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Biophysical Perspective
Major Determinants of Nucleosome Positioning
R�azvan V. Chereji1,* and David J. Clark1,*1Division of Developmental Biology, Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Institutes ofHealth, Bethesda, Maryland
ABSTRACT The compact structure of the nucleosome limits DNA accessibility and inhibits the binding of most sequence-spe-cific proteins. Nucleosomes are not randomly located on the DNA but positioned with respect to the DNA sequence, suggestingmodels in which critical binding sites are either exposed in the linker, resulting in activation, or buried inside a nucleosome,resulting in repression. The mechanisms determining nucleosome positioning are therefore of paramount importance for under-standing gene regulation and other events that occur in chromatin, such as transcription, replication, and repair. Here, we reviewour current understanding of the major determinants of nucleosome positioning: DNA sequence, nonhistone DNA-binding pro-teins, chromatin-remodeling enzymes, and transcription. We outline the major challenges for the future: elucidating the precisemechanisms of chromatin opening and promoter activation, identifying the complexes that occupy promoters, and understand-ing the multiscale problem of chromatin fiber organization.
INTRODUCTION
In eukaryotic cells, genomic DNA is highly compacted byhistones into chromatin, with contributions from nonhistoneproteins and RNA. The basic unit of chromatin structure isthe nucleosome. Nucleosomes comprise two moleculeseach of the four core histones (H2A, H2B, H3, and H4)and �147 bp of DNA tightly wrapped around the centralhistone octamer (1). Nucleosomes are connected by linkerDNA, the length of which varies between species and celltypes (2).
Nucleosomes serve multiple functions in the cell nucleus.First, they compact DNA; stretches of �50 nm (�150 bp)are wrapped into spools with diameter of �10 nm, contain-ing �1.7 superhelical left-handed turns. Nucleosomal DNAis sharply bent and overtwisted, and because the persistencelength of DNA is also �150 bp, significant bending energyis required (much larger than the typical thermal fluctuationenergy). This bending energy is compensated for by favor-able electrostatic interactions between the positivelycharged histone octamer and the negatively charged DNA(3,4) as well as by protein-protein interactions betweenthe core histones within the octamer. Secondly, nucleosomalDNA is occluded from interacting with many proteins, sonucleosomes can directly regulate access of DNA-bindingproteins to regulatory elements (e.g., promoters and en-
Submitted December 5, 2017, and accepted for publication March 8, 2018.
*Correspondence: [email protected] or [email protected]
Editor: Tamar Schlick.
https://doi.org/10.1016/j.bpj.2018.03.015
hancers). In particular, in vitro, nucleosomes completelyblock initiation by RNA polymerases and strongly inhibittranscript elongation (5). Thirdly, nucleosomes may containposttranslationally modified histones (e.g., methylation,acetylation, ubiquitination, etc.) and/or methylated DNA.They may also contain histone variants, such as H2A.Z orH3.3, instead of the more common H2A and H3, respec-tively. These epigenetic marks provide an additional layerof information above that of the genomic DNA sequenceand are indicative of transcriptional status (6). Therefore,learning the mechanisms that govern nucleosome posi-tioning is of paramount importance for understanding pro-tein binding and DNA-related processes in the context ofchromatin. Here, we discuss the most important determi-nants of nucleosome positioning and their relative impor-tance in establishing the chromatin landscape in cells.
Nucleosome mapping
Before discussing the factors that determine nucleosomeposition, a brief discussion of the current experimentalmethods for determining these positions in vivo is worth-while. The most popular technique for mapping nucleo-somes on a genomic scale involves digestion of chromatinwith micrococcal nuclease (MNase) followed by high-throughput sequencing (MNase-seq). Fig. 1 illustrates thebasic principle of MNase-seq. The chromatin is digestedby MNase, which preferentially destroys linker DNA, pro-ducing a discrete set of DNA fragments or a ‘‘DNA ladder’’
Biophysical Journal 114, 2279–2289, May 22, 2018 2279
FIGURE 1 Level of digestion influences the set of mononucleosomal
fragments obtained in MNase-seq experiments. Digestion of chromatin
by MNase produces DNA fragments of various lengths, which can be sepa-
rated by gel electrophoresis. An example of a gel shows the abundance of
the bands (mononucleosomes (1n), dinucleosomes (2n), trinucleosomes
(3n), and so forth) obtained by increasing levels of digestion by MNase.
