Corrections

BIOPHYSICS AND COMPUTATIONAL BIOLOGYCorrection for “Structural insights into the histone H1-nucleo-some complex,” by Bing-Rui Zhou, Hanqiao Feng, HidenoriKato, Liang Dai, Yuedong Yang, Yaoqi Zhou, and Yawen Bai,which appeared in issue 48, November 26, 2013, of Proc NatlAcad Sci USA (110:19390–19395; first published November 11,2013; 10.1073/pnas.1314905110).The authors note that, due to a printer’s error, references

41–50 appeared incorrectly. The corrected references follow.

41. Schalch T, Duda S, Sargent DF, Richmond TJ (2005) X-ray structure of a tetranucleo-some and its implications for the chromatin fibre. Nature 436(7047):138–141.

42. Clore GM, Tang C, Iwahara J (2007) Elucidating transient macromolecular interactionsusing paramagnetic relaxation enhancement. Curr Opin Struct Biol 17(5):603–616.

43. Dominguez C, Boelens R, Bonvin AM (2003) HADDOCK: a protein-protein dockingapproach based on biochemical or biophysical information. J Am Chem Soc 125(7):1731–1737.

44. Thakar A, et al. (2009) H2A.Z and H3.3 histone variants affect nucleosome structure:biochemical and biophysical studies. Biochemistry 48(46):10852–10857.

45. Vogler C, et al. (2010) Histone H2A C-terminus regulates chromatin dynamics, re-modeling, and histone H1 binding. PLoS Genet 6(12):e1001234.

46. Wong H, Victor JM, Mozziconacci J (2007) An all-atom model of the chromatin fibercontaining linker histones reveals a versatile structure tuned by the nucleosomal re-peat length. PLoS ONE 2(9):e877.

47. Lee KM, Hayes JJ (1998) Linker DNA and H1-dependent reorganization of histone-DNA interactions within the nucleosome. Biochemistry 37(24):8622–8628.

48. Boulikas T, Wiseman JM, Garrard WT (1980) Points of contact between histone H1and the histone octamer. Proc Natl Acad Sci USA 77(1):127–131.

49. Travers AA, Muyldermans SV (1996) A DNA sequence for positioning chromatosomes.J Mol Biol 257(3):486–491.

50. Goytisolo FA, et al. (1996) Identification of two DNA-binding sites on the globulardomain of histone H5. EMBO J 15(13):3421–3429.

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DEVELOPMENTAL BIOLOGYCorrection for “Organ-specific function of adhesion G protein-coupled receptor GPR126 is domain-dependent,” by ChinmoyPatra, Machteld J. van Amerongen, Subhajit Ghosh, FilomenaRicciardi, Amna Sajjad, Tatyana Novoyatleva, Amit Mogha,Kelly R. Monk, Christian Mühlfeld, and Felix B. Engel, whichappeared in issue 42, October 15, 2013, of Proc Natl Acad Sci USA(110:16898–16903; first published September 30, 2013; 10.1073/pnas.1304837110).The authors note that on page 16902, right column, third full

paragraph, lines 24–25 “5’-CGGGTTGGACTCAAGACGATAG-3’” should instead appear as “5’-ACAGAATATGAATACCTGA-TACTCC-3’.”

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PHYSICSCorrection for “Stable three-dimensional metallic carbon with in-terlocking hexagons,” by ShunhongZhang, QianWang, XiaoshuangChen, and Puru Jena, which appeared in issue 47, November 19,2013, of Proc Natl Acad Sci USA (110:18809–18813; first publishedNovember 4, 2013; 10.1073/pnas.1311028110).The authors note: “Our paper unfortunately missed refer-

ence to an earlier suggestion of the T6 structure (43). This workentitled ‘A hypothetical dense 3,4-connected carbon net andrelated B2C and CN2 nets built from 1,4-cyclohexadienoidunits’ by M. J. Bucknum and R. Hoffmann was published inJ Am Chem Soc 116:11456–11464 (1994), where the electronicstructure of a hypothetical 3,4-connected tetragonal allotrope ofcarbon is discussed. The results in this article are consistent withwhat we find. The same group had also suggested a metalliccarbon structure (44) that was published in J Am Chem Soc105:4831–4832 (1983), which we also missed to cite. We thankProf. Hoffmann for bringing these papers to our attention.”The complete references appear below.

