Histone H3 phosphorylation near the nucleosomedyad alters chromatin structureJustin A North1 Marek Simon1 Michelle B Ferdinand2 Matthew A Shoffner1

Jonathan W Picking1 Cecil J Howard23 Alex M Mooney1 John van Noort4

Michael G Poirier123 and Jennifer J Ottesen23

1Department of Physics The Ohio State University Columbus OH 43210 USA 2Department of Chemistry andBiochemistry The Ohio State University Columbus OH 43210 USA 3Ohio State Biochemistry ProgramThe Ohio State University Columbus OH 43210 USA and 4Huygens-Kamerlingh Onnes LaboratoryLeiden University The Netherlands

Received April 18 2013 Revised January 27 2014 Accepted February 3 2014

ABSTRACT

Nucleosomes contain 146 bp of DNA wrappedaround a histone protein octamer that controlsDNA accessibility to transcription and repaircomplexes Posttranslational modification (PTM) ofhistone proteins regulates nucleosome function Todate only modest changes in nucleosome structurehave been directly attributed to histone PTMsHistone residue H3(T118) is located near the nucleo-some dyad and can be phosphorylated This PTMdestabilizes nucleosomes and is implicated in theregulation of transcription and repair Here wereport gel electrophoretic mobility sucrosegradient sedimentation thermal disassemblymicrococcal nuclease digestion and atomic forcemicroscopy measurements of two DNAndashhistonecomplexes that are structurally distinct from nu-cleosomes We find that H3(T118ph) facilitates theformation of a nucleosome duplex with two DNAmolecules wrapped around two histone octamersand an altosome complex that contains one DNAmolecule wrapped around two histone octamersThe nucleosome duplex complex forms withinshort 150 bp DNA molecules whereas altosomesrequire at least 250 bp of DNA and form repeatedlyalong 3000 bp DNA molecules These results are thefirst report of a histone PTM significantly altering thenucleosome structure

INTRODUCTION

Eukaryotic DNA is organized into chromatin whichconsists of nucleosomes containing 146 bp wrapped

165 times around H2A H2B H3 and H4 histoneprotein octamers (1) This structure helps organize eukary-otic genomes within the cell nucleus and controls DNAndashprotein interactions to regulate DNA processing such astranscription replication and repair Nucleosome struc-ture and function are regulated by a number of factorsincluding histone posttranslational modifications (PTMs)(2) chromatin remodeling complexes (34) and histonechaperones (56)Over 100 histone PTMs have been reported (7)

A majority of histone PTMs are located on disorderedN-terminal histone tails which appear to function byproviding binding sites for additional protein complexes(8) and by impacting higher order chromatin compaction(910) A number of histone PTMs have been identifiedwithin the structured nucleosome core (11) including10ndash20 histone PTMs that have been identified within theDNAndashhistone interface (12ndash15) These are poised todirectly alter DNAndashhistone interactions Many of thesemodifications impact nucleosome stability mobility andunwrapping (16ndash20) demonstrating that histone PTMscan modulate nucleosome physical propertiesFurthermore lysine acetylation in the nucleosome dyadis reported to facilitate transcription in human cells bydestabilizing nucleosomes (21) These studies support anadditional model of histone PTM function in whichmodifications regulate transcription and DNA repairby directly controlling nucleosome stability andordynamics (22)There have been a number of nucleosome crystal struc-

tures solved with histone PTMs (23) and histone mutantsincluding H3(T118A) H3(T118I) and H3(T118H) (2425)but none of these studies have demonstrated a significantimpact of a single histone residue on the overall nucleo-some structure While these changes appear to influencethe free energy of DNAndashhistone interactions they have

To whom correspondence should be addressed Tel +614 292 4525 Fax +614 292 1685 Email ottesen1osueduCorrespondence may also be addressed to Michael G Poirier Tel +614 688 0742 Fax +614 292 7557 Email mpoiriermpsohio-stateedu

Nucleic Acids Research 2014 1ndash12doi101093nargku150

The Author(s) 2014 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicensesby-nc30) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly cited For commercialre-use please contact journalspermissionsoupcom

Nucleic Acids Research Advance Access published February 21 2014 by guest on February 23 2014

httpnaroxfordjournalsorgD

ownloaded from

not been reported to alter the overall structure of thenucleosomeThreonine 118 of histone H3 is located in the DNAndash

histone interface near the nucleosome dyad symmetry axis(Figure 1A) where significant DNAndashhistone interactionsoccur and was determined by mass spectrometry to be asite of phosphorylation [H3(T118ph)] (12) Genetic studiesfound that either a glutamic acid or alanine substitutionfor this residue is lethal in haploid yeast while low-levelexpression of H3(T118E) and H3(T118A) resulted indefects in transcriptional silencing (27) In additionH3(T118I) is a SWISNF INdependent (SIN) mutationwhere this substitution relieves the requirement of theSWItchSucrose NonFermentable (SWISNF) chromatinremodeling complex for mating type switching in buddingyeast (28) and either mutation H3(T118I) (29) orH3(T118H) (30) lowers the transcriptional barrier forPol II Our recent studies of single nucleosomes containing

H3(T118ph) found that this phosphorylation dramaticallyenhanced nucleosome mobility reduced DNAndashhistonebinding and facilitated nucleosome disassembly by SWISNF (20) This suggested that phosphorylation within theDNAndashhistone interface significantly impacts nucleosomestability In addition we noted that an additional DNAndashhistone complex was detected by electrophoretic mobilitygel shift assay (EMSA) following nucleosome reconstitu-tion in our previous studies we purified these away bysucrose gradient centrifugation (20)

Here we report EMSA sucrose gradient sedimentationthermal disassembly micrococcal nuclease (MNase) diges-tion and atomic force microscopy (AFM) measurements ofthese additional DNAndashhistone complexes that containH3(T118ph) These measurements support a model inwhich phosphorylation of H3(T118) induces the formationof two alternate DNAndashhistone complexes that involveDNA wrapping around two complete histone octamers

C

Nuc

Nuc Duplex

A

Nuc Duplex

DNuc Duplex

Altosome

E F

Nuc Duplex

DNAunmodT118ph

Flu

ore

scen

ce(A

U)

0

05

10B

Sucrose Fraction Number

Figure 1 Phosphorylated H3(T118) forms altered DNAndashhistone complexes (A) Nucleosome crystal structure (26) with histone H3 in blue andH3(T118ph) in red (B) Integrated fluorescence intensity of each fraction for sucrose gradient purification of mp2-247 DNA alone (gray) mp2-247reconstituted with unmodified histone octamer (HO) (red) and mp2-247 reconstituted with HO containing H3(T118ph) (blue) Fraction numbers in(B) correspond to the fractions resolved by EMSA in Supplementary Figure S1C for the H3(T118ph) sample (C) EMSA of NPS-192 DNAsreconstituted with either unmodified or H3(T118ph) histone octamer (D) EMSA of DNAndashhistone complexes with NPS-147 DNAs containingeither the mp2 or L variegatus 5S NPS reconstituted with unmodified H3(T118E) or H3(T118ph) HO The gray sphere superimposed on theDNA pictogram indicates a nucleosome and its position on the DNA (E) EMSA of NPS-187 DNA reconstituted with unmodified H3(T118E) orH3(T118ph) HO (F) EMSA of NPS-247 DNAs reconstituted with unmodified HO or HO containing H3(T118ph)