More extensive digestion trims the linker DNA but also invades the core
particles. Marker: 50 bp DNA ladder. To see this figure in color, go online.
Chereji and Clark
corresponding to mononucleosomes, dinucleosomes, trinu-cleosomes, etc., as the regularly spaced beads-on-a-stringstructure is digested to its constituent beads. Mononucleoso-mal DNA fragments are purified and sequenced from bothends using paired-end sequencing. The two reads origi-nating from each DNA fragment are aligned to the genomesequence to determine the position of the correspondingnucleosome, approximated by the center of the fragment.The number of nucleosome sequences needed for a high-resolution positioning analysis depends on the genomesize. About 10 million nucleosome sequences is goodcoverage for the yeast Saccharomyces cerevisiae (genome�12 Mb), but 500 million is barely adequate for mouse orhuman cells (genome �3000 Mb).
A complication in MNase-seq experiments is that thelengths of the nucleosomal DNA fragments obtained dependon the level of digestion because of sequence-dependentvariation in the rates of digestion in linkers and within nu-cleosomes by the endonuclease and exonuclease activitiesof MNase. This problem also affects the subset of nucleo-somes that are recovered in the mononucleosome band; inthe early stages of digestion, the mononucleosomes are rela-tively enriched in A/T nucleotides, whereas later in diges-tion, mononucleosomes are relatively enriched in G/Cnucleotides (7). This effect is presumably due to the prefer-ence of MNase for A/T-rich sequences over G/C-rich se-quences (8,9). Because the mononucleosome band in alight digest contains only a small fraction of the totalDNA (Fig. 1), any conclusions obtained by analyzing mono-nucleosomes from light digests should not be extrapolatedto the entire population of nucleosomes. Accordingly,most researchers sequence mononucleosomes obtained
2280 Biophysical Journal 114, 2279–2289, May 22, 2018
from a complete digestion (where the mononucleosomalband contains >80% of the DNA), although care must betaken to avoid overdigestion.
Although protection of �150 bp of DNA from MNase isconsistent with the detection of a nucleosome, other protein-DNA complexes can also protect about the same amount ofDNA (e.g., the RNA polymerase (Pol) III transcriptioncomplex (10)). A variant of MNase-seq called MNase-ChIP-seq involves an extra step in the protocol, in whichthe MNase digest is subjected to immunoprecipitation usingan antihistone antibody before purifying and sequencing theDNA. This method distinguishes nucleosomes from othercomplexes.
MNase-seq is the most frequently used method of map-ping nucleosomes, and a very large number of MNase-seqdata sets are currently available (11). However, the sequencepreference of MNase (8,9) has caused much controversy(7,12–15). To avoid the complications introduced byMNase, some alternative methods for profiling chromatinorganization have been developed. They include histoneChIP-seq (16,17), which uses sonication to fragment chro-mosomal DNA followed by immunoprecipitation using anantihistone antibody and DNA sequencing. The resolutionof this method is poor because it does not identify the loca-tion of the cross-link within the DNA fragment. A refinedversion of this method called ChIP-exo (18), in which theimmunoprecipitated DNA fragments are trimmed up to thehistone-DNA cross-link using an exonuclease, has muchhigher resolution. Another MNase-independent method formapping nucleosomes involves the use of methidium-propyl-EDTA, which preferentially cuts the linker DNAbetween nucleosomes, followed by sequencing (MPE-seq)(19). DNase-seq (20) and ATAC-seq (21) use probes (DNaseI or Tn5 transposase, respectively) for accessible chromatinand can yield useful nucleosome-positioning data butpotentially suffer from sequence preference issues likeMNase-seq.
The most important alternative method is chemical map-ping (22), which makes use of a targeted mutation in histoneH4 (S47C). This residue is very close to the nucleosomaldyad axis of symmetry in the structure (1,22). The sul-phydryl group of the cysteine reacts with a copper-chelatinglabel, which generates hydroxyl radicals that in turn cleavethe DNA in its vicinity. A map of these cleavage sites yieldsa high-resolution map of nucleosome dyads. The methodworks very well in budding yeast, but higher organismsgenerally have many copies of the histone genes, whichmakes it too difficult to introduce the S47C mutation inthese species (because all of the H4 genes would have tobe mutated). Potential problems with the chemical mappingmethod include DNA cleavage due to cysteine residues inthe DNA-binding domains of nonhistone proteins and back-ground DNA cleavage by the reagent. An improved protocolinvolving a Q85C mutation in histone H3 virtually elimi-nates these problems because it is based on detecting the
Biophysical Perspective
�51 bp fragments predicted by the crystal structure of thenucleosome (the Q85 residues in the two H3 moleculesare separated by 51 bp) (23). This improved protocol pro-vides a very precise nucleosome map, confirming the exis-tence of alternative nucleosome positions and that linkerDNA lengths are discrete, differing by multiples of the heli-cal twist (�10 bp) (23).
