New and Notable

Nucleosomes form the fundamentalrepeating unit of eukaryotic chromatin.Subtle modifications in nucleosomestructureandhistonetailsregulatechro-matin states; hence, a comprehensiveunderstanding of alterations in nucleo-some structure is of fundamental im-portance in chromatin biophysics. Thenature of core histone organization andnucleosome dynamics have been ex-tensively studied using biophysical ex-periments, multiscale computationalmodels, and simulations ((1,2) andreferences therein). However, the pre-cisemechanismsofhowDNAsequencemediates nucleosome structure and sta-bility remains to be completely under-stood.The eviction of H2A/H2B core his-tones from the histone octamer isknown to expose nucleosomal DNAfor transcription. In this issue of Bio-physical Journal, Kelbauskas et al. (3)have investigated the sequence depen-dence of H2A/H2B eviction mediatedby histone chaperone yNAP-1 usingsingle-molecule Fo¨rster resonance en-ergy transfer (FRET) and fluorescencecorrelation spectroscopy (FCS) exper-iments. Single molecule experimentshave gained a newfound interest inprobing chromatin dynamics, in part,because of their ability to study subtlevariations in chromatin dynamics at asingle nucleosome level, rather thanprobing population-averaged proper-ties (1,4–6). H2A/H2B dimers interactwith nearly 30 basepairs of nucleoso-mal DNA, thus depletion of H2A/H2Bdimers from the nucleosomes results insignificantexposureofthenucleosomalDNA. Understanding H2A/H2B deple-tion is of special significance in chro-matin biophysics as loss of H2A/H2Bhistones is associated with importantcellular processes, including transcrip-tion elongation, ATP-dependent nucle-osome remodeling, and function ofRNA polymerase.In a previous study (7), Kelbauskasetal.usedFRETandFCSassaystostudythe sequence dependence of mono-nucleosome structure and stability ofchromatin reconstitution in bulk solutionconditions. This sequence dependencewas tested in nucleosomes having threephysiologically relevant DNA se-quences: 1), sea urchin 5S rDNA; 2),DNA from yeast GAL10 promoter se-quence; and 3), DNA Mouse MammaryTumor Virus promoter DNA (MMTV-B). While GAL10 and MMTV-B nucle-osomes are known to exhibit significantconformational dynamics associatedwith gene activation in vivo, sea urchin5S rDNA is extensively used as atemplate DNA for in vitro studies ofnucleosome dynamics. Nucleosome sta-bility was tested by analyzing the con-formation-dependent FRET response inthethreenucleosomes,withFRETdonorand acceptor fluorophores attached tonucleosomal DNA.The authors showed that upon dilu-tion to subnanomolar concentrations(corresponding to single-molecule stud-ies), the efficiency of FRET signalsfrom MMTV-B and GAL10 nucleo-somes is reduced by as much as 35%,while that from 5S nucleosomes re-mains undiminished. The in vitro FCSassaysshowedthattherelativediffusioncoefficient for 5S nucleosomes washigher than that of MMTV-B andGAL10 nucleosomes, suggesting thatunder ambient conditions, 5S nucleo-somes are more compact, as comparedto MMTV-B and GAL10 nucleosomes.Similarresultsareobservedforthethreesets of nucleosomes at elevated temper-atures (42 C) and upon treatment withhigh salt concentrations. These obser-vationssuggestthat5Snucleosomesaremore stable than MMTV-B and GAL10nucleosomes. In coherence with ge-nomic regulatory mechanisms, thesedifferencesinnucleosomestructureandstability may mediate nucleosome rec-ognition and histone modifications.In this issue, Kelbauskas et al. (3)report that treatment with histone chap-erone yNAP-1 causing H2A/H2B his-tone eviction leads to the same FRETchanges as observed in correspondingsubnanomolar dilution assays. Notably,inbothassays,theextentofreductioninFRET signal was also similar forMMTV-B and GAL10 nucleosomes.Furthermore, salt stability assays sug-gest that while yNAP-1 histone chap-erone was acting on each of the threenucleosomes, 5S nucleosomes exhibitno FRET change. These results rein-force the previous studies that H2A/H2Breleasecausessequence-dependentFRETresponses.TheyNAP1assayalsosuggests that the three mono-nucleo-somes follow two-state dynamics withfolded and unfolded states. While thetwo-state behavior is most pronouncedin 5S nucleosomes, the rate of nucleo-somal DNA dynamics is enhanced inGAL10 and MMTV-B nucleosomes.Simulations of a nucleosome core par-ticle having Xenopus laevis core his-tones and a palindromic DNA fromhumana-satellitesequence(2)alsosug-gest specific interactions between H2A-H2B histones and nucleosomal DNA.Functionally important interactions arereported at interface of H4-H2A andH4-H2B histones (2), suggesting thatthe sequence-dependent eviction of H2A-H2B dimers may also be evolutionarilyconserved in higher eukaryotes.Kelbauskas et al. (3) analyze twopossible hypotheses for the biophysicalmechanism leading to differences inFRET response in these nucleosomesundersubnanomolarconcentrations:thefirst hypothesis suggesting differences