2 Nucleic Acids Research 2014

by guest on February 23 2014httpnaroxfordjournalsorg

Dow

nloaded from

arranged edge-to-edge We observe a lsquonucleosome duplexrsquocomplex that involves two short DNA molecules wrappedaround two histone octamers and an lsquoaltosomersquo complexthat involves one DNA molecule wrapping around twohistone octamers We refer to these complexes as nucleo-some duplexes and altosomes because of their similarityto structures formed by SWISNF chromatin remodeling(31ndash33) We find that nucleosome duplexes form with shortDNA molecules (147 187 and 247 bp) whereas altosomesform only with DNA molecules longer than 247 bp andrepeatedly form on 3 kb DNA molecules These resultssuggest that phosphorylation within the nucleosome dyadcan significantly impact nucleosome structure which couldplay a role in the regulation of RNA transcription andDNA repair

MATERIALS AND METHODS

DNA constructs

The mp2-147 mp2-187 mp2-192 and mp2-247 DNA mol-ecules were prepared as previously described (34) from thepMP2 plasmid (35) The 5S-147 5S-187 5S-192 and 5S-247molecules were prepared by polymerase chain reactionfrom the plasmid p5S in which the mp2 positioningsequence in pMP2 was replaced with the Lytechinusvariegatus 5S RNA positioning sequence (36) The 5SX-192 Pho5-N1 Pho5-N2 and Gal4-192 molecules wereprepared from plasmids containing the Xenopus borealis5S RNA positioning sequence (37) the Pho5 promotercontaining the first (N1) and second (N2) nucleosome pos-itions of the upstream activator sequence (UASg) (38) andthe Gal1-Gal10 UASg containing the nucleosome pos-itioning sequence over the Gal4 binding site (39) respect-ively The 601-17mer array was prepared as previouslydescribed (16)

Preparation of DNAndashhistone complexes

Nucleosomes nucleosome duplexes and altosomes wereprepared by salt double dialysis (34) Purified histoneoctamer and DNA were mixed at a molar ratio of 1125for single nucleosome positioning sequence (NPS) tem-plates and at a mass ratio of 1125 and 1133 for 601-2mer and 601-17mer templates respectively in 2M NaCl5mM Tris pH 80 05mM ethylenediaminetetraaceticacid (EDTA) and 1mM benzamidine Samples weredialyzed into 5mM Tris pH 80 1mM EDTA and1mM benzamidine and purified by sucrose gradient cen-trifugation (34) Addition of 05mM MgCl2 was requiredto maintain stability during the purification ofH3(T118ph) nucleosomes H3(T118ph) was prepared byexpressed protein ligation as previously described (2040)Wild-type histones H3(T118E) H2A(C0) and H4(C0)were prepared by recombinant expression in Escherichiacoli and purified as previously described (41)

Phosphatase treatment

Dependence of nucleosome duplex and altosome forma-tion on H3(T118) phosphorylation was determined bytreating unmodified or H3(T118ph)-containing HO with

Antarctic phosphatase (AP New England Biolabs)Octamers at 1mgml were incubated with 4Uml AP in25mM TrisndashHCl 1mM MgCl2 01mM ZnCl2 1MNaCl pH 80 at 37C for 30 min Reactions werequenched by addition of EDTA to 25mM final concen-tration Treated samples were desalted by ZipTipC18(EMD Millipore) before assay by MALDI-TOF MS(Bruker Daltonics Microflex) Spectra were processedwith SavitzkyndashGolay smoothing and peaks were pickedusing the Centroid algorithm in Bruker FlexAnalysissoftware unprocessed data are shown in Figure 2Treated samples were used for nucleosome reconstitutionwith mp2-187 and mp2-247 DNA according to aboveprotocols

Fluorescence measures of relative histone and DNAcontent

The ratio of H2AH2B heterodimer to DNA or H3H4tetramer to DNA of unmodified nucleosomes comparedwith H3(T118ph)-containing nucleosomes nucleosomeduplexes and altosomes were quantified using Cy5-labeled H2A Alexa488-labeled H4 and Cy3-labeledDNA as follows H2A(C0-Cy5) and H4(C0-Alexa488)were labeled before refolding into the histone octamer aspreviously reported (17) (Supplementary Figure S8A)Labeling efficiency was 50ndash60 as determined by ultra-violet-visible absorbance and MALDI-TOF MS (data notshown) fluorescent histones from the same preparationwere used to refold both H3(T118ph)-containing and un-modified octamer to control for labeling efficiencyFluorescent octamer was reconstituted onto Cy3-end-labeled mp2-147 mp2-187 and mp2-247 resolved by 5native polyacrylamide gel electrophoresis (PAGE) in 03Tris-borate-EDTA (TBE) at 20Vcm imaged on aTyphoon 8600 variable mode imager (GE Healthcare)and quantified using ImageQuant Alexa488 fluorescenceis resolved by 488 nm laser excitation and imaging with a520plusmn20nm bandpass emission filter Cy3 by 532 nm ex-citation laser and 610 nmplusmn20nm emission filter Cy5 by633 nm excitation laser and 670plusmn20nm emission filterReconstituted samples were verified for absence of fluor-escence resonance energy transfer between the three fluor-escent tags on a Fluoromax 3 spectrofluorometer (Horiba)(Supplementary Figure S8C) To validate that the threefluorescent dyes are spectrally separable by the Typhoonimager three different DNA molecules were end-labeledwith Alexa488 Cy3 and Cy5 and resolved by nativePAGE gel before imaging (Supplementary Figure S8B)

Thermal disassembly

Purified nucleosome duplexes and altosomes containingmp2-187 or mp2-247 DNA were diluted to 50 nM in20mM Tris (pH 80) and heated at 53C for 0 and 90min Reactions were quenched by transfer of 1 ml of theheated nucleosomes into 6 ml of 3 sucrose in 02 TBESamples were analyzed by PAGE with 02 TBE The gelwas pre-run for 3 h before running the samples for 3 h at20 Vcm at 4C with continuous buffer recirculation Gelswere imaged with a Typhoon 8600 variable mode imager(GE Healthcare) and quantified by ImageQuant

Nucleic Acids Research 2014 3

by guest on February 23 2014httpnaroxfordjournalsorg

Dow

nloaded from

Nap1 assembly

Unmodified and H3(T118ph)-containing nucleosomeswere assembled from DNA and purified octamer byyNap1 His6-tagged yNap1 (generous gift from ToshioTsukiyama) was expressed and purified as previouslyreported (42) DNA at 125 ngml and HO at 125 ngmlwere incubated with 0ndash72 mM yNap1 dimer at 30C for60 min in 130mM NaCl 05mM MgCl2 75mM Tris pH75 025mM EDTA 025mM dithiothreitol 01mgmlbovine serum albumin 25 glycerol 001 NP40 and001 Tween20 Reactions were then resolved by PAGEwith 5 polyacrylamide and 03 TBE at 20Vcm at 4Cwith continuous buffer recirculation

