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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Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251–260 (1997).
Radman-Livaja, M. & Rando, O. J. Nucleosome positioning: how is it established, and why does it matter? Dev. Biol. 339, 258–266 (2010).
Struhl, K. & Segal, E. Determinants of nucleosome positioning. Nat. Struct. Mol. Biol. 20, 267–273 (2013).
Groth, A., Rocha, W., Verreault, A. & Almouzni, G. Chromatin challenges during DNA replication and repair. Cell 128, 721–733 (2007).
Zaret, K. S. & Carroll, J. S. Pioneer transcription factors: establishing competence for gene expression. Genes Dev. 25, 2227–2241 (2011).
Teif, V. B. et al. Genome-wide nucleosome positioning during embryonic stem cell development. Nat. Struct. Mol. Biol. 19, 1185–1192 (2012).
Valouev, A. et al. Determinants of nucleosome organization in primary human cells. Nature 474, 516–520 (2011).
Sperling, L. & Weiss, M. C. Chromatin repeat length correlates with phenotypic expression in hepatoma cells, their dedifferentiated variants, and somatic hybrids. Proc. Natl Acad. Sci. USA 77, 3412–3416 (1980).
Berkowitz, E. M. & Riggs, E. A. Characterization of rat liver oligonucleosomes enriched in transcriptionally active genes: evidence for altered base composition and a shortened nucleosome repeat. Biochemistry 20, 7284–7290 (1981).
Jakob, K. M., Ben Yosef, S. & Tal, I. Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo. Nucleic Acids Res. 12, 5015–5024 (1984).
Szerlong, H. J. & Hansen, J. C. Nucleosome distribution and linker DNA: connecting nuclear function to dynamic chromatin structure. Biochem. Cell Biol. 89, 24–34 (2011).
Wallrath, L. L. & Elgin, S. C. Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev. 9, 1263–1277 (1995).
Sun, F. L., Cuaycong, M. H. & Elgin, S. C. Long-range nucleosome ordering is associated with gene silencing in Drosophila melanogaster pericentric heterochromatin. Mol. Cell Biol. 21, 2867–2879 (2001).
Mavrich, T. N. et al. Nucleosome organization in the Drosophila genome. Nature 453, 358–362 (2008).
Voong, L. N. et al. Insights into nucleosome organization in mouse embryonic stem cells through chemical mapping. Cell 167, 1555–1570.e15 (2016).
Henikoff, J. G., Belsky, J. A., Krassovsky, K., MacAlpine, D. M. & Henikoff, S. Epigenome characterization at single base-pair resolution. Proc. Natl Acad. Sci. USA 108, 18318–18323 (2011).
Kent, N. A., Adams, S., Moorhouse, A. & Paszkiewicz, K. Chromatin particle spectrum analysis: a method for comparative chromatin structure analysis using paired-end mode next-generation DNA sequencing. Nucleic Acids Res. 39, e26 (2011).
Jain, M., Olsen, H. E., Paten, B. & Akeson, M. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 17, 239 (2016).
Noll, M. & Kornberg, R. D. Action of micrococcal nuclease on chromatin and the location of histone H1. J. Mol. Biol. 109, 393–404 (1977).
Van Holde, K. Chromatin (Springer-Verlag, NY, 1988).
Lu, X. et al. Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure. Genes Dev. 23, 452–465 (2009).
Chereji, R. V. et al. Genome-wide profiling of nucleosome sensitivity and chromatin accessibility in Drosophila melanogaster. Nucleic Acids Res. 44, 1036–1051 (2016).
Kharchenko, P. V. et al. Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 471, 480–485 (2011).
Wu, C., Wong, Y. C. & Elgin, S. C. The chromatin structure of specific genes: II. Disruption of chromatin structure during gene activity. Cell 16, 807–814 (1979).
Albert, I. et al. Translational and rotational settings of H2A.Z nucleosomes across the Saccharomyces cerevisiae genome. Nature 446, 572–576 (2007).
Yuan, G. C. et al. Genome-scale identification of nucleosome positions in S. cerevisiae. Science 309, 626–630 (2005).
Pointner, J. et al. CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S. pombe. EMBO J. 31, 4388–4403 (2012).