Many computational tools are available for the analysis ofnucleosome positioning data (11), but these are outside thescope of this article.
General features of chromatin organization
To describe chromatin organization in a quantitative way, itis useful to define two quantities. We define nucleosomeoccupancy (or coverage) as the probability of a basepairto be occupied by any nucleosome. To estimate this proba-bility, one would need to sequence all nucleosomes from apopulation of cells and to compute the fraction of cellsthat have nucleosomes occupying each specific basepair(Fig. 2). In practice, this is impossible to do because the pre-cise total number of cells is not known in an experiment, and
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Nucleosome occupancy
Dyad distribution
Nucleosome footprint
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FIGURE 2 Nucleosome occupancy versus nucleosome dyad distribution.
(A) Different cells contain nucleosomes at different positions. Each nucle-
osome footprint (blue oval) covers 147 bp of DNA, whose center corre-
sponds to a nucleosome dyad (red diamond). (B) By stacking all
nucleosome sequences and dividing by the number of cells, we obtain the
nucleosome occupancy, i.e., the probability of each basepair to be covered
by any nucleosome. The probability that any nucleosome dyad is located at
a given basepair represents the nucleosome dyad distribution, and this is
computed by stacking the single-basepair footprints of the nucleosome cen-
ters and dividing by the number of cells. Although we always compute the
above probabilities by analyzing the ensemble of nucleosome configura-
tions within a population of cells (i.e., computing ensemble averages),
assuming that the system is ergodic, we can also think of these probabilities
as time averages in a single cell. For example, when we map the nucleo-
somes (i.e., at a specific time), we may detect that a given basepair was
covered by a nucleosome in 80% of the cells so that the nucleosome occu-
pancy of that position is 0.80. Assuming the cell nucleus is an ergodic sys-
tem, we may also say that if we follow the dynamics of a given cell
throughout time, the same position will be covered by a nucleosome 80%
of the time. To see this figure in color, go online.
multiple steps of the nucleosome-sequencing experimentsintroduce different additional unknowns (e.g., the polymer-ase chain reaction amplification factor of each sequence, theprobability of each DNA fragment to be sequenced by thesequencer, etc.). Because of all these unknowns, severalways of normalizing the raw read counts have beendescribed in the literature. Taking the raw occupancy profileobtained by stacking and counting the number of nucleo-some sequences, assuming that at least one region in eachchromosome is almost always occupied by a nucleosome,one can divide the raw occupancy by the maximal countand obtain an estimation of the occupancy probability(24–26). To avoid making this assumption, one can usethe raw occupancy profile and normalize it by the genomicaverage (17) or by the average occupancy for each individ-ual chromosome (7,27).
Another useful quantity that describes nucleosome posi-tioning is the nucleosome dyad distribution (Fig. 2). Similarto the occupancy, the dyad distribution can be representedeither as a probability distribution (the probability of a givenbasepair to be occupied by any nucleosome center or dyad)or as a (normalized) dyad counts profile (which counts thenumber of nucleosome dyads that occupy each basepairand may be normalized by the genomic average or by theaverage dyad density per individual chromosome). Thenucleosome dyad distribution, D, and nucleosome occu-pancy, O, are not independent distributions. They are relatedby a simple convolution, OðnÞ ¼ ðD � kÞðnÞ, where theconvolution kernel, k, is trivial; kðmÞ ¼ 1 for�73 % m% 73 and kðmÞ ¼ 0 for jm j > 73: In thisarticle, we use only the normalized nucleosome occupancyand dyad distributions. Both are normalized by their corre-sponding chromosome average, and we refer to them simplyas nucleosome occupancy and dyad density.
Nucleosomal arrays are often interrupted by nucleosome-depleted regions (NDRs) (28,29), also called nucleosome-free regions (30), which usually correspond to DNase Ihypersensitive sites (31). These accessible regions are typi-cally found at regulatory and functional regions, such aspromoters, enhancers, and origins of DNA replication(32–37).