Exonuclease III mapping

The nucleosome positions within the mp2-187 and mp2-247 DNA molecules were determined with Exonuclease III(ExoIII) mapping as previously reported (34) Reactionswere carried out in an initial volume of 50 ml with 10 nMnucleosomes and 50Uml of ExoIII (New EnglandBiolabs) in 20mM Tris 05mM MgCl2 pH 80 at 16C

MNase footprinting

Histone protection of the mp2-187 and mp2-247 DNAmolecules was determined by MNase digestionReactions were carried out in an initial volume of 10 mlwith 10 nM nucleosomes and 0ndash40 Uml of MNase (NewEngland Biolabs) in 20mM Tris pH 80 05mM CaCl2 at37C to prevent H3(T118ph) nucleosome disassemblyAfter 20 min reactions were quenched with 15mMEDTA final concentration and resolved by PAGE Gelswere stained with SYBR Gold (Invitrogen) and imaged bya Typhoon 8600 variable mode imager (GE Healthcare)

AFM imaging

For imaging of mp2-147 and mp2-247 purified nucleo-somes were fixed by dilution to 05 nM in 05 TrisEDTA 05mM MgCl2 and 001 glutaraldehyde and in-cubation on ice for 30 min for 601-2mer and 601-17merpurified arrays were diluted to 05 nM in 05 Tris EDTASamples were then deposited on poly-D-lysinendashtreatedmica surface as previously described (16) Samples wereimaged with a Dimension Icon with ScanAsyst SPM(Bruker) using Peak Force Mode and ScanAsyst Air tips(Bruker) with a scan rate of 1Hz and 01 pN peak forceImages were processed and analyzed with Gwyddion 219open source software

RESULTS

EMSA analysis indicates that H3(T118ph) induces theformation of nucleosome duplexes and altosomesindependent of DNA sequence

During our initial studies of the impact of H3(T118ph) onnucleosome stability and dynamics we carried out nucleo-some reconstitutions by salt dialysis (41) In addition tocanonical nucleosomes we observed the formation ofDNAndashhistone complexes with a significantly altered elec-trophoretic mobility which sediment about two timesfurther on a sucrose gradient than canonical nucleosomes(Figure 1 and Supplementary Figure S1) consistent withthe formation of a DNAndashhistone complex twice the size ofa canonical nucleosome We used sucrose gradient centri-fugation to separate and purify canonical nucleosomesand these alternate complexes which allowed us to char-acterize the impact of H3(T118ph) on the stabilitymobility and remodeling of canonical nucleosomes (20)

C

AP - - + +

780076007400mz

A unmod- AP

T118ph- AP

T118ph+ AP

mp2-247

Nuc Duplex

Altosome

B

AP - - + +

mp2-187

Nuc Duplex

+2

+2

+2

Figure 2 Formation of nucleosome duplexes and altosomes are H3(T118ph)-dependent (A) (Top unmodAP) mass spectrum of unmodified HO(H3 [M+2H]2+ mz expected 7637 observed 7637) (Middle T118phAP) H3(T118ph) HO before AP treatment (H3(T118ph) [M+2H]2+ mzexpected 7677 observed 7676) and (Bottom T118ph+AP) H3(T118ph) HO after AP treatment (H3(T118) ([M+2H]2+ mz expected 7637observed 7634) (B and C) EMSA of mp2-187 and mp2-247 DNArsquos respectively reconstituted with unmodified HO without AP treatment (lane1) H3(T118ph) HO without AP (lane 2) unmodified HO after AP (lane 3) and H3(T118ph) HO after AP (lane 4)

4 Nucleic Acids Research 2014

by guest on February 23 2014httpnaroxfordjournalsorg

Dow

nloaded from

Here we have investigated the nature of these alternatemobility complexes that were consistently observed byEMSA of nucleosome reconstitutions carried out withH3(T118ph)

Alterations in electrophoretic mobility within polyacryl-amide gels are sensitive to DNA length DNA sequenceand position of the histone octamer along the DNAmolecule (4344) To rule out DNA effects on the forma-tion of these alternate mobility complexes we firstinvestigated the influence of DNA sequence on the elec-trophoretic mobility of these complexes We reconstitutednucleosomes with either unmodified H3 or H3(T118ph)onto 192 bp DNA with six different NPS We consistentlyobserved low mobility bands form with each of the NPSbut only in reconstitutions carried out with H3(T118ph)(Figure 1C) This indicates that the DNA sequence is notresponsible for the alteration in mobility or the formationof this low mobility complex which we refer to as a nu-cleosome duplex (32)

We also investigated the influence of DNA length on theformation of nucleosome duplexes by salt dialysis with147 187 and 247 bp DNA molecules containing acenter-positioned mp2 (Figure 1A SupplementaryFigure S1A) or 5S (data not shown) NPS The mp2NPS is a variant of the 601 NPS (3545) We found thatthe nucleosome duplex is formed irrespective of DNAlength (Figure 1DndashF) In addition we observed an add-itional band in EMSA of samples prepared with 247 bpDNA molecules This band has a faster electrophoreticmobility than center-positioned nucleosomes Howeveralthough the electrophoretic mobility is similar to thatof a depositioned nucleosome this DNAndashhistonecomplex sediments through a sucrose gradient twice asfar as canonical nucleosomes similar to the nucleosomeduplex (Supplementary Figure S1) We refer to the highmobility complex as an altosome (31) based on the add-itional experiments discussed below

Phosphorylation at H3(T118) is sufficient and necessaryfor nucleosome duplex and altosome formation

H3(T118) phosphorylation could potentially alter nucleo-some formation by generating a misfolded state of thehistone octamer structure To test this model we treatedrefolded histone octamer containing H3(T118ph) with APto remove the phosphate group from T118 within thefolded histone octamer The removal of the phosphatewas confirmed by mass spectrometry (Figure 2A) Wefind that reconstitutions with the dephosphorylatedhistone octamer (Figure 2B and C) or with H3(T118E)(Figure 1D and E) only formed canonical nucleosomesThis indicates that the phosphate group on H3(T118) isnecessary and sufficient for the formation of nucleosomeduplexes and altosomes

The histonendashDNA ratio of nucleosome duplexes andaltosomes are different

Different ratios of histone proteins within the protein coreor relative to DNA within the nucleosome duplex andaltosome could be an explanation for the altered electro-phoretic mobility and sucrose gradient sedimentation

