Asymmetric unwrapping of nucleosomal DNApropagates asymmetric opening anddissociation of the histone coreYujie Chena,1, Joshua M. Tokudaa,1, Traci Toppingb, Steve P. Meisburgera,2, Suzette A. Pabita, Lisa M. Glossb,3,and Lois Pollacka,3
aSchool of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853; and bSchool of Molecular Biosciences, Washington State University,Pullman, WA 99164
Edited by Taekjip Ha, Johns Hopkins University, Baltimore, MD, and approved December 6, 2016 (received for review July 8, 2016)
The nucleosome core particle (NCP) is the basic structural unit forgenome packaging in eukaryotic cells and consists of DNA woundaround a core of eight histone proteins. DNA access is modulatedthrough dynamic processes of NCP disassembly. Partly disassembledstructures, such as the hexasome (containing six histones) and thetetrasome (four histones), are important for transcription regulationin vivo. However, the pathways for their formation have beendifficult to characterize. We combine time-resolved (TR) small-angleX-ray scattering and TR-FRET to correlate changes in the DNAconformations with composition of the histone core during salt-induced disassembly of canonical NCPs. We find that H2AH2B his-tone dimers are released sequentially, with the first dimer beingreleased after the DNA has formed an asymmetrically unwrapped,teardrop-shape DNA structure. This finding suggests that the octa-some-to-hexasome transition is guided by the asymmetric unwrap-ping of the DNA. The link between DNA structure and histonecomposition suggests a potential mechanism for the action of pro-teins that alter nucleosome configurations such as histone chaper-ones and chromatin remodeling complexes.
contrast variation SAXS | FRET | nucleosomes | hexasome | time resolved
Genome access is highly regulated through the hierarchicalorganization of proteins and nucleic acids within the cellnucleus. The nucleosome core particle (NCP) is the first level of thishierarchy (1) and contains two dimers of H2AH2B histones and an(H3H4)2 tetramer that is assembled as a dimer of dimers. Aroundthis symmetric octamer core, 146 base pairs of dsDNA are wrap-ped in 1.7 superhelical turns (1, 2). The NCP structure physicallyimpedes access to DNA, but is dynamically modulated by numerousmechanisms: posttranslational modification (PTM) of histones, in-corporation of histone variants, DNA sequence-dependent effects,and the actions of extrinsic protein factors (e.g., histone chaperones,ATP-dependent remodeling complexes, and histone PTM bindingproteins) (3, 4).Studies of the intrinsic properties and dynamics of NCPs are
critical for understanding how nuclear machinery gains DNA ac-cess in vivo (3, 5, 6). Insight into the nature of partially unfoldedNCP structures has been gleaned from in vitro studies of NCPassembly and disassembly. Intermediate species with partiallyunwrapped DNA (5, 7), disrupted histonehistone interfaces (8, 9),and dissociation of one (hexasomes) or two (tetrasomes) H2AH2B dimers have been reported (1012). Some of these NCP in-termediates have been directly connected to chromatin functionin vivo. For example, the hexasome is formed by the action ofRNA Pol II (13) and the essential histone chaperone FACT (14).In addition to equilibrium studies, the kinetics of nucleosome
assembly and disassembly have been characterized by bulk andsingle-molecule methods, including Frster resonance energytransfer (FRET) (7, 8, 1517), atomic force microscopy (AFM)(9, 18), force spectroscopy (1921), and small-angle X-ray scat-tering (SAXS) (10, 22). Many studies focused primarily on specificDNAhistone contacts and local conformational changes. Few, if
any studies, use complementary methods to directly compare, onsimilar kinetic time scales, the structural changes of the DNA andhistone components of the NCP. A major gap in our understand-ing of NCP disassembly arises from our limited knowledge ofthe coordination between DNA conformation and histone corecomposition.Because the NCP proteinDNA complex is stabilized predomi-
nantly by polycationpolyanion interactions, the in vitro equilibriumand kinetic properties can be manipulated by ionic solvent condi-tions. NaCl has been widely used to study partially assembled, bi-ologically relevant NCP species that are marginally populated underphysiological conditions (5, 16, 23, 24). The use of recombinanthistones and the Widom 601 DNA sequence (selected for its abilityto form stable, well-positioned nucleosomes) (25, 26) allows pro-duction of large amounts of homogeneous NCPs (601-NCP) forbiophysical studies. Fig. 1 shows the NaCl-induced disassemblypathway for 601-NCPs (7, 8, 23, 24). Whereas the various speciesshown in Fig. 1 have been detected at equilibrium, much less isknown about the kinetics of NCP disassembly, including the rele-vant transition times and pathways between states, or the potentialfor coordination between DNA unwrapping and disruption ofhistonehistone interfaces.Our recent time-resolved SAXS (TR-SAXS) study of salt-
induced NCP dissociation revealed asymmetric DNA releasefrom the histone octamer (22). In kinetic jumps from 01.9 M
Nucleosomes are fundamental proteinDNA structures throughwhich eukaryotes package and organize DNA inside the nucleus.Nucleosomes are disassembled to gain access to the critical in-formation stored in DNA. Here, we describe a new experimentalapproach that characterizes the kinetics of nucleosome disassem-bly and the synergy between DNA conformation and proteincomponents. Using NaCl to disrupt electrostatic interactions, weidentify kinetic pathways and transient intermediates that revealhow DNA unwrapping and protein dissociation are linked in thismacromolecular complex. These dynamic structures may providenew insight into the regulation of DNA access during transcription,replication, and repair.
Author contributions: Y.C., J.M.T., T.T., L.M.G., and L.P. designed research; Y.C., J.M.T.,T.T., S.P.M., S.A.P., and L.P. performed research; Y.C., J.M.T., T.T., and L.M.G. analyzeddata; and Y.C., J.M.T., L.M.G., and L.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the Small AngleScattering Biological Data Bank (SASBDB), https://www.sasbdb.org/ (accession nos.SASDBS7 and SASDBT7).1Y.C. and J.M.T. contributed equally to this work.2Present address: Department of Chemistry, Princeton University, Princeton, NJ 08544.3To whom correspondence may be addressed. Email: LP26@cornell.edu or email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1611118114/-/DCSupplemental.
334339 | PNAS | January 10, 2017 | vol. 114 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1611118114