Fig. 3 A shows the average nucleosome occupancy anddyad density for all genes relative to the transcription startsite (TSS) in S. cerevisiae. There is a prominent NDR atthe promoter, just upstream of the TSS, that is flanked bypeaks of high nucleosome occupancy. The downstream nu-cleosomes on the genes are numbered sequentially such thatthe first nucleosome is the þ1 nucleosome. Similarly, thefirst nucleosome upstream is the �1 nucleosome. This posi-tioning of regularly spaced nucleosomal arrays on each generelative to a fixed point in the DNA sequence (the TSS inthis case) results in ‘‘nucleosome phasing.’’ Phasing is notthe same as spacing, which refers only to the averagedistance between adjacent nucleosomes and does not neces-sarily depend on a fixed point in the DNA sequence. Phasing
Biophysical Journal 114, 2279–2289, May 22, 2018 2281
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-5 -4 -3 -2 -1 0 1
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0 1 2
Convergent
Tandem
Divergent
Tandem
FIGURE 3 Nucleosome organization near the gene ends in S. cerevisiae. (A) Average nucleosome occupancy (top) and nucleosome dyad distributions
(bottom) at the TSS (left) and TTS (right) are shown. Profiles obtained for three levels of MNase digestion are shown. (B) A heat map shows the nucleosome
dyad distribution near all TTSs, sorted according to the distance to the promoter (NDR; thick blue stripe) of the downstream gene. Only about one-third of the
TTSs are located in nucleosome-depleted regions. These NDRs correspond to the promoters of the downstream genes in the tandem gene pairs. Inset:
a scheme of the gene alignments is shown. All yeast genes (red arrows) are aligned at their TTSs and sorted according to the distance to the promoter
(NDR) of the downstream gene (gray arrows). The aligned NDRs corresponding to the downstream genes (gray arrows) generate a blue stripe in the
heat map, which is flanked by phased nucleosome arrays on both sides. The promoters corresponding to the genes aligned at their TTSs (the first in each
pair of genes; red arrows) are not in phase with each other (because of different gene lengths) and do not generate a blue stripe in the heat map. (C) The
heat maps show the dyad distributions near both gene ends: (left) 50 ends are aligned at the þ1 nucleosome and are sorted according to the distance to
the promoter (NDR) of the upstream gene, and (right) 30 ends are aligned at the TTS and are sorted according to the distance to the promoter (NDR) of
the downstream gene. Genes are split into two groups according to the relative orientation of the neighboring gene: divergent/tandem and convergent/tandem,
respectively. Insets: schemes of the alignments are shown; all yeast genes (red arrows) are aligned either at their 50 (left panel) or 30 ends (right panel) andsorted according to the distance to the NDR of the upstream and downstream genes (gray arrows), respectively. When the 50 ends and the NDRs of the genesare aligned, a blue stripe appears in the heat map, indicating the locations of the NDRs. Annotations forþ1 nucleosome position, NDR center, TSS, and TTS
were obtained from (23), and MNase-seq data were obtained from (7).
Chereji and Clark
requires that regularly spaced nucleosome arrays are simi-larly positioned in all cells. This stereotypical chromatinorganization has been reported many times for yeast(28,29,38) and other organisms (35,39–41). ProminentNDRs are also observed at yeast origins of replication(autonomously replicating sequence elements) and attRNA genes (10,36).