To investigate this possibility we used fluorescence to de-termine the ratio of H2AndashH2B heterodimers and H32ndashH42tetramers relative to DNA molecules We labeled H2Awith Cy5 and H4 with Alexa488 using cysteine residuesinserted at the N-terminus by site-directed mutagenesisHistone octamers were refolded with fluorophore-labeledH2A and H4 and either unmodified H3 or H3(T118ph)(Supplementary Figure S8A) Following gel filtrationpurification nucleosomes were reconstituted with eachlabeled histone octamer and Cy3-labeled DNAThe canonical nucleosomes nucleosome duplexes and

altosomes were analyzed by EMSA and the fluorophoreemissions were detected with a Typhoon scanner(Figure 3AndashC) We confirmed that none of thesecomplexes undergo fluorescence resonance energy transferand that each fluorophore was spectrally separable(Supplementary Figure S8B and C) We found that thecanonical nucleosomes and nucleosome duplexes showedthe same relative emissions ratio for H2AndashH2Bheterodimer H32ndashH42 tetramerDNA implying that nu-cleosome duplexes contain one intact histone octamer perDNA In contrast altosomes maintain an equivalent ratioof heterodimers to tetramer but twice the ratio of H2AndashH2B heterodimers and H3ndashH4 tetramers relative to DNAequivalent to two intact histone octamers per DNA(Figure 3D)

Nucleosome duplexes are assembled by the Nap1 histonechaperone

Salt dialysis reconstitution is the most common approachto assemble nucleosomes in vitro (41) However itremained possible that these altered structures might bean artifact of this experimental approach Therefore weused the histone chaperone Nap1 to assemble DNAndashhistone complexes with both unmodified H3 andH3(T118ph) Nap1 can deposit both H32ndashH42 tetramersand H2AndashH2B heterodimers to form nucleosomes in vitro(56) We found that deposition of unmodified histonesonto mp2-187 by Nap1 results in nucleosomes that havethe same electrophoretic mobility as nucleosomes formedby salt dialysis EMSA of H3(T118ph)-containingcomplexes deposited by Nap1 onto mp2-187 results inbands with the same electrophoretic mobility as the ca-nonical nucleosomes and the nucleosome duplexesformed by salt dialysis (Figure 4) This result demonstratesthat this structure is not an artifact of salt dialysisassembly We also carried out Nap1-mediated DNAndashhistone assemblies with mp2-247 DNA We observe aband with the same mobility as altosomes formed bysalt dialysis (Supplementary Figure S2) However Nap1assembled unmodified depositioned nucleosomes withsimilar electrophoretic mobilities to altosomesTherefore our data are consistent with altosome forma-tion by Nap1 but we could not explicitly rule out that theband was a depositioned nucleosomeNucleosome assembly or disassembly is an equilibrium

process that sets up a competition between histonendashDNAcomplexes and histonendashNap1 complexes The dependenceof nucleosome duplex formation on Nap1 concentrationcan therefore be used as a measure of stability We find

Nucleic Acids Research 2014 5

by guest on February 23 2014httpnaroxfordjournalsorg

Dow

nloaded from

that a significantly lower concentration of Nap1 is

required to disassemble canonical nucleosomes when

H3(T118ph) is present than when H3 remains unmodified

In fact while 18mM Nap1 is sufficient for the formation

of positioned canonical nucleosomes both with and

without H3(T118ph) (Figure 4A and B and Supplemen-

tary Figure S2A and B) an increase in Nap1 concentra-

tion to 36 mM maintains well-positioned unmodified

canonical nucleosomes while H3(T118ph) structures dis-

assemble (Figure 4C and Supplementary Figure S2C)

Second H3(T118ph)-containing canonical nucleosomes

nucleosome duplexes and altosomes all disassemble at

similar concentrations of Nap1 (Figure 4C and

Supplementary Figure S2C) This suggests that the nu-

cleosome duplexes and altosomes containing

H3(T118ph) have a similar stability to canonical nucleo-

somes containing H3(T118ph) and that the stability of

these complexes is dependent on H3(T118) phosphoryl-

ation These results are compatible with our prior

studies of H3(T118ph) canonical nucleosomes (20)

Nucleosome duplexes and altosomes have different thermalstabilities and DNA footprints

We previously found that H3(T118ph) alters the thermalstability and mobility of canonical nucleosomes (20)Therefore we investigated the stability of nucleosomeduplexes to thermal disassembly by incubating them at53C and characterizing the products by EMSA Wefind that nucleosome duplexes converted to canonical nu-cleosomes following 30-min incubation at 53C irrespect-ive of DNA length (Figure 5A and B and SupplementaryFigure S3) In contrast the electrophoretic mobility ofaltosomes did not change after incubation at 53C andwe did not observe any separation of altosomes into ca-nonical nucleosomes or free histones and naked DNA(Figure 5C and D) This suggests the altosome species isthermally stable However it should be noted that EMSAwould not necessarily separate altosome species in whichwrapping subtly changed and that the separation of onealtosome into two canonical nucleosomes would requireacceptor DNA

These thermal disassembly studies suggest that nucleo-some duplexes contain miswrapped nucleosomes that

Ex 532nmEm 610nm

Ex 488nmEm 520nm

Ex 633nmEm 670nm

C

A

0

1

2

His

ton

e C

on

ten

t R

elat

ive

to U

nm

od

ifie

d N

ucl

eoso

mes H2ADNA

H4DNAH2AH4

D

Nuc Duplex

Altosomes

Ex 532nmEm 610nm

Ex 488nmEm 520nm

Ex 633nmEm 670nm

B

Nuc Duplex

mp2-187

mp2-247

Cy3

Alexa488

Alexa488

Cy5Cy5

Figure 3 Relative DNA and histone content of nucleosome duplexes and altosomes (A) Crystal structure (26) of nucleosome illustrating 50 end-labeled DNA with Cy3 (green) Alexa488-labeled H4(C0) (blue) and Cy5-labeled H2A(C0) (red) H3(T118ph) in yellow The histone tails are flexibleand largely unstructured EMSA of (B) Cy3-labeled mp2-187 and (C) Cy3-labeled mp2-247 DNAs reconstituted with fluorescent-labeled unmodifiedand H3(T118ph) HO Each EMSA gel was imaged by the Cy3-DNA label (left excitation at 532 nm emission at 610plusmn10nm) Alexa488-H4 label(middle excitation at 488 nm emission at 520plusmn10nm) and Cy5-H2A label (right excitation at 633 nm emission at 670plusmn10nm) (D) Fluorescenceratio of two fluorophore-labeled components (blue Cy5-H2A versus Cy3-DNA orange Alexa488-H4 versus Cy3-DNA pink Cy5-H2A versusAlexa488-H4) for H3(T118ph) canonical nucleosomes (Nuc) nucleosome duplex (ND) and altosomes (Alto) species relative to the same twofluorophore-labeled components of the unmodified nucleosome species for each indicated DNA length in base pairs Error bars are the standarddeviation of three independent reconstitutions

6 Nucleic Acids Research 2014

by guest on February 23 2014httpnaroxfordjournalsorg

Dow

nloaded from

convert to correctly wrapped nucleosomes upon heating to53C To investigate these species we determined theDNA footprint of nucleosome duplexes and altosomeswith MNase digestion (Supplementary Figure S4) andExoIII nucleosome mapping (Supplementary Figures S5and S6) By each approach we find that the nucleosomeduplex containing H3(T118ph) on 187 bp DNA had a basepair footprint that is similar to the 147 bp footprint ofcanonical nucleosomes both before and after 30 min at53C In contrast the DNAndashhistone complexes formedwith H3(T118ph) on 247 bp DNA molecules whichcontain both nucleosome duplexes and altosomes aftersucrose gradient purification had both a 147- and 247-bp DNA footprint and increased protection from diges-tion when compared with canonical nucleosomes formedwith unmodified H3 or H3(T118ph) on 247 bp DNA Weconfirmed by EMSA that both the nucleosome duplexesand altosomes did not disassemble under the conditions ofthe digestions (data not shown) These results suggest thatthe nucleosome duplexes contain DNA that is wrappedaround the histone octamer similarly to nucleosomeswhile the altosomes contain a DNA organization thatappears to be distinct from nucleosomal DNA organiza-tion with a larger DNA footprint

