Determinants of nucleosome organization in primary human cells

Abstract

Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.

Figure 1. Global parameters of cell-specific nucleosome phasing and positioning in human

a, In vivo granulocyte distogram (calculation explained in Supplementary Fig. 3A). X axis represents the range of recorded distances. Y axis represents frequencies of observed distances within 1-pile (blue) and 3-pile (red) subsets. 1-pile subset represents the entire dataset, 3-pile subset represents a subset of sites containing 3 or more coincident read starts. b, Distogram of the in vitro reconstituted nucleosomes showing 1-and 3-pile subsets as in (a). c, In vivo granulocyte phasogram (calculation explained in Supplementary Fig. 3B). X axis shows the range of recorded phases. Y axis shows frequencies of corresponding phases. Plotted are phasograms of 1-, 3-, and 5-pile subsets. Inset, linear fit to the positions of the phase peaks within 3-pile subsets (slope = 193 bp). d, Phasograms of blood cell types. Inset, linear fits in CD4+ T-cells (203 bp) and granulocytes (193 bp). e, Phasograms of 1-, 3-and 5-pile subsets in the in vitro data.

Figure 2. Transcription and chromatin modification-dependent nucleosome spacing

a, Nucleosome spacing as a function of transcriptional activity. X axis represents gene expression values binned according to RPKM values. Internucleosome spacing is plotted along the Y axis. Dashed lines represent genome-wide average spacing for each cell type. b, Nucleosome spacing within genomic regions marked by specific histone marks in CD4+ T-cells. Bar height plots estimated nucleosome spacing for each histone modification. Bar colors differentiate chromatin types (euchromatin vs heterochromatin).

Figure 3. Sequence signals that drive nucleosome positioning

a, Sequence signals within sites containing moderately positioned in vitro nucleosomes (stringency > 0.5). Distance from the positioned dyad to a given dinucleotide are plotted along the X axis; Y axis represents frequency of a given k-mer divided by its genome-wide expectation. The 147 bp footprint of a nucleosome is indicated by an orange band. b, Changes in AA dinucleotide usage with increasing positioning stringency. X and Y axes same as in (a). Shown are curves of AA usage within the sites of increasingly positioned dyads (stringency cutoffs of 0.4, 0.5, 0.6, 0.7). c, Sequence signals within sites containing in vitro positioned nucleosomes (stringency > 0.5) that also have high in vivo stringency (stringency > 0.4). X and Y axes same as in (a). d, Schematic depiction of the container site positioning mechanism. The C/G-rich core area (green) favors occupancy, but does not precisely position the nucleosome (top). Adding flanking A/T-rich repelling elements (purple, bottom) restricts the position of the nucleosome. e, Nucleosome organization around container sites in vivo and in vitro. X axis represents distances from the dyads to container sites(based on 300,000 container sites). Frequencies of nucleosome dyads around those sites are plotted along the Y axis. The upper plot shows distribution of in vivo dyads across CD4+ cells, CD8+ cells, and granulocytes. The ovals depict hypothetical nucleosome positions across the site with color intensities reflecting their positioning strength. The lower plot shows distribution of dyads in vitro and in MNase control.

Figure 4. Influence of gene regulatory function on nucleosome positioning

a, Comparison of sequence preferences of nucleosomes in vivo and in vitro. Normalized nucleosome core coverage in vivo (granulocytes) for a given sequence 4-mer are plotted along the X axis. In vitro core coverage is plotted along the Y axis. Each data point on the plot represents one of the 256 possible 4-mers (colored according to their G/C content). The diagonal line depicts the positions in the plot for which sequence-based preferences of nucleosomes would be the same in vivo and in vitro. b, Nucleosome core coverage over CpG islands in vivo and in vitro. X axis represents coordinates within CpG islands (0–100%) and flanking upstream of the transcriptional start sites (TSS) (left) and downstream of the TSS (right). Normalized frequencies of nucleosome cores in vivo (upper plot) and in vitro (lower plot)are plotted along the Y axis. c, In vivo CD4+ T-cell nucleosome organization around promoters. X axis represents distance from the TSS (blue arrow). Normalized frequencies of nucleosome dyads are plotted along the Y axis. Shown are nucleosome arrangements within four gene groups (not expressed 0–0.1 RPKM, low expressed 0.1–1 RPKM, moderately expressed 1–8 RPKM, highly expressed >8 RPKM). Pie chart depicts distribution of RPKM values across gene groups. d, RNA Pol II binding signal within highly expressed genes (orange curve) and H3K4me3-marked nucleosome dyad frequency (green curve) within highly expressed genes(>8 RPKM). Nucleosomes show consistent positions, indicated by grey lines pointing to nucleosome centers. e, Schematic depiction of nucleosome organization around promoters of repressed and active genes. Promoters of repressed genes do not have a well-defined nucleosome organization, while promoters of active genes have a nucleosome free region (NFR, blue), RNA Pol II (orange) localized at the NFR boundary, and positioned nucleosomes (red) radiating from the NFR. Height of the ovals represents nucleosome frequency(inferred from c). f, Nucleosome distribution around the top 1000 NRSF sites in vivo and in vitro. Distances from the NRSF binding sites are plotted along the X axis. Y axis represents the normalized frequency of nucleosome dyads. Blue ovals depict hypothetical nucleosome positions. NRSF binding site is shown by the green rectangle.