43. Bucknum MJ, Hoffmann R (1994) A hypothetical dense 3,4-connected carbon netand related B2C and CN2 nets built from 1,4-cyclohexadienoid units. J Am Chem Soc116(25):11456–11464.

44. Hoffmann R, Hughbanks T, Kertesz M, Bird PH (1983) Hypothetical metallic allotropeof carbon. J Am Chem Soc 105(14):4831–4832.

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CELL BIOLOGYCorrection for “Visualization of repetitive DNA sequences inhuman chromosomes with transcription activator-like effectors,”by Hanhui Ma, Pablo Reyes-Gutierrez, and Thoru Pederson,which appeared in issue 52, December 24, 2013, of Proc NatlAcad Sci USA (110:21048–21053; first published December 9,2013; 10.1073/pnas.1319097110).The authors note that, due to a printer’s error, references

25–29 appeared incorrectly. The corrected references are:

25. Miyanari Y, Ziegler-Birling C, Torres-Padilla ME (2013) Live visualization of chromatindynamics with fluorescent TALEs. Nat Struct Mol Biol 20(11):1321–1324.

26. Sanjana NE, et al. (2012) A transcription activator-like effector toolbox for genomeengineering. Nat Protoc 7(1):171–192.

27. Ma H, et al. (2012) A highly efficient multifunctional tandem affinity purificationapproach applicable to diverse organisms. Mol Cell Proteomics 11(8):501–511.

28. Uetake Y, et al. (2007) Cell cycle progression and de novo centriole assembly aftercentrosomal removal in untransformed human cells. J Cell Biol 176(2):173–182.

29. Jacobson MR, Pederson T (1997) RNA traffic and localization reported by fluorescencecytochemistry. Analysis of mRNA Formation and Function, ed Richter JD (Academic,New York), pp 341–359.

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Structural insights into the histoneH1-nucleosome complexBing-Rui Zhoua, Hanqiao Fenga, Hidenori Katoa, Liang Daib,c, Yuedong Yangb,c,1, Yaoqi Zhoub,c,1, and Yawen Baia,2

aLaboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892;and bSchool of Informatics and cCenter for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indiana University-PurdueUniversity Indianapolis, Indianapolis, IN 46202

Edited by S. Walter Englander, The University of Pennsylvania, Philadelphia, PA, and approved October 22, 2013 (received for review August 6, 2013)

Linker H1 histones facilitate formation of higher-order chromatinstructures and play important roles in various cell functions.Despite several decades of effort, the structural basis of how H1interacts with the nucleosome remains elusive. Here, we investi-gated Drosophila H1 in complex with the nucleosome, using solu-tion nuclear magnetic resonance spectroscopy and other biophysicalmethods. We found that the globular domain of H1 bridges thenucleosome core and one 10-base pair linker DNA asymmetrically,with its α3 helix facing the nucleosomal DNA near the dyad axis.Two short regions in the C-terminal tail of H1 and the C-terminaltail of one of the two H2A histones are also involved in the for-mation of the H1–nucleosome complex. Our results lead to a resi-due-specific structural model for the globular domain of theDrosophila H1 in complex with the nucleosome, which is differentfrom all previous experiment-based models and has implicationsfor chromatin dynamics in vivo.

Eukaryotic genomic DNA is packaged into chromatin throughassociation with positively charged histones to form the nu-cleosome, the structural unit of chromatin (1–3). The nucleo-some core consists of an octamer of histones with two copiesof H2A, H2B, H3, and H4, around which ∼146 bp of DNA windsin ∼1.65 left-handed superhelical turns (4). At this level ofthe DNA packaging, chromatin resembles a beads-on-a-stringstructure, with the nucleosome core as the beads and the linkerDNA between them as the strings (5). At the next level of DNApackaging, H1 histones bind to the linker DNA and the nucle-osome to further condense the chromatin structure (6, 7). H1-mediated chromatin condensation plays important roles in cel-lular functions such as mitotic chromosome architecture andsegregation (8), muscle differentiation (9), and regulation ofgene expression (10, 11).Linker H1 histones typically are ∼200 amino acid residues in