Although omnipresent in genome-wide studies of nucleo-some organization, average profiles can be misleading, as
2282 Biophysical Journal 114, 2279–2289, May 22, 2018
the average pattern does not offer much information aboutthe gene-to-gene variability. For example, the transcriptiontermination site (TTS) appears to be another phasing point,although the NDR is much weaker and so is the phasingrelative to the TTS (Fig. 3 A) (30). However, only about athird of yeast genes possess a true NDR at their TTS; themajority of TTSs do not have an NDR (Fig. 3 B). Moreover,only the TTSs that overlap with the promoter of a down-stream gene contain a true NDR (tandem gene pairs). This
A
B
C
FIGURE 4 Multiple determinants of nucleosome positioning. (A) An Integrative Genomics Viewer browser snapshot shows the nucleosome distribution on
yeast chromosome IV (550,400–558,160). Nonhistone barriers (e.g., GRFs Reb1, Rap1, and Abf1) bind to DNA (light blue rectangles) and compete with
histones, resulting in nucleosome-depleted regions. Weakly transcribed genes (light pink rectangles) are characterized by well-positioned nucleosomes form-
ing regular arrays, whereas highly transcribed genes (red rectangles) are characterized by poorly positioned nucleosomes, i.e., increased cell-to-cell vari-
ability. Pol II occupancy (Rpb3 subunit) shows the relative transcription level. MNase-seq data and ChIP-seq data (Reb1, Rap1, Abf1, and Rpb3) are
shown in individual Integrative Genomics Viewer tracks. For illustrative purposes, to distinguish linkers between individual nucleosomes, MNase-seq reads
were trimmed symmetrically to 101 bp. ChIP-seq data show Reb1 (114), Rap1 (115), Abf1 (116), and Rpb3 (16). MNase-seq data from (7) are shown. (B)
DNA sequence alone is not able to properly position nucleosomes at the gene promoters. The left heat map shows nucleosome dyads reconstituted in vitro by
salt gradient dialysis (data from (52)) and the lack of nucleosome-depleted regions and nucleosome phasing at promoters. The other four heat maps show the
distribution of nucleosome dyads in vivo in different remodeler mutants (data from (27)). A triple mutant in which three nucleosome-spacing enzymes have
been deleted (chd1D isw1D isw2D) and a double mutant in which the two most important spacing enzymes are deleted (chd1D isw1D) both show vastly
disrupted nucleosome phasing compared to that of wild-type cells (rightmost heat map). CHD1 is active in the single mutant isw1D, which has regular nucle-
osome arrays, shorter spacing, and weaker phasing compared to that of wild-type cells (27). All heat maps contain yeast promoters aligned at the NDR and
sorted according to NDR width from wild-type cells. (C) The most important determinants of nucleosome organization include the following. 1) Stable bar-
rier complexes prevent nucleosome formation to create NDRs at multiple regions along the genome. Promoters may be occupied by transcription-preinitia-
tion complexes, transcription factors (TFs), and/or chromatin remodelers. tRNA genes are occupied by a stable preinitiation complex containing Pol III TFs
TFIIIB and TFIIIC; replication origins are occupied by the origin recognition complex (ORC). 2) In combination with barrier complexes, chromatin remod-
elers (spacing enzymes) form regular nucleosome arrays between NDRs. 3) Pol II disrupts nucleosome arrays from gene bodies, whereas remodelers restore
(legend continued on next page)
Biophysical Perspective
Biophysical Journal 114, 2279–2289, May 22, 2018 2283
Chereji and Clark
becomes obvious when convergent or tandem gene pairs areanalyzed separately; the TTSs corresponding to the conver-gent genes are not generally nucleosome-depleted (Fig. 3 C,right panel). On the other hand, most yeast promoters arenucleosome-depleted, independent of the orientation ofthe upstream gene (Fig. 3 C, left panel). This shows that,unlike promoters, TTSs are not generally nucleosome-freeper se; they are located within an NDR only if they are adja-cent to a promoter.
In summary, promoters are flanked by phased arrays ofnucleosomes. In yeast, the average spacing of the nucleo-somes in these arrays is �165 bp, although individual genesdisplay a range of values (27). In higher organisms, thespacing is usually longer. During the last decade, a greatamount of effort has been put into understanding the deter-minants of nucleosome positioning and how these cangenerate the complex nucleosome organization patternseen in Fig. 3 C.
Determinants of nucleosome positioning
Multiple factors affect nucleosome positioning in vivo,including DNA sequence, other DNA-binding proteins,transcription, and chromatin remodelers. Below, we discussthe most important determinants of nucleosome positioning.
DNA sequence
The translational position of a nucleosome refers to its loca-tion on the DNA, which can be defined by the chromosomalcoordinates of its boundaries or, more commonly, by thechromosomal coordinate corresponding to its dyad (nucleo-somal DNA midpoint; Fig. 2). The importance of DNAsequence in determining nucleosome positions is clearfrom experiments involving reconstitution of nucleosomesusing only purified DNA and core histones. They showthat different DNA sequences have different affinities forthe histone octamer because the free energy required tobend the DNA sharply around the histone octamer dependson the sequence. Early studies involving nuclease digestionof native and reconstituted nucleosomes noted that manynucleosomal sequences contain quasiperiodic nucleotidedistributions (42–44). These reflect the anisotropic bendingpreferences of the DNA, which predetermine the orientationof the DNA relative to the histone octamer, with A/T-richminor grooves preferentially facing in and G/C-rich minorgrooves preferentially facing out (43).