AFM images of the nucleosome duplexes and altosomesreveal they have twice the volume as canonicalnucleosomes

The observation that both nucleosome duplexes andaltosomes sediment twice as far within a sucrosegradient as canonical nucleosomes suggests the mass ofthe particles could be larger than a canonical nucleosomeTo investigate the size of nucleosome duplexes andaltosomes we used AFM We prepared by sucrosegradient purification unmodified nucleosomes canonicalnucleosomes containing H3(T118ph) and a mixture ofboth nucleosome duplexes and altosomes with mp2-247DNA (Figure 6) or only nucleosome duplexes with mp2-147 (Supplementary Figure S7) DNA Because nucleo-some duplexes and altosomes sediment similarly throughsucrose gradients they could not be separated and weretherefore imaged together by AFMFollowing AFM imaging of these complexes we

quantified the average area and height of 200 particlesof each sample type Analysis of these imagesdemonstrated that the height distribution of unmodifiedcanonical nucleosomes H3(T118ph) canonical nucleo-somes nucleosome duplexes and altosomes with eithermp2-247 or mp2-147 had a maximum of 3 nm This isthe canonical mononucleosome height measured by AFM(46) In contrast we found that the area distributionmaximum of nucleosome duplexes and altosomes withmp2-247 was twice as large as both unmodified andH3(T118ph)-containing canonical nucleosomes withmp2-247 (Figure 6) In addition the area distribution ofthe nucleosome duplexes with the mp2-147 DNA moleculehad two peaks (Supplementary Figure S7) The larger areapeak maximum was approximately double the area distri-bution maximum of canonical nucleosomes The smallerarea peak maximum was equal to the canonical nucleo-some distribution maximum We attribute this secondpeak to canonical nucleosomes generated by the destabil-ization of the nucleosome duplexes when they are dilutedfor AFM imaging similar to the thermal destabilizationobserved by EMSA The observation that nucleosomeduplexes and altosomes are the same height and twicethe area of canonical nucleosomes implies that thevolume of these complexes is double that of canonicalnucleosomes We also found that the shapes of thenucleosome duplexes and altosomes were elliptical whilecanonical nucleosomes containing either unmodified H3or H3(T118ph) were circularThese results combined with our observation that nu-

cleosome duplexes can be converted to canonical nucleo-somes by heat and contain equal numbers of DNAmolecules and histone octamers suggest that the nucleo-some duplexes contain two DNA molecules and twohistone octamers where the two DNA molecules partiallywrap around each of the histone octamers In contrast theAFM analysis of altosomes combined with the observa-tions that altosomes cannot be converted to canonical nu-cleosomes that they have an increased DNA footprintand that they contain twice as many histone octamers asDNA molecules suggests that the altosomes contain oneDNA molecule wrapped around two histone octamers

R

BA

mp2-187 unmod mp2-187 T118ph

Nuc duplex

[Nap1] (microM)

0

02

04

06

08

10

Ban

d F

ract

ion

C

0 2 4 6 8[Nap1] (microM)

0

02

04

06

08

10

Fra

ctio

n C

entr

ally

Po

siti

on

ed N

ucl

eoso

mes

D

0 2 4 6 8[Nap1] (microM)

unmod Nucunmod DNA

T118ph NucT118ph DNA unmod Nuc

T118ph NucT118ph ND

[Nap1] (microM)

0 R 0

Figure 4 Nucleosome duplexes are assembled by yNap1 EMSA of (A)unmodified HO and (B) H3(T118ph) containing HO reconstituted onmp2-187 by salt dialysis (lane 1 lsquoRrsquo) unmodified HO mixed with DNAin the absence of yNap1 (lane 2 lsquo0rsquo) and unmodified HO assembled onDNA in the presence of increasing amounts of yNap1 as indicated foreach lane (C) Quantification of the fraction of yNap1 assembled nu-cleosomes (lsquonucrsquo squares) nucleosome duplexes (lsquoNDrsquo triangles) andremaining free DNA (lsquoDNArsquo diamonds) as a function of [Nap1] forunmodified (black) and H3(T118ph) HO (gray) from the data in (A)and (B) (D) Fraction of centrally positioned versus depositioned nu-cleosomes assembled by yNap1 as a function of [Nap1] for unmodifiednucleosomes (black square) and H3(T118ph) nucleosomes (graysquares)

Nucleic Acids Research 2014 7

by guest on February 23 2014httpnaroxfordjournalsorg

Dow

nloaded from

Altosomes form on 3000 bp DNA molecules

Our observation that increasing the DNA length to 247 bpallowed for two histone octamers to form on one DNAmolecule raised the question of whether a further increasein DNA length would result in larger complexesTherefore we reconstituted nucleosomes with unmodifiedH3 or H3(T118ph) on two different extended DNA con-structs either a 364 bp DNA molecule containing two601-like NPSs (2-mer array Supplementary Figure S7F)or a 3-kb DNA molecule that contained a tandem repeatof seventeen 601-like NPSs (16) (17-mer arraySupplementary Figure S1A) We analyzed the DNAndashhistone complexes by AFM For the 2-mer arrays weobserved two nucleosomes with dimensions similar tomononucleosomes with unmodified H3 but only oneDNAndashhistone complex that was significantly larger thana single nucleosome when H3(T118ph) was used(Supplementary Figure S7G and H) For the 17-merarrays we used limiting amounts of histone octamerwith a ratio of 1 histone octamer to 2 NPS to preventaggregation As anticipated we find that nucleosomesformed with unmodified histone octamers have dimen-sions similar to mononucleosomes (Figure 6F) Incontrast the 3-kb DNA molecule reconstituted withH3(T118ph)-containing histone octamer forms numerous

DNAndashhistone complexes that are significantly larger thansingle nucleosomes (Figure 6G) Interestingly comparedwith unmodified H3 fewer H3(T118ph) complexes formedper DNA molecule This confirms that the altosome struc-tures are not restricted to short DNA segments andsuggests that the altosomes that form on mp2-247 DNAmolecules can also form on significantly longer DNAmolecules

DISCUSSION

We find that H3(T118ph) significantly influences DNAwrapping around the histone octamer We observe twotypes of altered DNAndashhistone complexes a nucleosomeduplex with low electrophoretic mobility and analtosome with a high electrophoretic mobility Howeverthere are key differences between the nucleosome duplexand the altosome The nucleosome duplex has the sameratio of histone octamer to DNA has the same DNAfootprint as canonical nucleosomes and can convert tocanonical nucleosome when heated In contrast thealtosome contains two equivalents of histone octamerper DNA has a larger DNA foot print compared withcanonical nucleosomes and is thermally stable comparedwith the nucleosome duplex Both complexes have a

