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Large-scale ATP-independent nucleosome unfolding by a histone chaperone

Nature Structural & Molecular Biology volume 23, pages 1111–1116 (2016)Cite this article

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Abstract

DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy. FACT binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.

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Figure 1: Experimental approach to analyze the effect of FACT on nucleosome structure and dynamics.
Figure 2: Nucleosome unfolding by yFACT is extensive and reversible.
Figure 3: Nucleosome unfolding by yFACT involves the majority of nucleosomal DNA.
Figure 4: Uncoiling of nucleosomal DNA by FACT occurs via an all-or-none mechanism.
Figure 5: Conditional mutations in FACT(Spt16-11) affect nucleosome unfolding in vitro.
Figure 6: FACT-dependent uncoiling of nucleosomal DNA.

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Acknowledgements

We thank D. Gaykalova for help with designing the fluorescent probes. This work was supported by NIH grants GM58650 to V.M.S. and R01GM064649 to T.F., and by the Program of the Presidium of the Russian Academy of Sciences 'Basic Research for the Development of Biomedical Technologies' (FIMT-2014-011). Part of this work was performed with the equipment of the Center for Collective Use 'Genom' of the Institute of Molecular Biology, Russian Academy of Sciences (http://www.eimb.ru/RUSSIAN_NEW/INSTITUTE/ccu_genome_c.php/) supported by the Ministry of Education and Science of the Russian Federation (agreement no. 14.621.21.0001, unique project identification number RFMEFI62114X0001). Development and applications of spFRET were supported by the Russian Science Foundation grant 14-24-00031. Facilities of the Supercomputing Center of Lomonosov Moscow State University were used for the modeling of FRET in nucleosomes. LSM710-Confocor3 microscope was granted by the M.V. Lomonosov Moscow State University Program of Development.

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Authors and Affiliations

  1. Biology Faculty, Lomonosov, Moscow State University, Moscow, Russia

    Maria E Valieva, Grigoriy A Armeev, Kseniya S Kudryashova, Nadezhda S Gerasimova, Alexey K Shaytan, Olga I Kulaeva, Mikhail P Kirpichnikov, Vasily M Studitsky & Alexey V Feofanov

  2. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia

    Kseniya S Kudryashova, Mikhail P Kirpichnikov & Alexey V Feofanov

  3. Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA

    Olga I Kulaeva & Vasily M Studitsky

  4. Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah, USA

    Laura L McCullough & Tim Formosa

  5. Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia

    Pavel G Georgiev & Vasily M Studitsky

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Contributions

M.E.V. constructed templates, designed and performed spFRET experiments, analyzed spFRET data, performed gel-shift and native gel experiments and wrote the manuscript; G.A.A. designed and performed computer modeling, developed a program for spFRET raw data analyses and contributed to writing the manuscript; K.S.K. designed and managed spFRET experiments; N.S.G. designed and performed fluorescence-marker gel-shift experiments, interpreted results and designed some experiments; A.K.S. designed computer modeling and wrote the manuscript; O.I.K. designed templates and analyzed the data; L.L.M. purified yFACT and some histones; T.F. wrote the manuscript and interpreted data; P.G.G. interpreted data; M.P.K. interpreted data; V.M.S. purified donor chromatin, designed experiments, interpreted results and wrote the manuscript; A.V.F. designed spFRET experiments, interpreted results and wrote the manuscript.

Corresponding authors

Correspondence to Vasily M Studitsky or Alexey V Feofanov.

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Integrated supplementary information

Supplementary Figure 1 Analysis of FRET efficiency in unfolded nucleosomes by molecular modeling.

On the left: Color-coded maps of predicted FRET efficiencies for molecular models of labeled nucleosomes with different degree of DNA uncoiling (see Online Methods). The red and blue areas on the color maps for all labels correspond to the structures where all three label pairs are in low or high FRET conformations, respectively. Shaded rectangles correspond to native structures, in which conformations are close to those expected from the crystal structure. On the right: Models of nucleosome conformations corresponding to positions numbered in cyan on the graphs.

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Supplementary Figure 1 (PDF 224 kb)

Supplementary Data Set 1

Uncropped gel images (PDF 385 kb)

Models of FACT-dependent uncoiling of nucleosomal DNA

Previously suggested models of nucleosome unfolding. Unfolding of intact nucleosomes could occur via: DNA uncoiling from an intact histone octamer, DNA uncoiling accompanied by octamer disassembly, without or with disruption of the H3:H3 dimer interface, opening of the (H3-H4) dimer-dimer interface without further DNA uncoiling. (MOV 8027 kb)

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Valieva, M., Armeev, G., Kudryashova, K. et al. Large-scale ATP-independent nucleosome unfolding by a histone chaperone. Nat Struct Mol Biol 23, 1111–1116 (2016). https://doi.org/10.1038/nsmb.3321

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