When a histone octamer binds to a short DNA molecule,it must choose between several overlapping translationalpositions specified by the bending properties of the DNA
the regular organization. 4) The DNA sequence affects nucleosome positions eith
cleotide distributions favor specific rotational positions, whereas poly(dA:dT) t
other proteins that prevent nucleosome formation (e.g., the motif shown in the p
from this locus).
2284 Biophysical Journal 114, 2279–2289, May 22, 2018
sequence. Thus, nucleosomes formed on a population ofshort DNA molecules occupy one of several alternativepositions. These translational positions are usually shiftedrelative to one another by �10 bp, the helical repeat ofDNA, and are referred to as rotationally related translationalpositions because, for all of these positions, the same side ofthe DNA helix faces inward and is in contact with the his-tones (the result of the anisotropic bending mentionedabove). We illustrate this concept further by consideringtheþ1 nucleosome on a specific gene; its position in a givencell is one of several rotationally related possibilities (23).Each of these alternative positions for the þ1 nucleosomecontributes to the occupancy of the þ1 nucleosome in pro-portion to the fraction of cells that adopt that position(Fig. 2). The range of rotational positions for the þ1 nucle-osome and their relative occupancies determine their‘‘fuzziness.’’
A comparison of high-resolution nucleosome maps on thesame DNA sequence in vivo and reconstituted in vitro re-veals that very similar translational positions are adoptedin vitro and in vivo. However, the relative occupancies ofeach position are quite different. This reflects, at least inpart, the fact that nucleosomes in reconstituted chromatinare not regularly spaced, unlike nucleosomes in vivo. Theseobservations led to the proposal that genomic DNA mayencode nucleosome positions (45–47). More precisely, itwas proposed that the genomic DNA sequence specifiessets of overlapping translational positions from which thecell must choose. Recently, it has been shown that the þ1and �1 nucleosomes preferentially occupy equivalent rota-tional positions, both in wild-type and in cells depleted ofthe ‘‘Remodels the structure of chromatin’’ (RSC) complex,and it is only the relative occupancy of each of these alter-native rotational positions that is different in the mutantcells. This suggests that, at the single-nucleosome scale, his-tone octamers sample the possible rotational positions deter-mined by the genomic sequence and preferentially select thebest alternative positions, separated by multiples of the he-lical twist (23). This hypothesis is also supported by in vitrostudies (48).
Later studies essentially confirm that DNA sequence is animportant determinant of nucleosome positioning in vivo butemphasize that additional factors, such as chromatinremodelers, are needed to account for the nucleosome occu-pancies observed in vivo (49–52) (Fig. 4). The strongesteffect of DNA sequence on nucleosome positioning isthat of poly(deoxyadenylic-deoxythymidylic) (poly(dA:dT))tracts, which intrinsically disfavor nucleosome formationbecause of their particular intrinsic bending properties(53). Poly(dA:dT) tracts can also guide chromatin
er directly through its DNA-bending properties (e.g., periodic WW/SS dinu-
racts disfavor nucleosome formation) or indirectly through the binding of
romoter is recognized by the Gcn4 activator, which activates transcription
Biophysical Perspective
remodelers to slide nucleosomes (54) or create NDRs (52).Other DNA motifs are recognized by sequence-specific tran-scription factors and general regulatory factors (GRFs),which in turn may recruit chromatin remodelers. Theseproteins together can determine the width of the NDR andinfluence the positioning of the flanking nucleosomes. Inconclusion, DNA sequence is an important determinant innucleosome positioning, but additional factors are neededto determine long-range chromatin organization (48).
Nonhistone barriers
A critical factor in determining chromatin organization isthe NDR. It has been proposed that nonhistone proteinsbound within the NDR act as a barrier to nucleosome forma-tion, directing nucleosomes into flanking positions. Nucleo-somes formed on the DNA between two such barriers are‘‘statistically positioned’’ (55,56) similarly to a gas of hardspheres interacting through a hard-core potential (57,58).The distribution of nucleosomes on a genomic scale hassince been studied using the model of a gas of one-dimen-sional hard rods (24–26,59–64), and it has been shownthat competition with nonhistone barriers and steric occlu-sion can suffice to explain the salient features of nucleosomepositioning (23).