0 5 15 30 60 90Time (min)

A

0

02

04

06

08

10

Ban

d F

ract

ion

0 15 30 45 60 75 90Time (min)

Nuc Duplex

mp2-187 T118ph B

Nuc Duplex

0 5 15 30 60 90Time (min)

C

0

02

04

06

08

10

Ban

d F

ract

ion

0 15 30 45 60 75 90Time (min)

Nuc Duplexmp2-247 T118ph D

Altosome

Nuc Duplex

Altosome

Figure 5 Nucleosome duplexes are decoupled into mononucleosomes (A) EMSA of purified nucleosome duplexes containing H3(T118ph) HO andmp2-187 after heating at 53C for the indicated amount of time Nucleosome duplexes convert to positioned and depositioned mononucleosomes asdetermined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4) (B) Quantification of fraction of nucleosome duplexes(squares) nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time Error bars are the standard deviation of threeindependent experiments (C) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53Cfor the indicated amount of time Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNaseand ExoIII nucleosome mapping (see Supplementary Figure S5) (D) Quantification of the fraction of nucleosome duplexes (squares) altosomes(triangles) nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time Error bars are the standard deviation of threeindependent experiments

8 Nucleic Acids Research 2014

by guest on February 23 2014httpnaroxfordjournalsorg

Dow

nloaded from

similar height but are twice the volume relative to canon-ical nucleosomes and have an elliptical shape Weconclude that the nucleosome duplex contains twohistone octamers positioned side by side with two DNAmolecules wrapped around the two histone octamers andthat the altosome contains one DNA molecule wrappedaround two histone octamers in a similar configuration(Figure 7) While nucleosome duplexes containingH3(T118ph) convert to canonical nucleosomesH3(T118ph) containing altosomes do not This suggeststhat the altosome structure containing H3(T118ph) is en-ergetically favorable relative to canonical nucleosomescontaining H3(T118ph) or is kinetically trapped in thataltosome state This work appears to represent the firstdirect demonstration of a single histone PTM causing alarge alteration in nucleosome structure

The DNAndashhistone interactions near the nucleosomedyad contribute significantly to nucleosome stability(34) with implications for nucleosome assembly and dis-assembly (1621) Our results suggest that in addition to animpact on nucleosome stability modified contacts withinthe dyad region can influence nucleosome structure TheDNA phosphate backbone is within 3 A of the side chainhydroxyl of H3(T118) (26) such that the addition of aphosphate at this residue could introduce a strong

electrostatic repulsion between the phosphate on thethreonine and the DNA phosphate in addition to asteric conflict between the protein and DNA phosphatemoieties The alternate structures are stabilized bysubmillimolar concentrations of Mg2+ while metal ionstypically stabilize compacted chromatin through inter-actions with the phosphate backbone it is also possible

Surface Area (nm2)0 100 200 300 400 500

0

02

04

06

Rel

ativ

e F

req

uen

cy

DUnmod NucT118ph NucT118ph ND and Alto

A

B

C

Height (nm)0 2 4 6000 222 444 666

0

02

04

06

Rel

ativ

e F

req

uen

cyE

F

G

601-17mer Unmod

601-17mer T118ph

200nm

200nm

200nm 200nm

200nm

247 UnmodCanonical Nucs

247 T118phCanonical Nucs

247 T118phNuc DuplexesAltosomes

Figure 6 Nucleosome duplexes and altosomes are twice the size of mononucleosomes AFM images of purified (A) mp2-247 nucleosomes withunmodified HO (247 Unmod Canonical Nucs) (B) mp2-247 nucleosomes with H3(T118ph) HO (247 T118ph Canonical Nucs) and (C) mp2-247nucleosome duplexes with H3(T118ph) HO (247 T118ph Nuc Duplexesaltosomes) Inset is 60 nm in width Histograms of (D) surface area and (E)height of mp2-247 containing unmodified nucleosomes (black square n=173) H3(T118ph) canonical nucleosomes (blue circle n=140) andH3(T118ph) nucleosome duplexesaltosomes (red triangle n=175) AFM images of 601-17mer arrays reconstituted with (F) unmodified(112plusmn05 nucleosomes per molecule n=27) and (G) H3(T118ph) HO (71plusmn04 discrete complexes per molecule n=32) Inset is 400 nm in width

A BNuc Duplex Nuc Duplex

Altosome Altosome

Figure 7 Models of nucleosome duplexes and altosomes (A) Model ofDNA-histone wrapping without DNA crossing of a nucleosome duplexcontaining two DNA molecules and two histone octamer componentsand an altosome containing one DNA molecules and two histoneoctamers (B) Model of DNAndashhistone wrapping with DNA crossingsfor a nucleosome duplex and an altosome

Nucleic Acids Research 2014 9

by guest on February 23 2014httpnaroxfordjournalsorg

Dow

nloaded from

that recruitment of a metal ion by phosphothreoninecould further alter local contacts in this region In priorwork we demonstrated a dramatic destabilization of ca-nonical nucleosomes bearing this phosphate modification(20) Notably a Thr to Glu substitution is insufficient toreplicate the effects of phosphothreonine on either canon-ical nucleosome destabilization in our prior studies or theformation of nucleosome duplexes and altosomesobserved here suggesting that electrostatic effects arenot sufficient and the steric effect or other specificproperties of the side chain phosphorylation are necessaryOur results suggest that in the nucleosome duplexes and

altosomes electrostatic and steric clash between thephosphothreonine side chain and the DNA is avoidedby partial wrapping of the DNA around two separatehistone octamers aligned edge-to-edge For short DNAmolecules our results suggest that two DNA moleculesare required to maintain DNAndashhistone contacts whileDNA molecules of 250 bp and longer can do this withone DNA molecule (Figure 7) As the DNA extends fromone octamer to the other the DNA could remain on theoutside of the complex (Figure 7A) or could cross betweenoctamers (Figure 7B) The complexes that do not involveDNA crossing are not topologically constrained andreduce potential steric interactions between DNA standsTherefore we anticipate that complexes without DNAcrossings to be energetically preferredAlternate large DNAndashhistone complexes have previ-

ously been observed in vitro Chromatin remodeling bySWISNF and Remodels the Structure of Chromatin(RSC) can result in nucleosome disassembly or slidingbut also in persistent altered histone-DNA particles (31ndash3347ndash49) Remodeling of single nucleosomes results in theformation of a nucleosome duplex that has similar char-acteristics to the nucleosome duplex formed withH3(T118ph) (3133) Nucleosome arrays formed withlonger DNA molecules can be converted by SWISNFto arrays of structures termed as altosomes that containone DNA molecule wrapped around two histone octamers(31) AFM images of altosomes formed by chromatin re-modeling appear to be similar to the structures formedwith H3(T118ph) (50) such that we categorize thealtered structures that we observe as altosomes althoughtheir formation is not dependent on chromatin remodelingactivity The in vivo role of altosomes remains unclearwith competing views of these structures as kinetic inter-mediates (5152) or as functional products of SWISNF-induced chromatin remodeling for example with alteredDNA site exposure to transcription factor binding (32) Inthis context the thermal stability of H3(T118ph) altosomestructures (Figure 5) stands in contrast to the increasedrate of H3(T118ph) nucleosome disassembly by SWISNFin our previous studies (20)Dinucleosome-like particles have also been reported to

form by manipulating the DNA sequence to overlap twostrong NPS by 44 bp to simulate invasion of a neighboringnucleosome for example during chromatin remodeling(53) These cylindrical particles contain two H3H4 tetra-mers but only three H2AH2B heterodimers protect250 bp of DNA and are thought to stack histoneoctamers face-to-face with an extended superhelical