length, with a short N-terminal region, followed by a ∼70–80-amino acid structured globular domain (gH1) and a ∼100-aminoacid unstructured C-terminal domain that is highly enriched inLys residues. H1 stabilizes the nucleosome and facilitates foldingof nucleosome arrays into higher-order structures (12–15). gH1alone confers the same protection from micrococcal nucleasedigestion to the nucleosome as the full-length H1 does (16). TheN-terminal region of H1 is not important for nucleosome binding(16, 17), whereas the C terminus is required for H1 bindingto chromatin in vivo (18, 19) and for the formation of a stemstructure of linker DNA in vitro (17, 20, 21).The globular domain structures of avian H5 (22) and budding

yeast Hho1 (23), which are both H1 homologs, have been de-termined at atomic resolution and show similar structures. Inaddition, numerous studies have indicated that gH1/gH5 bindsaround the dyad region of the nucleosome (14, 24), leading tomany conflicting structural models for how the globular domainof H1/H5 binds to the nucleosome (SI Appendix, Fig. S1) (24–26). These models are divided into two major classes, symmetricand asymmetric, on the basis of the location of gH1/gH5 in thenucleosome. In the symmetric class, gH1/gH5 binds to the nu-cleosomal DNA at the dyad and interacts with both linker DNAs

(16, 17, 27, 28). In the asymmetric class, gH1/gH5 binds to thenucleosomal DNA in the vicinity of the dyad axis and to 10 bp(27, 29–32) or 20 bp (19, 29, 33, 34) of one linker DNA, oris located inside the DNA gyres, where it interacts with histoneH2A (35). In addition, Zhou and colleagues also characterizedthe orientation of gH5 in the gH5-nucleosome complex (29). Theuse of nonuniquely positioned nucleosomes and indirect meth-ods may have contributed to the differences in these models (SIAppendix, Fig. S1).Multidimensional nuclear magnetic resonance (NMR), and in

particular methyl-based NMR, provides a direct approach to thestructural characterization of macromolecular complexes (36,37). We have previously assigned chemical shifts of the methylgroups of the side chains of residues Ile, Leu, and Val in the corehistones (38) and the backbone amides in the disordered his-tone tails (39), which provide the fingerprints for investigatingthe interactions between H1 and the nucleosome. Here, we usedNMR, along with several other methods, to determine the loca-tion and orientation of the globular domain of a stable mutant ofDrosophila H1 on a well-positioned nucleosome.

ResultsHistone Regions Involved in the Formation of the H1–NucleosomeComplex. To identify the nucleosome-binding regions of H1, wefirst used a gel shift assay to examine the binding of several H1fragments, which contain gH1 and various lengths of C-terminalregions, to the nucleosome centered on 167 bp DNA with theWidom “601” sequence (40), which can uniquely position the

Significance

Linker H1 histones control the accessibility of linker DNA be-tween two neighbor nucleosomes to DNA-binding proteinsand regulate chromatin folding. We investigated the structureof the H1–nucleosome complex through a combination ofmultidimensional nuclear magnetic resonance spectroscopy,site-directed mutagenesis-isothermal-titration calorimetry andcomputational design/modeling. The results lead to a uniquestructural model for the globular domain of H1 in complex withthe nucleosome that contains residue-level information andhave implications for the dynamics of chromatin in vivo. Inaddition, our approach will be useful for testing the hypothesisthat the globular domain of H1 variants might have distinctbinding geometries within the nucleosome, and thereby con-tribute to the heterogeneity of chromatin structure.

Author contributions: B.-R.Z. and Y.B. designed research; B.-R.Z., H.F., L.D., Y.Y., and Y.Z.performed research; H.K. contributed new reagents/analytic tools; B.-R.Z., H.F., L.D., Y.Y.,Y.Z., and Y.B. analyzed data; and B.-R.Z. and Y.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1Present address: Institute for Glycomics and School of Informatics and CommunicationTechnology, Griffith University, Southport, QLD 4222, Australia.

2To whom correspondence may be addressed. E-mail: yawen@helix.nih.gov.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1314905110/-/DCSupplemental.

