A comparison of in vitro nucleosome positioning mapped with chicken, frog and a variety of yeast core histones

Abstract

Using high-throughput sequencing, we have mapped sequence-directed nucleosome positioning in vitro on four plasmid DNAs containing DNA fragments derived from the genomes of sheep, drosophila, human and yeast. Chromatins were prepared by reconstitution using chicken, frog and yeast core histones. We also assembled yeast chromatin in which histone H3 was replaced by the centromere-specific histone variant, Cse4. The positions occupied by recombinant frog and native chicken histones were found to be very similar. In contrast, nucleosomes containing the canonical yeast octamer or, in particular, the Cse4 octamer were assembled at distinct populations of locations, a property that was more apparent on particular genomic DNA fragments. The factors that may contribute to this variation in nucleosome positioning and the implications of the behavior are discussed.

Keywords: Cse4; chromatin; core histones; histone variants; nucleosome positioning.

Graphical abstract

Fig. 1

Nucleosomal DNA preparation. DNA recovered after micrococcal nuclease digestion of reconstitutes prepared with five different types of histone preparation was fractionated by agarose gel electrophoresis. DNAs cut from the gel are indicated by the red brackets. Selected marker DNA sizes are indicated.

Fig. 2

Sequence read numbers. (a) Numbers of paired-end sequence reads mapped onto the four DNA sequences are presented as a function of the type of core histone used for reconstitution. The total number of reads for each reconstitute was normalized to 15 × 10⁶. (b) The number of sequence reads per base pair for each of the DNA sequences averaged over the five types of reconstitute. (c) The number of sequence reads per base pair, for all five types of reconstitute, is plotted as a function of the G + C content of each DNA sequence. The correlation coefficient (R²) for the data is shown.

Fig. 3

Size distributions of the histone octamer binding sites. (a) The distributions of nucleosomal DNA lengths, obtained from paired-end sequencing of the DNAs recovered from the five different core histone reconstitutes (color coded) for each of the four DNA sequences, are shown. Numbers of molecules are presented as a fraction of the total number of molecules indicated by paired-end reads that aligned to each DNA in each reconstitute. (b) Average lengths of nucleosomal DNAs recovered from selected reconstitutes [all reconstitutes, chicken and frog reconstitutes averaged (CF) and Cse4 reconstitute] formed on each type DNA. (c) Average lengths of nucleosomal DNAs recovered from Phins and BLG or from all DNAs for all five histone types.

Fig. 4

Core histone octamer positioning on genomic DNA sequences. The locations and relative abundance of frog histone octamer binding sites on each genomic DNA are presented in terms of (i) sequencing coverage (black) and (ii) calculated nucleosome dyads (red; see Methods). The maps have been adjusted so that the total signal for each map is normalized (to unity). Schematic representations of the gene structures (transcribed sequences) within each of the genomic regions are indicated (arrows; blue for the overlapping gene on the opposite strand on YRO) and the locations of the ILPR (Phins), the Mos1 transposon (Mos1) and the late-firing replication origin (YRO) are identified by green rectangles.

Fig. 5

Relationships between histone octamer positioning site affinity maps. Scatter plots of the relative free-energy values (ΔG⁰) of binding sites, derived from the dyad profiles for selected pairs of histone type maps, are presented. R values derived from linear regression analysis (red line) are shown on each panel. In the context of the average for the four DNAs, the panels have been ranked from the highest (left) to the lowest (right) with respect to R.

Fig. 6

Relationships between histone octamer positioning site affinity maps. (a) R values, derived from linear regression analysis of scatter plots of the relative free-energy values (ΔG⁰) of positioning sites, for the coverage (top panel) and dyad (lower panel) profiles, are presented in color-coded format. YTD, yeast (tetramer/dimer); YO, yeast octamer; C, chick; F, frog; Y, an average of the yeast octamer and the yeast tetramer/dimer data; CF, an average of the chicken and frog data; Cse4, Cse4 yeast octamer. (b) The correlation values (R) from the analysis of the dyad profiles [lower panel in (a)] were used to determine the mean correlations for all analyses of selected histone-type comparisons (left panel) and for all analyses of each DNA type (right panel).

Fig. 7

Relationships between histone octamer binding site affinity maps and DNA sequence. Scatter plots of the relative free-energy values (ΔG⁰) of the binding sites identified in the dyad profiles, measured on each DNA reconstituted with the indicated type of core histone, and the base composition (G + C content) of the binding site, measured in a 111-bp window centered on the dyad of the site, are shown. For each scatter plot, the correlation (R) is indicated.

Fig. 8

Relationships between histone octamer binding site affinity maps and DNA sequence. (a) The relationship between the average relative free energy (ΔG⁰) of binding sites, derived from the dyad profiles, binned in respect of G + C content (5% bin size), shown for analyses using yeast histones (black), Cse4 histones (blue), chicken histones (red) and frog histones (green), is presented for each of the four DNA types. (b) The correlation (R) between the relative free-energy values (ΔG⁰) of binding sites and the G + C content within defined regions (windows) of the binding sites are presented as a function of the location of the base composition windows relative to the dyad of the binding site. G + C content profiles were generated for each sequence with (i) a central 11-bp window and then with (ii) a set of pairs of 10-bp windows centered on and straddling the central 11-bp window and moving out from the center in 10-bp steps. Analyses for reconstitutes prepared with yeast histones (black), Cse4 histones (blue), chicken histones (red) and frog histones (green) are shown for each of the four DNA types.

Fig. 9

Relationships between histone octamer binding site affinity maps and DNA sequence. The correlation (R) between the relative free energy (ΔG⁰) of binding sites, derived from the dyad profiles, and G + C content (upper graph) or AAAA frequency (lower graph) for data derived from reconstitutes prepared with chicken (blue), frog (red), yeast (yellow) and Cse4 (cyan) histones are presented as a function of DNA type. The average correlation (R) and standard error for each group is shown.