Electrostatic encoding of genome organization principles within single native nucleosomes

Abstract

The eukaryotic genome, first packed into nucleosomes of about 150 bp around the histone core, is organized into euchromatin and heterochromatin, corresponding to the A and B compartments, respectively. Here, we asked if individual nucleosomes in vivo know where to go. That is, do mono-nucleosomes by themselves contain A/B compartment information, associated with transcription activity, in their biophysical properties? We purified native mono-nucleosomes to high monodispersity and used physiological concentrations of biological polyamines to determine their condensability. The chromosomal regions known to partition into A compartments have low condensability and vice versa. In silico chromatin polymer simulations using condensability as the only input showed that biophysical information needed to form compartments is all contained in single native nucleosomes and no other factors are needed. Condensability is also strongly anticorrelated with gene expression, and especially so near the promoter region and in a cell type dependent manner. Therefore, individual nucleosomes in the promoter know whether the gene is on or off, and that information is contained in their biophysical properties. Comparison with genetic and epigenetic features suggest that nucleosome condensability is a very meaningful axis onto which to project the high dimensional cellular chromatin state. Analysis of condensability using various condensing agents including those that are protein-based suggests that genome organization principle encoded into individual nucleosomes is electrostatic in nature. Polyamine depletion in mouse T cells, by either knocking out ornithine decarboxylase (ODC) or inhibiting ODC, results in hyperpolarized condensability, suggesting that when cells cannot rely on polyamines to translate biophysical properties of nucleosomes to control gene expression and 3D genome organization, they accentuate condensability contrast, which may explain dysfunction known to occur with polyamine deficiency.

Fig. 1:. Condense-seq measures single-nucleosome condensability genome-wide.

a, Overall workflow of condense-seq. b, The total amount of nucleosome core particles (NPC) or nucleosomal DNA remaining in the supernatant was measured by UV-VIS spectrometry (left) and their integrity was checked by running gels (right, lane 1 is for DNA NEB Low Molecular Weight ladder) for different concentrations of condensing agents. c, Genome segmentation into chromatin states based on histone PTM ChIP-seq data (right). All mono-nucleosomes of chromosome 1 were categorized, and their condensability distribution for each chromatin state is shown. d, RNA-seq data (red) and condensability (blue) over the entire chromosome 1 (bin size 100 kb). e, All genes in chromosome 1 were grouped into five quantiles according to the transcription level (quantile 1 through 5 for increasing transcription). (top) Condensability, AT content and H3K25ac along the transcription unit coordinate averaged for each quantile. (bottom) Heat maps show the same quantities for each gene, rank ordered with increasing condensability. TSS (transcription start site), TTS (transcription termination site). f, Promoter condensability (averaged over 5kb window around TSS) for H1-hESC and GM12878. Each gene is colored according to their relative expression levels in the two cell types. Black symbols are for embryonic stem cell marker genes.

Fig. 2:. 3D genome compartmentalization information is encoded in native mono-nucleosomes.

a, Nucleosome–nucleosome pair-wise interaction energies, ε_ij, were derived from the condense-seq measurement based on the Flory–Huggins theory. Chromatin polymer simulation was performed using these interaction energies to predict the three-dimensional chromatin structure solely from nucleosome condensability. b, Comparison of contact probability matrix between Hi-C data of GM12878 (lower triangle) and the polymer simulation (upper triangle). In the bottom panel, the A/B compartment scores were computed using the Hi-C data or polymer simulation with interaction energies based on the condensability (phi). TAD insulation scores were also computed for Hi-C data and polymer simulation. c, The contact probability vs genomic distance from Hi-C data (orange) and polymer simulation (blue). d,A/B compartment score vs condensability in 100 kb bin. Black line is a logistic curve fit. e, Condensability vs chromatin accessibility (ATAC score) in 1kb bin. f, Condensability and ATAC score vs ChromHMM chromatin state.

Fig. 3:. Identification of the biophysical driving force of chromatin condensation and its genetic/epigenetic determinants.

a, Correlation of condensability scores across condensing agents tested: spermine (sp⁴⁺), spermidine (spd³⁺), Cobalt hexamine (CoH³⁺), polyethylene glycol molecular weight 8000 (PEG), Ca2+, HP1α and HP1β/tSUV39H1 (HP1bSUV). b, Conditional correlations between condensability and various genetic/epigenetic factors for spermine (top) and HP1α (bottom). c, Condensability profiles vs gene unit position averaged over each of the five quantiles, from weakly expressed to highly expressed genes for spermine (top) and HP1α (bottom). d–f, condense-seq results of PTM library. The effects of single PTMs on nucleosome condensation are depicted in the cartoon for spermine (d) and HP1α (f). Each symbol represents a PTM of a specific type as shown in the legend and its size is proportional to the strength of the effects. The colors of the marks indicate the direction of the effect (red: decrease condensability, blue: increase condensability) compared with the unmodified control. e, All condensability scores of the PTM library using spermine as a condensing agent. The library members were sorted based on the lowest to highest condensability scores from top to bottom. On the left panel, the ladder-like lines represent each histone peptide from N-terminal (left) to the C-terminal (right). Each mark on the line indicates the location of the PTMs, and the shape of the marks represents the PTM type (ac: acetylation, me: methylation, cr: crotonylation, ub: ubiquitylation, ph: phosphorylation, GlcNAc: GlcNAcylation, mut: amino acid mutation, var: histone variant). On the right panel,the change in condensability scores of the various modified nucleosomes compared to the control nucleosomes without any PTMs are shown.

Fig. 4:. Polyamine deficiency globally hyperpolarizes but locally disorganizes the chromatin condensability.

a, Ornithine decarboxylase (ODC) is a key enzyme in polyamine biogenesis and is inhibited by DFMO. b, Mouse CD8⁺ T cells were activated in vitro before condense-seq. wild type (WT), DFMO treated (+DFMO), and ODC knockout (ODC KO). c, Mono-nucleosome condensability distribution for WT, +DFMO and ODC KO in various chromatin states classified using ChromHMM. d, Condensation point (c_1/2) for chromosome 1, showing larger dynamic ranger and hyperpolarization for +DFMO and ODC KO. e, Condensability over gene units averaged over genes belonging to five quantiles of gene expression (FPKM: Fragments Per Kilobase of transcript per Million mapped reads). f,g, Gene set enrichment analysis of polyamine deficient conditions (f: +DFMO, g: ODC KO) compared with the wild type. Genes were ordered by their Δz, z-score of condensability relative to the wild type, shown above. In the graph, each row corresponds to the GO biological process strongly enriched for strongly positive or strongly negative Δz values, and genes belonging to that gene set are localized by tick marks. The enriched GO biological processes are clustered by their biological functions (red: developmental, green: cell signaling, orange: immunologically related). h, For each quantile of Δz near TSS, averaged Δz vs transcription unit position is shown for ODC KO VS WT (upper left) and +DFMO VS WT (upper right), and averaged ChIP-seq signals are shown for H3K4me3 (lower left), and H3K27me3 (lower right) i, Polyamine deficiency induces global hyperpolarization of chromatin compartmentalization but disrupts local chromatin organization, especially genomic locations enriched with H3K27me3 marks.