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nucleosome. To be consistent with later NMR experiments, weused a stable mutant of H1 in which four residues in the hy-drophobic core of gH1 are replaced with the correspondingresidues in gH5 (22) (Fig. 1 A and B and SI Appendix, Fig. S2).This stable H1 mutant is necessary to allow NMR experimentsto be conducted at the high temperature (35 °C) required toobserve the methyl groups in the H1-nucleosome complex (38).In this study, H1 refers to this stable mutant unless specifiedotherwise. We found that all fragments of H1 shift the positionof the nucleosome in the agarose gel (Fig. 1C). In particular,the fragment containing residues 37–211 shifted the nucleo-some as efficiently as H137–256. Using isothermal titration calo-rimetry (ITC), we further showed that H11–256, H137–211, andthe wild type full-length H11–256 have similar binding affinitiesfor the nucleosome (SI Appendix, Fig. S3 and Table S1). There-fore, to avoid signal overlaps in NMR spectra (SI Appendix, Fig.S4A), we chose H137–211 for subsequent NMR studies. Using15N/13C-labeled H137–211, we assigned the observable residuesof H137–211 in complex with the nucleosome (SI Appendix, Fig.S4B). The unobserved residues of H137–211, which are pre-sumably folded into the nucleosome, include gH1 (45–118),residues 119ASAKKEK125 immediately following the globulardomain, and the Lys-rich region 164KKPKAKKAVAT174 in themiddle of the C-terminal tail (Fig. 1 D and E). We furtherdemonstrated that H137–211 and the wild-type full-length H11–256used the same regions to bind to the nucleosome (SI Appendix,Fig. S4C). Similar results were observed when the nucleosomewith 208 bp DNA was used (SI Appendix, Fig. S4D), indicatingthat H1 binds within the 10 bp of the linker DNAs that enter/exitthe nucleosome core particle.To determine whether the histone core of the nucleosome

interacts with H137–211, we compared the methyl spectra of thenucleosome histones in the absence or presence of H137–211. Wefound that H137–211 did not perturb the methyl spectra (SI Ap-pendix, Fig. S5A), indicating that H137–211 and the histone coreare not in direct contact. We then tested whether the C-terminaltails of H2A and the N-terminal tails of H3 interact with H137–211,as they are close to the linker DNA (Fig. 2A). The peak intensities/

volumes of the C-terminal residues (119–122) of the 15N-labeledH2A decreased by about half in the 1H-15N heteronuclear singlequantum correlation (HSQC) spectra on binding of H137–211or the wild-type full-length H11–256, with little changes in theirchemical shifts (Fig. 2 B–E), whereas those in 15N-labeled H3 andthe N-terminal tails of 15N-labeled H2A remain unchanged in bothpeak intensities and chemical shifts (Fig. 2 B–E and SI Appendix,Fig. S5 B and C). Similar results were observed for H137–211 incomplex with the nucleosome array, which has a longer linker DNA(SI Appendix, Fig. S6). These results indicate that one of the twodisordered H2A C-terminal tails in the nucleosome and two shortregions in the disordered C-terminal tails of the wild-type full-lengthH11–256 became folded upon binding of H1 to the nucleosome.

Determination of the Orientation of gH1 on the Nucleosome by PRE.We next performed paramagnetic relaxation enhancement (PRE)experiments to determine the orientation of gH1 in the H137–211-nucleosome complex. In the PRE experiments, residues imme-diately outside the two ends of gH1 (P44 and A119) and on thesurface (L60, A83, and K109) were chosen to minimize the per-turbation to the gH1 structure (Fig. 1B). These residues wereeach individually mutated to Cys and linked to the para-magnetic compound [(S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanethiosulfonate) (MTSL) or ((S-meth-anethiosulfonylcysteaminyl)ethylenediamine-N,N,N′,N′-tetraaceticacid) (MTS-EDTA-Mn2+)] through a disulfide bond. Binding ofparamagnetic spin-labeled H137–211 reduced the peak intensities ofthe observable backbone amide 1H-15N signals in the nucleosomein a manner dependent on distance from the paramagnetic center(42). The peak intensities of residues in the C-terminal tails ofH2A were only weakly affected by MTSL but were strongly af-fected by MTS-EDTA-Mn2+ (Fig. 3 A–D and SI Appendix, Fig.S7), consistent with the anticipated PRE effects from the twotypes of spin labels (42). The intensity changes in the observableresidue T119 in H2A, which is near the folded region of H2A,indicate it is closer to H137–211 residues A83 and K109 than toH137–211 residues L60, A119, and P44 (Fig. 3 A–D, Fig. 1B, andFig. 2A). These results indicate that in the H137–211–nucleosome