Endow, S. A., Polan, M. L. & Gall, J. G. Satellite DNA sequences of Drosophila melanogaster. J. Mol. Biol. 96, 665–692 (1975).
Lohe, A. R., Hilliker, A. J. & Roberts, P. A. Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. Genetics 134, 1149–1174 (1993).
Hergeth, S. P. & Schneider, R. The H1 linker histones: multifunctional proteins beyond the nucleosomal core particle. EMBO Rep. 16, 1439–1453 (2015).
Woodcock, C. L., Skoultchi, A. I. & Fan, Y. Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length. Chromosome Res. 14, 17–25 (2006).
Blank, T. A. & Becker, P. B. Electrostatic mechanism of nucleosome spacing. J. Mol. Biol. 252, 305–313 (1995).
Fan, Y. et al. H1 linker histones are essential for mouse development and affect nucleosome spacing in vivo. Mol. Cell Biol. 23, 4559–4572 (2003).
Allan, J., Hartman, P. G., Crane-Robinson, C. & Aviles, F. X. The structure of histone H1 and its location in chromatin. Nature 288, 675–679 (1980).
Hayes, J. J. & Wolffe, A. P. Preferential and asymmetric interaction of linker histones with 5S DNA in the nucleosome. Proc. Natl Acad. Sci. USA 90, 6415–6419 (1993).
Simpson, R. T. Structure of the chromatosome, a chromatin particle containing 160 base pairs of DNA and all the histones. Biochemistry 17, 5524–5531 (1978).
Bednar, J. et al. Structure and dynamics of a 197 bp nucleosome in complex with linker histone H1. Mol. Cell 66, 729 (2017).
Weintraub, H. Histone-H1-dependent chromatin superstructures and the suppression of gene activity. Cell 38, 17–27 (1984).
Bresnick, E. H., Bustin, M., Marsaud, V., Richard-Foy, H. & Hager, G. L. The transcriptionally-active MMTV promoter is depleted of histone H1. Nucleic Acids Res. 20, 273–278 (1992).
Kim, A. & Dean, A. A human globin enhancer causes both discrete and widespread alterations in chromatin structure. Mol. Cell Biol. 23, 8099–8109 (2003).
Tsukiyama, T. & Wu, C. Purification and properties of an ATP-dependent nucleosome remodeling factor. Cell 83, 1011–1020 (1995).
Varga-Weisz, P. D. et al. Chromatin-remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II. Nature 388, 598–602 (1997).
Ito, T., Bulger, M., Pazin, M. J., Kobayashi, R. & Kadonaga, J. T. ACF, an ISWI-containing and ATP-utilizing chromatin assembly and remodeling factor. Cell 90, 145–155 (1997).
Emelyanov, A. V. et al. Identification and characterization of ToRC, a novel ISWI-containing ATP-dependent chromatin assembly complex. Genes Dev. 26, 603–614 (2012).
Hanai, K., Furuhashi, H., Yamamoto, T., Akasaka, K. & Hirose, S. RSF governs silent chromatin formation via histone H2Av replacement. PLoS Genet. 4, e1000011 (2008).
Fyodorov, D. V., Blower, M. D., Karpen, G. H. & Kadonaga, J. T. Acf1 confers unique activities to ACF/CHRAC and promotes the formation rather than disruption of chromatin in vivo. Genes Dev. 18, 170–183 (2004).
Corona, D. F. et al. ISWI regulates higher-order chromatin structure and histone H1 assembly in vivo. PLoS Biol. 5, e232 (2007).
Scacchetti, A. et al. CHRAC/ACF contribute to the repressive ground state of chromatin. Life Sci. Alliance 1, e201800024 (2018).
Sovic, I. et al. Fast and sensitive mapping of nanopore sequencing reads with GraphMap. Nat. Commun. 7, 11307 (2016).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Krietenstein, N. et al. Genomic nucleosome organization reconstituted with pure proteins. Cell 167, 709–721.e12 (2016).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Grant, C. E., Bailey, T. L. & Noble, W. S. FIMO: scanning for occurrences of a given motif. Bioinformatics 27, 1017–1018 (2011).
Cherbas, L. et al. The transcriptional diversity of 25 Drosophila cell lines. Genome Res. 21, 301–314 (2011).
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.
The authors declare no competing interests.
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Integrated supplementary information
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).
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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