The question arises as to the nature of the proteins formingbarriers at NDRs. It has been proposed that there is a special,nuclease-accessible, ‘‘fragile’’ nucleosome in some NDRs,but this is controversial (7,65). It is likely that sequence-spe-cific proteins are required for barrier formation becauseNDRs are formed at specific genomic locations, such as pro-moters, enhancers, tRNA genes, and replication origins.Indeed, it is clear that yeast tRNA genes are occupied bythe stable Pol III transcription factor (TFIIIB-TFIIIC) com-plex (10) and that yeast replication origins (autonomouslyreplicating sequence elements) are occupied by stable originrecognition complexes (ORC) (66). The barrier complexesformed at Pol II promoters have not yet been identified,but by analogy with tRNA genes, they may include transcrip-tion factors and GRFs, such as Reb1, Abf1, or Rap1 (Fig. 4A). Other general regulators of gene expression, such asMediator, are also found at promoter NDRs (67).
Most sequence-specific DNA-binding proteins areoccluded from their target sites if they are occupied by anucleosome, although transcription factors can displace thenucleosome if it contains multiple sites for the same factoror for different factors (68). However, there are transcriptionfactors (‘‘pioneer’’ factors) that are capable of high-affinitybinding to nucleosomal sites (69,70). Transcription-factorbinding may lead to nucleosome remodeling (71,72).
Transcription
In vitro, Pol II can transcribe through a nucleosome, result-ing in the loss of a histone H2A-H2B dimer or, if multiplepolymerases are present on the same template, in the com-
plete displacement of the histones from the DNA (73).This also seems to be true in vivo (17), and in general, nucle-osomal arrays are disrupted on the most highly transcribedgenes in yeast (Fig. 4 A). Apart from having poorly posi-tioned nucleosomes, highly transcribed yeast genes showextreme nucleosome spacing. The average spacing for the200 most-transcribed yeast genes is �159 bp, compared tothe genomic average of �165 bp (3). A correlation betweenhigher transcription levels and shorter nucleosome spacingis also observed in human T cells (41). Transcription rateis therefore an important factor in chromatin organization.Additional disordering effects are likely to result from thestochastic activation and inactivation of promoters that leadsto the discontinuous production of mRNA (transcriptionalbursting) (74–76).
The effect of transcription, or at least the potentiation fortranscription, on chromatin organization is more evident inhigher organisms. Although different cell types from thesame organisms contain the same DNA sequence, theirchromatin organization can be very different, reflecting theirvarious differentiation states. Generally, there is a cleardifference between the promoter chromatin structures ofactive and silent genes: active promoters are nucleosome-depleted and flanked by phased nucleosome arrays, whereassilent promoters are covered by nucleosomes, which areincorporated into regular arrays but are not phased relativeto the TSS (13,15,40). This suggests that nonhistone barriercomplexes like those found at yeast promoters, which act asphasing points for neighboring nucleosomes, might also bepresent at active gene promoters in higher organisms andmight be absent from silent gene promoters. If this barriercomplex sets the chromatin organization to potentiate tran-scription, then it could also constitute a mechanism of tran-scriptional memory (77) both by bookmarking the activegene promoter during the first round of transcription andby facilitating gene looping (78). Consistent with the genelooping hypothesis, it was recently found that Mediator,which is one possible component of the promoter barriercomplex, interacts with multiple factors that are involvedin mRNA 30-end processing (67).
ATP-dependent chromatin remodelers
The ATP-dependent chromatin remodelers (79) play a crit-ical role in determining chromatin organization and nucleo-some positioning. Both yeast and higher organisms havemany such enzymes, which have been intensively studied(27,52,80–85). Their activities include shifting nucleosomesalong DNA, spacing nucleosomes, removing or transferringnucleosomes, and switching histone variants. Remodelerscan convert stable chromatin into a dynamic structure usingthe free energy available from ATP hydrolysis.
The combination of their activities is sufficient to accountfor the stereotypical chromatin organization on genes,although the details of the interplay between different
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remodelers and how they relate to epigenetic marks are notyet fully understood. The SWI/SNF and RSC classes ofenzymes are capable of shifting and removing nucleosomesin vitro and play a role in determining the size of the NDR.RSC is particularly important for positioning theþ1 and�1nucleosomes (81,84). The INO80 chromatin remodeler alsohas a role in establishing the NDR and in positioning theflanking nucleosomes (86). The ISW1 and CHD1 classesof enzymes space nucleosomes in vitro and on genes in vivo(83,87–89) (Fig. 4 B). Indeed, in yeast, the CHD1 and ISW1enzymes compete to set short or longer nucleosome spacingon genes (27). This effect increases heterogeneity at allnucleosome positions because the þ2 nucleosome willhave more than one position to choose from relative tothe þ1 nucleosome, depending on which enzyme is settingthe spacing. This heterogeneity in linker length is predictedto affect the folding of the chromatin fiber (90,91). Anotherclass of enzymes, exemplified by the SWR complex, recog-nizes an NDR and then exchanges H2A-H2B dimers in theflanking nucleosomes for H2A.Z-H2B dimers (86,92),although H2A.Z exchange does not affect nucleosome posi-tioning (81).