Fig. 1. The globular domain and two discrete regions of linker histone H1 bind to the nucleosome. (A) Sequence of H1, highlighting the globular domain(cyan), the quadruple mutations (green and bold), and the C-terminal regions (red) that are involved in the binding of the nucleosome, and secondarystructures. (B) The structural model of the globular domain, which is modeled using the B chain of the gH5 globular domain structure as the template (22).The mutated residues are shown as green sticks. The balls indicate the positions for spin labeling, except that positions 44 and 119 outside the gH1 areindicated with residues 45 and 118 in gH1, respectively. (C) Gel shift assay results for different fragments of H1. The nucleosome contains 167 bp DNAcentered with the 601 sequence. (D) Overlay of 1H-15N HSQC spectra of H1 in free form (blue) and in complex with the nucleosome (black). In this experiment,the ratio of H137–211 to the nucleosome in the complex is ∼0.7. The disappearance of peaks indicates that many residues in H137–211 become folded on bindingof the nucleosome. (E) Deviation of Cα chemical shifts of observable residues of H1 in complex with the nucleosome from random coil values.

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complex, the α3 helix of gH1, which is within the region 83–109, is closer to the nucleosome core than the gH1 N- and C-terminal border residues (P44 and A119) and loop 1 residueL60 (Fig. 1B).

Determination of the Location of gH1 on the Nucleosome by PRE.Wefurther used the methyl groups of Ile, Val, and Leu residues ingH1 as probes to determine the location of gH1 on the nucleo-some. We first assigned most of the methyl groups in gH1 bycombining three approaches (Fig. 3E and SI Appendix, Fig. S8):mutation of specific residues, comparison of the peak positionsof the methyl groups of gH1 in the free form and in complex with

the nucleosome, and spin-labeling effects on the methyl groupsin gH1 in the absence or presence of the nucleosome. Using theassigned methyl groups, we examined the PRE effects on themethyl groups of gH1 with MTSL labeled at position T119 ofH2A or at position K37 of H3 in the nucleosome (Fig. 2A). Theresidues at these positions are disordered in the nucleosome butare close to the nucleosome core, with relatively fixed locations.The spin labels have little effects on the chemical shifts of themethyl groups in gH1, indicating that they do not perturb thestructure of the gH1–nucleosome complex (Fig. 3 E–G). The spin-label at position 119 of H2A has large PRE effects on the methylgroups of L97, V98, and V99 at the C-terminal region of the α3helix of gH1, whereas the spin-label at position 37 of H3 stronglyaffected not only these methyl groups but also L60 in loop 1 andL103 and I104 in the C-terminal region of the β1 strand (Fig. 3 F–I).Because there are two H3 K37 residues in the nucleosome

structure that are separated by ∼65 Å (Fig. 2A), the observationthat methyl groups in one region of gH1 are strongly affected bythe MTSL labels at the H3 K37 sites further indicates that gH1binds to the nucleosome asymmetrically. This is also consistentwith the folding of only one of the two H2A C-terminal tails inthe nucleosome on H137–211 binding (Fig. 2 B and C). Further-more, as MTSL spin labels in gH1 have only small effects on thebackbone amide NMR signals from the disordered H2A T119(Fig. 3A), gH1 must be distant from this disordered H2A C-terminal tail in the H137–211–nucleosome complex. Therefore,the large PRE effects on some methyl groups of gH1 from MTSLlabels at H2A T119 (Fig. 3H) indicate that gH1 is close to thefolded H2A C-terminal tail in the H1–nucleosome complex.

Important H1 Residues for Nucleosome Binding.We used site-directedmutagenesis and ITC to identify gH1 residues that are importantfor the binding of H137–211 to the nucleosome. Positively chargedLys residues on the surface of gH1 were each mutated to Ala, andthe effects of the mutations on the dissociation equilibrium con-stant (KD) were measured by ITC (Fig. 4A and SI Appendix, TableS1). Mutation of each of the six residues (K58, K91, K95, K102,K107, and K116) showed larger effects (> a factor of 2.5) thanothers. These mutations are located on two distinct surfaces onnearly opposite sides of the gH1 structure (Fig. 4B). One surfaceincludes K91 and K95 in the α3 helix. The other surface includesresidues K58 at the C-terminal region of the α1 helix and residuesK102, K107, and K116 in the two β strands. In addition, ITCexperiments showed that gH1 binding to the nucleosome was∼12 times weaker than H137–211 (SI Appendix, Fig. S9). Theseresults are consistent with the NMR observation that residues119ASAKKEK125 and 164KKPKAKKAVAT174 in the middle ofthe C-terminal tail of H11–256 are folded.

