sectional images using ImageJ. For scanning electron microscopy imaging, additional samples were fixed and dehydrated in ethanol (30,50,70, and 100%, 60 min per step) and then hexamethyldisilane for terminal dehydration under vacuum.…”

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

“…How might assembly of the deacetylase sub-complex with CHD4 to form intact NuRD influence transcription? Histone acetylation is thought to lead to a looser packing between nucleosomes and chromatin decompaction [61][62][63][64][65] , but here, by studying compaction in the immediate vicinity of the stably bound NuRD complex, we unexpectedly find that assembly of the intact NuRD complex leads to a more open chromatin structure where enhancers can diffuse faster and explore more of the nucleus (see Figure 5e). The chromatin decompaction and faster movement of enhancers in the presence of intact NuRD provides a molecular explanation for the increase in intermediaterange chromatin interactions (Figure 1b), the blurring of TAD and A/B compartment boundaries (Figure 1c), the increase in longer range enhancer-promoter interactions (Figure 1d), and the changes in enhancer interactions with promoters that are regulated by NuRD (Figure 1e).…”

“…The decrease in anomalous exponent unexpectedly suggests that in the absence of the intact NuRD complex chromatin is more condensed, whilst the diffusion coefficient and length of confinement show that CHD4 molecules diffuse more slowly and explore a smaller nuclear volume. We had expected to find that in the absence of deacetylation by NuRD, that the more acetylated chromatin would be decondensed [48][49][50][51][52] and that CHD4 would explore a greater nuclear volume. Notably, however, in the absence of MBD3 we observed a significant increase in the proportion of CHD4 molecules in the fast F2 state exhibiting directed motion (18 % vs 7.4 %, see Figure 3d, Extended Data Figure 10).…”

“…The increase in anomalous exponent in wild-type cells unexpectedly suggests that in the presence of the intact NuRD complex chromatin is less condensed, whilst the increased apparent diffusion coefficient and length of confinement show that CHD4 molecules diffuse more rapidly and explore a larger nuclear volume. We had expected to find that recruitment of the deacetylase by CHD4 would lead to less acetylated chromatin and greater condensation [61][62][63][64][65] -and that the bound CHD4 molecules in wild-type cells would thus explore a smaller nuclear volume. In addition, in the presence of intact NuRD we also observed a significant decrease in the proportion of CHD4 molecules in the fast F2 state exhibiting directed motion (7.4 % in wild-type cells vs 18% in Mbd3-ko cells; see Figure 4d and Extended Data Figure 8d).…”