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Genome-wide measurement of local nucleosome array regularity and spacing by nanopore sequencing

Abstract

The nature of chromatin as regular succession of nucleosomes has gained iconic status. However, since most nucleosomes in metazoans are poorly positioned it is unknown to which extent bulk genomic nucleosome repeat length reflects the regularity and spacing of nucleosome arrays at individual loci. We describe a new approach to map nucleosome array regularity and spacing through sequencing oligonucleosome-derived DNA by Illumina sequencing and emergent nanopore technology. In Drosophila cells, this revealed modulation of array regularity and nucleosome repeat length depending on functional chromatin states independently of nucleosome positioning and even in unmappable regions. We also found that nucleosome arrays downstream of silent promoters are considerably more regular than those downstream of highly expressed ones, despite more extensive nucleosome phasing of the latter. Our approach is generally applicable and provides an important parameter of chromatin organization that so far had been missing.

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Fig. 1: Outline and initial characterization of the Array-seq strategy.
Fig. 2: Array-seq analysis of BG3-c2 chromatin.
Fig. 3: Nucleosome array regularity as a function of transcription activity.
Fig. 4: Nucleosome array regularity at heterochromatin repeats and as a function of histone H1.
Fig. 5: Illumina-based sequencing of long fragments allows determining NRL along the genome.

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Acknowledgements

This project was supported by grant Be1140/8-1 of the German Research Council (DFG) to P.B.B. We thank T. Straub, T. Schauer and D. Jain for suggestions for data processing; A. Zabel for help with chromatin preparations and MNase digests; A. Hauser for raw data processing of nanopore reads; and P. Korber for critical reading of the manuscript and many suggestions to improve it.

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Contributions

S.B. devised the project, performed the experiments and analyzed the data under the supervision of and with input from P.B.B. S.K. performed all sequencing under the supervision of H.B. S.B. and P.B.B. wrote the manuscript.

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Correspondence to Peter B. Becker.

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Integrated supplementary information

Supplementary Figure 1 Nanopore sequencing of different MNase digestion degrees.

a, NRL for BG3-c2 cells determined from the maxima of peaks corresponding to oligonucleosome-sized fragments in the Bioanalyzer profile of MNase-digested chromatin shown in Fig. 1b. b, Bioanalyzer tracks of BG3-c2 chromatin digested with increasing amounts of MNase (low to high). c, NRLs determined for the different digestion degrees shown in b. NRL is determined from the maxima of peaks corresponding to oligonucleosome-sized fragments in the Bioanalyzer profiles. d, Size distributions of Array-seq reads derived from different MNase digestion degrees. e, NRL for the different digestion degrees shown in b determined from the maxima of peaks corresponding to oligonucleosome-sized fragments in d.

Supplementary Figure 2 MNase digestion does not introduce major biases in the representation of chromatin states.

a, High-molecular-weight genomic DNA was purified from BG3-c2 cells and digested with MNase to achieve a fragment length distribution comparable to the one obtained in Array-seq experiments. b, MNase-digested genomic DNA was sequenced on a MinION device and mapped to the genome. Different chromatin states are represented similarly to their genomic ratios. Representation of chromatin states in MNase-digested chromatin closely resembles the one of digested genomic DNA c, Read coverage on parts of chromosome 2 L for MNase-digested and MinION-sequenced genomic DNA (blue) and BG3-c2 chromatin digested with increasing MNase amounts (gray).

Supplementary Figure 3 Replicate of an Array-seq experiment on Drosophila BG3-c2 cells and mapping of MNase-digested genomic DNA to chromatin states.

a, Replicate of an Array-seq experiment on BG3-c2 cells with 107,571 reads, analogous to the one shown in Fig. 2. Size distributions of reads mapped to the nine-state chromatin model. b, Size distributions of reads from MNase-digested genomic DNA mapped to the nine-state chromatin model. c, Size distributions for the BG3-c2 Array-seq replicate mapped to the whole genome (black), the gene bodies of the 25% least expressed genes (bottom 25%; blue), or the gene bodies of the 25% most expressed genes (top 25%; red).

Supplementary Figure 4 Invariance of results to variation of sequence read numbers.

a, Size distributions of Array-seq reads mapped to the nine-state chromatin model. Read numbers for each of the states were reduced to the one for the state with the least number of reads (state 5, 19,612 reads) to exclude effects based on different sample sizes. b, Regularity scores for a.

Supplementary Figure 5 RNA interference controls and global changes in NRL for knockdowns, influence of MNase digestion degree on tetranucleosomal fragment length.

a, Western blot documenting efficient RNA interference against H1 expression (α-H1) in Kc cells. Detection of lamin serves as a loading control. b, Global NRLs in control and H1-knockdown cells determined by the slopes of the fitted lines through the maxima of the N1–N5 peaks of the Bioanalyzer tracks. c, Western blot documenting efficient RNA interference against Iswi expression (α-ISWI) in Kc cells. Detection of lamin serves as a loading control. d, Global NRLs in control and ISWI-knockdown cells determined by the slopes of the fitted lines through the maxima of the N2–N4 peaks of the Bioanalyzer tracks. e, Size distributions of Illumina-sequenced tetranucleosomal fragments derived from BG3-c2 chromatin digested with increasing amounts of MNase. Indicated in the top right are apparent NRLs when calculated as [N4 fragment length – 4 × 147]/3 + 147. f, Apparent NRLs for the different chromatin states as determined by Illumina sequencing of tetranucleosomal fragments derived from different MNase digestion degrees. As a comparison, apparent NRLs from ISWI-depleted cells are shown in red triangles.

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Baldi, S., Krebs, S., Blum, H. et al. Genome-wide measurement of local nucleosome array regularity and spacing by nanopore sequencing. Nat Struct Mol Biol 25, 894–901 (2018). https://doi.org/10.1038/s41594-018-0110-0

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