A Structural Model for the gH1–Nucleosome Complex. Taking all thedata together, our experimental results suggest that gH1 uses thetwo positively charged surfaces to bridge the nucleosome coreand the linker DNA asymmetrically. GH1 is close to one of thetwo H2A C-terminal tails and one of the two H3 N-terminal tails,with its α3 helix facing the nucleosome core. Using these re-straints and the HADDOCK program (43), we docked the gH1onto the nucleosome by forcing the K91 and K95 residues in theα3 helix of gH1 to interact with the nucleosomal DNA near thedyad region and the K58, K102, K107, and K116 residues tointeract with one of the nearby 10-bp linker DNAs (SI Appendix,Fig. S10). After initial rigid body docking and subsequent energyminimization that allows contacting residues to adopt differentconformations, the calculated low-energy structures were clus-tered. We found that one of the clusters was most consistent withall of the experimental results (Fig. 5 A and B and SI Appendix,Fig. S10D), including the mutation effects on the binding affinitybetween H137–211 and the nucleosome (Fig. 5C), PRE effects onH2A T119 in the nucleosome by spin labels in gH1 (Fig. 5D),

Fig. 2. Asymmetric binding of H137–211 to the nucleosome. (A) Nucleosomestructure, highlighting residues H2A T119 and H3 K37. The DNA structure istaken from the tetra-nucleosome structure with 10 bp DNA at both entryand exit regions (41): H2A is in orange, H3 is in blue, and H4 is in green. (B)Overlay of 1H-15N HSQC spectra of the nucleosome with 15N-labeled H2A inthe absence (black) or the presence (orange) of mutant H137–211. (C) Same asin B except the wild-type full-length H11–256 (magenta) was used instead ofmutant H137–211. Note that only strong peaks in the spectra are shown (SIAppendix, Fig. S7). In this experiment, the ratio of H137–211 to the nucleo-some is 1.0. The dashed lines indicate that the volumes of the peaks in theboxes are integrated together. (D) Ratios of peak volumes of T119, K121,and the region that include K118, E120, and K122 of the H2A tails in thenucleosome in complex with mutant H137–211 to those in the free nucleo-some. (E) Same as in D except that the wild-type full-length H11–256 wasused instead of mutant H137–211. The asterisks in B–E indicate that thepeaks in the dashed box were integrated together when the peak vol-umes were measured.

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and PRE effects on the methyl groups of gH1 by spin labelsat H2A T119 (Fig. 5E) and H3 K37 (Fig. 5F). In the model,although gH1 directly interacts with only one linker DNA, it isvery close to the other linker DNA (Fig. 5B and SI Appendix, Fig.S10E). A slight shift of this linker DNA toward the nucleosomecore could also lead to its interaction with gH1. Therefore, itis possible that gH1 may interact with both DNA linkers, onestrongly and the other weakly (SI Appendix, Fig. S6A).

The C-Terminal Tail of Histone Variant H2A.Z Disfavors H1 Binding.Earlier studies have shown that the nucleosome containing his-tone variant H2A.Z affects H1 binding (44), and the C-terminaltail of H2A.Z may play a role in the reduced binding affinity(45). The amino acid sequence of the very C-terminal region(119TEKKA123) of H2A is different from corresponding residues(123EETVQ127) of H2A.Z. To test the role of the C-terminal tailof H2A.Z in H1 binding, we measured the binding affinity ofH137–211 to the nucleosome containing H2A with the last sixresidues replaced by the corresponding residues of H2A.Z. Wefound that the binding was reduced to an undetectable level byITC (SI Appendix, Fig. S11), whereas deletion of the last fiveresidues of H2A increased KD by a factor of ∼10. These resultsfurther confirm the earlier NMR observation that one of theC-terminal tails of H2A is involved in the formation of theH1–nucleosome complex.