DNA wrapping to generate a cylindrical particleHowever these structures are structurally distinct fromnucleosome duplex and altosomes structures formed byeither chromatin remodeling or H3(T118ph)

Currently the in vivo functions of histone PTMs nearthe nucleosome dyad are not well understood Acetylationof H3(K115) and H3(K122) histone PTMs known tooccur in vivo (12) near the nucleosome dyad has beenshown to destabilize nucleosomes in vitro (1634)Following these reports it was then found that nucleo-some destabilization by H3(K122) is a significant regula-tor of transcription (21) demonstrating an in vivo functionof nucleosome dyad modifications Interestingly massspectrometry indicates that H3(T118ph) occurs withH3(K122) acetylation (12) These studies combined withour results suggest that H3(T118ph) could function withH3(K122) acetylation to destabilize the nucleosome struc-ture and form altosomes to regulate transcription Recentreports suggest that acetylation of the centromeric H3homolog CENP-A at K124 in the dyad is correlatedwith conversion of octameric nucleosomes in the centro-mere to a four-histone hemisome structure which ishypothesized to play a role in regulation of DNA replica-tion (46) Taken in aggregate these studies hint at majorroles for the dyad region in regulating aspects of nucleo-some structure However in vivo studies similar to thereport on H3(K122) acetylation (21) will be required todetermine the physiological function of H3(T118ph)

There are four additional histone residues within theDNAndashhistone interface that have been identified as phos-phorylation sites (12ndash15) To date there is little under-standing of these buried phosphorylations Futurestudies will be required to determine whether these add-itional histone PTMs impact nucleosome structure simi-larly to H3(T118ph) and how these modifications functionin vivo Our studies here indicate that phosphorylation inthe DNAndashhistone interface have the potential to signifi-cantly impact nucleosome structure and DNA wrapping

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online

ACKNOWLEDGEMENTS

The authors thank Karolin Luger Mekonnen Dechassaand members of the Ottesen and Poirier laboratories forhelpful discussions

FUNDING

American Heart Association Predoctoral Fellowship[0815460D to JAN 10PRE3150036 to AMM]OSUCCC and the James Pelotonia Fellowship [toJAN] American Cancer Society [IRG-6700344 seedfunding to JJO] National Institutes of Health (NIH)[GM083055 to MGP and JJO] Career Award in theBasic Biomedical Sciences from the Burroughs WellcomeFund [to MGP] National Science Foundation[MCB0845695 to JJO] Human Frontier Science

10 Nucleic Acids Research 2014

by guest on February 23 2014httpnaroxfordjournalsorg

Dow

nloaded from

Program [RGY00572009 to JvN] Funding for openaccess charge NIH [R01GM083055]

Conflict of interest statement None declared

REFERENCES

1 LugerK MaderAW RichmondRK SargentDF andRichmondTJ (1997) Crystal structure of the nucleosome coreparticle at 28 A resolution Nature 389 251ndash260

2 SuganumaT and WorkmanJL (2011) Signals and combinatorialfunctions of histone modifications Ann Rev Biochem 80473ndash499

3 HotaSK and BartholomewB (2011) Diversity of operation inATP-dependent chromatin remodelers Biochim Biophys Acta1809 476ndash487

4 ClapierCR and CairnsBR (2009) The biology of chromatinremodeling complexes Ann Rev Biochem 78 273ndash304

5 ParkYJ and LugerK (2008) Histone chaperones in nucleosomeeviction and histone exchange Curr Opin Struct Biol 18282ndash289

6 AvvakumovN NouraniA and CoteJ (2011) Histonechaperones modulators of chromatin marks Mol Cell 41502ndash514

7 KouzaridesT (2007) Chromatin modifications and their functionCell 128 693ndash705

8 YunM WuJ WorkmanJL and LiB (2011) Readers ofhistone modifications Cell Res 21 564ndash578

9 Shogren-KnaakM IshiiH SunJM PazinMJ DavieJR andPetersonCL (2006) Histone H4-K16 acetylation controlschromatin structure and protein interactions Science 311844ndash847

10 LiG and ReinbergD (2011) Chromatin higher-order structuresand gene regulation Curr Opin Genet Dev 21 175ndash186

11 MersfelderEL and ParthunMR (2006) The tale beyond thetail histone core domain modifications and the regulation ofchromatin structure Nucleic Acids Res 34 2653ndash2662

12 ZhangL EugeniEE ParthunMR and FreitasMA (2003)Identification of novel histone post-translational modifications bypeptide mass fingerprinting Chromosoma 112 77ndash86

13 DawsonMA BannisterAJ GottgensB FosterSD BartkeTGreenAR and KouzaridesT (2009) JAK2 phosphorylateshistone H3Y41 and excludes HP1alpha from chromatin Nature461 819ndash822

14 HurdPJ BannisterAJ HallsK DawsonMA VermeulenMOlsenJV IsmailH SomersJ MannM Owen-HughesT et al(2009) Phosphorylation of histone H3 Thr-45 is linked toapoptosis J Biol Chem 284 16575ndash16583

15 Tweedie-CullenRY BrunnerAM GrossmannJ MohannaSSichauD NanniP PanseC and MansuyIM (2012)Identification of combinatorial patterns of post-translationalmodifications on individual histones in the mouse brain PLoSOne 7 e36980

16 SimonM NorthJA ShimkoJC FortiesRAFerdinandMB ManoharM ZhangM FishelR OttesenJJand PoirierMG (2011) Histone fold modifications controlnucleosome unwrapping and disassembly Proc Natl Acad SciUSA 108 12711ndash12716

17 ShimkoJC NorthJA BrunsAN PoirierMG andOttesenJJ (2011) Preparation of fully synthetic histone H3reveals that acetyl-lysine 56 facilitates protein binding withinnucleosomes J Mol Biol 408 187ndash204

18 NeumannH HancockSM BuningR RouthA ChapmanLSomersJ Owen-HughesT van NoortJ RhodesD andChinJW (2009) A method for genetically installing site-specificacetylation in recombinant histones defines the effects of H3 K56acetylation Mol Cell 36 153ndash163

19 BannisterAJ ZegermanP PartridgeJF MiskaEAThomasJO AllshireRC and KouzaridesT (2001) Selectiverecognition of methylated lysine 9 on histone H3 by the HP1chromo domain Nature 410 120ndash124

20 NorthJA JavaidS FerdinandMB ChatterjeeNPickingJW ShoffnerM NakkulaRJ BartholomewBOttesenJJ FishelR et al (2011) Phosphorylation of histoneH3(T118) alters nucleosome dynamics and remodeling NucleicAcids Res 39 6465ndash6474

