An alternative beads-on-a-string chromatin architecture in Thermococcus kodakarensis

Abstract

We have applied chromatin sequencing technology to the euryarchaeon Thermococcus kodakarensis, which is known to possess histone-like proteins. We detect positioned chromatin particles of variable sizes associated with lengths of DNA differing as multiples of 30 bp (ranging from 30 bp to >450 bp) consistent with formation from dynamic polymers of the archaeal histone dimer. T. kodakarensis chromatin particles have distinctive underlying DNA sequence suggesting a genomic particle-positioning code and are excluded from gene-regulatory DNA suggesting a functional organization. Beads-on-a-string chromatin is therefore conserved between eukaryotes and archaea but can derive from deployment of histone-fold proteins in a variety of multimeric forms.

Figure 1

Chromatin particle spectrum analysis sequences of MNase-digested T. kodakarensis and yeast chromatin recapitulate nuclease-protected DNA fragment sizes in paired-read end-to-end distance distributions. (A) Left panel shows ethidium-stained (negative image) gel separation of DNA purified from MNase-digested T. kodakarensis de-proteinized ‘naked’ genomic DNA and chromatin. The de-proteinized sample yields a smear of DNA fragments whereas chromatin yields a distinct ladder increasing in size in 30-bp intervals. Right panel shows frequency distribution of paired-read end-to-end size values after CPSA sequencing of T. kodakarensis MNase-digested naked DNA (supplementary Fig S1A online) and chromatin sample *. (B) Left panel shows gel separation of DNA purified from MNase-digested yeast (S. cerevisiae) chromatin showing characteristic eukaryotic 150-bp nucleosome ladder. Right panel shows frequency distribution of paired-read end-to-end size values after CPSA sequencing of material on gel. Peaks relating to mono-, di- and tri-nucleosome DNA fractions are indicated; TF marks trans-acting factor-bound species. CPSA, chromatin particle spectrum analysis; MNase, micrococcal nuclease.

Figure 2

CPSA of T. kodakarensis MNase-digested chromatin reveals positioned chromatin particles of variable sizes. (A) Naked DNA- and chromatin-derived paired-read mid-point distributions across 30 kb of the T. kodakarensis genome. Paired-read end-to-end distances were separated into size classes ranging from 30 to 510 bp in 60-bp steps. The naked DNA sample yields dense patterns of sequence read peaks and defines the underlying preference of MNase for sites within the T. kodakarensis genome. The chromatin-derived data set reveals distinct peaks of sequence reads different in distribution from the naked DNA control sample, suggesting the presence of positioned MNase-resistant particles protecting various different lengths of DNA. (B) Genome browser view of naked DNA- and chromatin-derived paired-read mid-point frequency distributions in the 150 bp and 330 bp size classes over 3 kb of the T. kodakarensis genome encompassing two genes. (C) Graph of paired read mid-point position frequencies at, and surrounding, a 150-bp particle at T. kodakarensis genome position 661,522 (peak summit position mapped in 1-bp bins). Lower frequency peaks at positions ∼15 bp either side of the main 150 bp peak are distinctly resolved in particle size classes increasing/decreasing by 30 bp. CPSA, chromatin particle spectrum analysis; MNase, micrococcal nuclease; ORF, open reading frame.

Figure 3

T. kodakarensis chromatin particles do not form regular arrays, but occur in proximity to larger and smaller sub-particles. (A) Summary of chromatin landscape analysis to map average chromatin particle distributions surrounding particular particle types. Particle positions in one size class are mapped as peaks in paired read mid-point frequency throughout the genome; frequency distributions are aligned according to these peak summits, then summed to produce a cumulative frequency distribution of that particle size class; the same summing process is applied to frequency distributions from surrounding size classes, and the data plotted as a surface graph resembling a landscape (x-axis=bp either side of original particle; y-axis=cumulative frequency; z-axis=size class). The original particles show up as a peak in the landscape at x=0. Any other peaks in the landscape indicate that other particles of a particular size occur, on average, in common positions relative to the original particle. (B) Chromatin landscape surrounding yeast 150-bp particles (nucleosomes) shows regular peaks in 150 bp, 300 bp and 450-bp size classes, reflecting the fact that the average eukaryotic nucleosome is part of a regular array of other nucleosomes. (C) Chromatin landscape surrounding T. kodakarensis 150-bp MNase-resistant particles show an almost flat landscape surrounding the main particle peak, apart from closely localized sub-peaks, which occur at 15-bp intervals either side of the main peak as the particle size class changes by 30 bp. (D) Landscape obtained when T. kodakarensis 150-bp MNase-resistant chromatin particle positions are plotted using the MNase-digested naked DNA control data set, confirming that the T. kodakarensis chromatin particle read peaks are not an artefact of MNase cleavage bias. MNase, micrococcal nuclease.

Figure 4

T. kodakarensis chromatin particles behave as dynamic multimers of a 30-bp DNA-associated sub-unit (A) A model for T. kodakarensis chromatin particles as linear multimers consisting of variable numbers of 30 bp-binding subunits accounting for the observed MNase-resistant particle distributions from CPSA data. A 120 bp MNase-resistant chromatin particle is depicted as a linear aggregate of four 30-bp subunits (grey boxes) in which gain and loss of single subunits from each end is possible. Particle sizes and predicted 15-bp shifts in particle mid-point position (red lines) relative to the original 120-bp entity are shown for particles resulting from gain or loss of one and two 30-bp subunits below the graphs. The graphs show cumulative chromatin particle frequency distribution values (as described in Fig 3A) for 120-bp particles and surrounding size classes aligned with the particle model. Peaks in predicted positions occur in all cases. (B) Model for the constitution and dynamic characteristics of T. kodakarensis chromatin compared with the case in eukaryotes and Haloferax. CPSA, chromatin particle spectrum analysis; MNase, micrococcal nuclease; NFR, nucleosome-free region.

Figure 5

T. kodakarensis chromatin particles are likely to consist of aggregates of archaeal histone dimers associated with a characteristic base composition profile and are excluded from gene-regulatory DNA. (A) Atomic force microscopy of T. kodakarensis histone protein assembled onto a 3 kb linear DNA reveals bead-like particles of variable size. (B) Average G/C content underlying and surrounding T. kodakarensis chromatin particles of various sizes. The number of mapped particles used in the calculation is given as n. (C) Chromatin particle landscapes (axes as described in Fig 3) surrounding yeast TSS show the characteristic NFR as a ‘valley’ in the graph. (D) Chromatin landscape surrounding the ATG of the first ORF from operon predictions for T. kodakarensis shows a CFR. (E) Chromatin landscape surrounding the positions of intergenic short DNA palindrome motifs in T. kodakarensis shows a CFR. CFR, chromatin-free region; NFR, nucleosome-free region; ORF, open reading frame; TSS, transcriptional start sites.