DiscussionOur structural model of the gH1–nucleosome complex differsfrom all previous experiment-based models in either the locationor orientation of gH1/H5 or the length of linker DNA involved.The observation that MTSL labels at H2A T119 or H3 K37 havelarge PRE effects on the methyl groups in only one region ofgH1 clearly shows that the gH1 binds to the nucleosome asym-metrically. For a symmetric binding, large PRE effects on methylgroups of gH1 from spin labels at H3 K37 are not expectedbecause they are separated by more than 20 Å (SI Appendix, Fig.S12A). It is interesting to note that Zhou and coworkers havealso found that gH5 binds to the nucleosome complex asym-metrically, involving 10-bp linker DNA (SI Appendix, Fig. S1)

(29). However, the orientation of gH5 in their model is oppositeto that of gH1 in ours: The α3 helix of gH5 in their model bindsto the linker DNA instead of the nucleosomal DNA, as in ourmodel (SI Appendix, Fig. S12 B and C). One possibility for thedifferences among various models of the gH1/H5–nucleosomecomplex might be that there are multiple gH1/H5 binding modesfor the nucleosome. Another possibility is that earlier models are

Fig. 3. Location and orientation of gH1 on the nucleosome determined by PRE effects. (A–D) PRE effects of spin labeling of H137–211 at positions 44, 60, 83,109, and 119 on the amide protons of H2A C-terminal tail residues T119 and K122 and N-terminal R3 and K5 residues in the nucleosome. (E–G) Methyl-spectraof gH1 in complex with the nucleosome (red) and the nucleosomes with MTSL labels at H2A T119 (orange) and H3 K37 (blue), respectively. (H and I) Bar graphsshowing PRE effects on the methyl groups of gH1 with MTSL label at H2A T119 (orange) and H3 K37 (blue), respectively.

Fig. 4. Effects of mutations in gH1 on the binding affinity of H137–211 to thenucleosome. (A) Effects of mutation of surface residues in gH1 on thebinding affinity between H137–211 and the nucleosome. (B) Structural illus-tration of the distribution of the gH1 residues whose Ala mutations lead toa large decrease in binding affinity.

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derived from less-controlled systems, using indirect methods (SIAppendix, Fig. S1).Among several computation-based models of the gH1/H5–

nucleosome complex (30–32, 46), two of them show similaritiesto our model in terms of both location and orientation of gH1 inthe complex (30, 31). In addition, our model is consistent withseveral other experimental observations. For example, the twoC-terminal tails of H2A in the Xenopus nucleosome cross-link todifferent positions of the DNA in the nucleosome on addition ofH1 (47). Calf H1 is cross-linked to the C-terminal region of H2A(48). The location of gH1 in our model is very close to the dis-ordered negatively charged C-terminal tail of HMGN2 in com-plex with the nucleosome, consistent with its role in inhibitingthe binding of H1 to the nucleosome (38). Binding of H1 to the10-bp linker DNA that neighbors the nucleosome core particlehas been used to explain the preferential “out-of-phase” pop-ulation of AT bases in the region (31, 49). The identification oftwo major discrete DNA-binding surfaces of gH1 in our muta-tion studies is in excellent agreement with earlier mutationstudies of the histone H5 globular domain (50) and Mouse H10,using fluorescence recovery after photo-bleaching methods (19)(SI Appendix, Fig. S13). In addition, within the limit of the res-olution, the tetra-nucleosome crystal structure can accommodategH1 in our model (41) with only small clashes with the linkerDNA that connects the neighboring nucleosomes (SI Appendix,Fig. S14), suggesting that a small structural rearrangement of thelinker DNA could allow H1 to condense chromatin to the two-start zigzag nucleosome high-order structure (7, 51).Our finding that two discrete regions in the C-terminal tail of