21 TropbergerP PottS KellerC Kamieniarz-GdulaKCaronM RichterF LiG MittlerG LiuET BuhlerM et al(2013) Regulation of transcription through acetylation of H3K122on the lateral surface of the histone octamer Cell 152 859ndash872

22 CosgroveMS BoekeJD and WolbergerC (2004) Regulatednucleosome mobility and the histone code Nat Struct MolBiol 11 1037ndash1043

23 LuX SimonMD ChodaparambilJV HansenJCShokatKM and LugerK (2008) The effect of H3K79dimethylation and H4K20 trimethylation on nucleosome andchromatin structure Nat Struct Mol Biol 15 1122ndash1124

24 IwasakiW TachiwanaH KawaguchiK ShibataT KagawaWand KurumizakaH (2011) Comprehensive structural analysis ofmutant nucleosomes containing lysine to glutamine (KQ)substitutions in the H3 and H4 histone-fold domainsBiochemistry 50 7822ndash7832

25 MuthurajanUM BaoY ForsbergLJEdayathumangalamRS DyerPN WhiteCL and LugerK(2004) Crystal structures of histone Sin mutant nucleosomesreveal altered protein-DNA interactions EMBO J 23 260ndash271

26 RichmondTJ and DaveyCA (2003) The structure of DNA inthe nucleosome core Nature 423 145ndash150

27 HylandEM CosgroveMS MolinaH WangD PandeyACotteeRJ and BoekeJD (2005) Insights into the role ofhistone H3 and histone H4 core modifiable residues inSaccharomyces cerevisiae Mol Cell Biol 25 10060ndash10070

28 KrugerW PetersonCL SilA CoburnC ArentsGMoudrianakisEN and HerskowitzI (1995) Amino acidsubstitutions in the structured domains of histones H3 and H4partially relieve the requirement of the yeast SWISNF complexfor transcription Genes Dev 9 2770ndash2779

29 HsiehFK FisherM UjvariA StuditskyVM and LuseDS(2010) Histone Sin mutations promote nucleosome traversal andhistone displacement by RNA polymerase II EMBO Rep 11705ndash710

30 BintuL IshibashiT DangkulwanichM WuYYLubkowskaL KashlevM and BustamanteC (2012)Nucleosomal elements that control the topography of the barrierto transcription Cell 151 738ndash749

31 UlyanovaNP and SchnitzlerGR (2005) Human SWISNFgenerates abundant structurally altered dinucleosomes onpolynucleosomal templates Mol Cell Biol 25 11156ndash11170

32 UlyanovaNP and SchnitzlerGR (2007) Inverted factor accessand slow reversion characterize SWISNF-altered nucleosomedimers J Biol Chem 282 1018ndash1028

33 SchnitzlerG SifS and KingstonRE (1998) Human SWISNFinterconverts a nucleosome between its base state and a stableremodeled state Cell 94 17ndash27

34 ManoharM MooneyAM NorthJA NakkulaRJPickingJW EdonA FishelR PoirierMG and OttesenJJ(2009) Acetylation of histone H3 at the nucleosome dyad altersDNA-histone binding J Biol Chem 284 23312ndash23321

35 PoirierMG BussiekM LangowskiJ and WidomJ (2008)Spontaneous access to DNA target sites in folded chromatinfibers J Mol Biol 379 772ndash786

36 SimpsonRT and StaffordDW (1983) Structural features of aphased nucleosome core particle Proc Natl Acad Sci USA 8051ndash55

37 RhodesD (1985) Structural analysis of a triple complex betweenthe histone octamer a Xenopus gene for 5S RNA andtranscription factor IIIA EMBO J 4 3473ndash3482

38 BergmanLW (1986) A DNA fragment containing the upstreamactivator sequence determines nucleosome positioning of thetranscriptionally repressed PHO5 gene of Saccharomycescerevisiae Mol Cell Biol 6 2298ndash2304

39 FedorMJ LueNF and KornbergRD (1988) Statisticalpositioning of nucleosomes by specific protein-binding to anupstream activating sequence in yeast J Mol Biol 204109ndash127

Nucleic Acids Research 2014 11

by guest on February 23 2014httpnaroxfordjournalsorg

Dow

nloaded from

40 ShimkoJC HowardCJ PoirierMG and OttesenJJ (2013)Preparing semisynthetic and fully synthetic histones h3 and h4to modify the nucleosome core Methods Mol Biol 981177ndash192

41 LugerK RechsteinerTJ and RichmondTJ (1999) Expressionand purification of recombinant histones and nucleosomereconstitution Methods Mol Biol 119 1ndash16

42 McBryantSJ ParkYJ AbernathySM LaybournPJNyborgJK and LugerK (2003) Preferential binding of thehistone (H3-H4)2 tetramer by NAP1 is mediated by the amino-terminal histone tails J Biol Chem 278 44574ndash44583

43 FlausA and Owen-HughesT (2003) Dynamic properties ofnucleosomes during thermal and ATP-driven mobilizationMol Cell Biol 23 7767ndash7779

44 PenningsS (1997) Nucleoprotein gel electrophoresis for theanalysis of nucleosomes and their positioning and mobility onDNA Methods 12 20ndash27

45 LowaryPT and WidomJ (1998) New DNA sequence rules forhigh affinity binding to histone octamer and sequence-directednucleosome positioning J Mol Biol 276 19ndash42

46 BuiM DimitriadisEK HoischenC AnE QuenetDGiebeS Nita-LazarA DiekmannS and DalalY (2012) Cell-cycle-dependent structural transitions in the human CENP-Anucleosome in vivo Cell 150 317ndash326

47 RoweCE and NarlikarGJ (2010) The ATP-dependentremodeler RSC transfers histone dimers and octamers through therapid formation of an unstable encounter intermediateBiochemistry 49 9882ndash9890

48 LorchY ZhangM and KornbergRD (2001) RSC unravels thenucleosome Mol Cell 7 89ndash95

49 LorchY CairnsBR ZhangM and KornbergRD (1998)Activated RSC-nucleosome complex and persistently altered formof the nucleosome Cell 94 29ndash34

50 SchnitzlerGR CheungCL HafnerJH SaurinAJKingstonRE and LieberCM (2001) Direct imaging of humanSWISNF-remodeled mono- and polynucleosomes by atomic forcemicroscopy employing carbon nanotube tips Mol Cell Biol 218504ndash8511

51 LorchY Maier-DavisB and KornbergRD (2006) Chromatinremodeling by nucleosome disassembly in vitro Proc Natl AcadSci USA 103 3090ndash3093

52 LiuN BallianoA and HayesJJ (2011) Mechanism(s) of SWISNF-induced nucleosome mobilization Chembiochem 12196ndash204

53 EngeholmM de JagerM FlausA BrenkR van NoortJand Owen-HughesT (2009) Nucleosomes can invade DNAterritories occupied by their neighbors Nat Struct Mol Biol16 151ndash158

12 Nucleic Acids Research 2014

by guest on February 23 2014httpnaroxfordjournalsorg

Dow

nloaded from