H1, 119ASAKKEK125 and 164KKPKAKKAVAT174, are involvedin nucleosome binding is consistent with the earlier experimentalobservations that the C-terminal tail of H1 plays an importantrole in nucleosome binding and chromatin structure condensa-tion (18, 20, 21). In particular, our results support the con-clusions that specific regions in the C-terminal tail of H1, ratherthan the distribution of positively charged residues, are re-sponsible for interaction with linker DNA (17, 18, 52, 53). In the

case of human H1.5, it has been shown that the seven residuesfollowing the globular domain, 121PKAKKAG127, are necessaryfor the formation of the linker DNA stem structure (17). Six ofthese seven residues are conserved in the 164KKPKAKKAVAT174region of the C-terminal tail of Drosophila H1. Our results showthat NMR provides a powerful tool for identifying specific nucle-osome-binding regions in the C-terminal tail of H1.It has been shown that the H2A.Z-containing nucleosome is

typically present at regions flanking the nucleosome-free region(54), near the DNA double-strand break (55), and at theboundary of euchromatin and heterochromatin, which preventsspread of heterochromatin regions (56). In general, these chro-matin regions are less condensed and more dynamic. Our findingthat the very last several residues at the H2A.Z C-terminal tailinhibit the binding of H1 to the H2A.Z nucleosome provides apossible mechanistic explanation for the dynamic features of theH2A.Z-enriched chromatin regions.Finally, our approach provides an experimental tool for testing

the hypothesis that the globular domains of individual H1 variantsmight have distinct binding geometries within the nucleosome thatcontribute to the heterogeneity of chromatin structure (9, 19, 57).

Materials and MethodsDesign of a Stable Mutant of gH1 and Preparation of Samples. The gH1 structuremodel was built using homology modeling and the structure of gH5. Proteindesign programs were used to select the mutation (V53I, S56A, C81V, and A97L)that stabilizes gH1 structure. The Drosophila H1 gene was synthesized andinserted into the pET42b vector.Mutationsweremade using theQuikChange kit.Proteins were expressed in Escherichia coli and purified using chromatography.MTSL or MTS-EDTA was linked to the protein, with the target site mutated toCys. Nucleosomeswere reconstituted by stepwise salt dialysis in the absence ofreducing agent, followed by HPLC to remove free DNA and immature nucle-osomes (SI Appendix, Materials and Methods and SI Appendix, Fig. S15).

NMR, PRE, and ITC Experiments. Isotope-labeledH1mixedwith the nucleosome,or vice versa. The spin-labeled H1 was mixed with nucleosomes containing 15N-labeled histones or vice versa. 1H-15N HSQC or 1H-13C heteronuclear multiplequantum correlation (HMQC) spectra were recorded. The NMR peak

Fig. 5. An asymmetrical structural model of the gH1-nucleosome complex. (A and B) Structural model of the gH1-nucleosome complex in surface and ribbonrepresentations. The rectangular frame approximately shows the enlarged regions in C–F. (C) Illustration of the Lys residues that show a large increase in KD(> a factor of 2.5) when mutated to Ala. (D) Illustration of the disordered residue H2A_T119 and residues P44 (H45), L60, A83, K109, and A119 (S118) in gH1 inthe gH1-nucleosome complex. (E) Illustration of the ordered residue H2A_T119 and the methyl groups in gH1. The methyl groups in red have an Ipara/Idia[Intensity (paramagnetic)/Intensity (diamagnetic)] of less than 0.3, whereas other methyl groups in cyan have larger values. (F) Illustration of one of the twoH3_K37 residues and the methyl groups in gH1. The methyl groups in red have Ipara/Idia of less than 0.3, whereas methyl groups in cyan have larger values.

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intensities or volumes were measured. ITC experiments were performedon a VP–ITC microcalorimeter (Microcal). The dissociation constant (KD)and the stoichiometry of binding (n) were determined by fitting theobserved binding curves to the model with independent n and KD values(SI Appendix, Materials and Methods).

Docking Calculation.We docked gH1 to the 167-bp DNA obtained from thetetra-nucleosome structure using HADDOCK program. Residues Lys91 andLys95 in gH1 were forced to interact with the nucleosomal DNA near the

dyad; the K58, K102, K107, and K106 residues were forced to interact withthe nearby linker DNA. A cluster of structures that is most consistent withthe experimental data were selected as the final model (SI Appendix,Materials and Methods).

ACKNOWLEDGMENTS. We thank Dr. Jemima Barrowman for editing themanuscript. This work is supported by the intramural research programof National Cancer Institute and National Institutes of Health (NIH)(to Y.B.) and by the National Institute of General Medical Sciences of theNIH (R01GM085003, to Y.Z.).

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