Overall, an attractive model of chromatin organizationinvolves interplay between the RSC, INO80, and barriercomplexes to create an NDR and to determine the locationof the þ1 nucleosome (52). This nucleosome acts as a refer-ence nucleosome for the ISW1 and CHD1 spacing enzymes,which track down the gene, adjusting the spacing. An inter-esting corollary of this model is that phase interference willoccur between nucleosomal arrays originating from adja-cent barriers, as spacing enzymes are recruited at both bar-riers and track toward one another (84). We propose that theremodelers organize chromatin by transferring nucleosomesbetween alternative positions specified by the DNAsequence, i.e., changing the occupancies of these positions.
DNA replication
Because DNA replication requires separation of the twoDNA strands, the passage of the helicase and the replicationmachinery results in a major disruption of chromatin.Assembly factors reconstitute old nucleosomes and assemblenew nucleosomes behind the replication fork (93). In yeast,origins of replication are clearly defined DNA sequencesbound by the ORC, which is sufficient to generate an NDRand to direct the positioning of neighboring nucleosomes.ORC is similar to a promoter barrier complex, constitutinga phasing point for the neighboring nucleosomes. On theother hand, the nucleosome organization of replication ori-gins has an important role in origin selection and function(36). The DNA replication process has been recently studiedboth experimentally (93–97) and theoretically (98,99). InDrosophila, the stereotypical nucleosome organization atpromoters and enhancers is rapidly lost during replicationand is relatively slow to recover (94).
2286 Biophysical Journal 114, 2279–2289, May 22, 2018
Modifications of DNA and histones
DNA methylation affects nucleosome positioning in multi-ple eukaryotes (100). Genomes of diverse eukaryotes (e.g.,Aureococcus anophagefferens, Emiliania huxleyi, Ostreo-coccus lucimarinus, and Micromonas pusilla) display aperiodic DNA methylation pattern in the linkers of genebodies, regardless of transcription levels, contributing tonucleosome positioning between methylated DNA regions(100). Covalent modifications of DNA, as 5-methylcytosineand 5-formylcytosine, affect DNA flexibility, nucleosomestability, and the positioning of nucleosomes (101,102).
Many amino acid residues of the histone tails are subjectto posttranslational modifications, such as acetylation,methylation, and phosphorylation. Histone modificationsmay affect nucleosome dynamics, stability, and other phys-ical properties of the nucleosome. Moreover, these modifiedresidues may also be important for recruiting remodelersthat can reorganize chromatin (103,104). For example,when DNA damage occurs, histone modifications are usedfor signaling the position of DNA damage and for recruitingchromatin remodelers to the site of damage that open thechromatin, allowing the repair proteins to access the siteof damage (105).
Future perspectives
The central importance of chromatin organization in generegulation is apparent from the rapidly increasing numberof links between chromatin proteins and disease, particu-larly cancer (see (106) for a recent review). It is fair tosay that there has been major progress in understandingthe basic chromatin organization of genes, particularly inyeast. It is obvious that nucleosome positions are deter-mined by an interplay of multiple factors (Fig. 4 C), noneof which is sufficient to establish the genome-wide nucleo-some organization alone, but there are still many details tobe resolved. Perhaps the most important challenges for thefuture include elucidating the mechanisms of chromatinopening and NDR formation, identifying the complexesthat occupy promoters and enhancers, and understandinghow chromatin organization is altered and maintained dur-ing differentiation and development.
Although the steady-state distribution of nucleosomesand other DNA-binding proteins can be observed in largeensembles of cells using currently available techniques,studying the dynamics of single-nucleosome remodelingevents in cells is both a major goal and challenge of futurework. Rapid advances in single-molecule tracking experi-ments will shed light on this interesting topic in thefollowing years and will allow us to elucidate the precisemechanisms of nucleosome positioning, chromatin organi-zation, and gene regulation.
As scientists attempt to solve the multiscale problems ofchromatin fiber organization (91,107,108), new opportunities
Biophysical Perspective
for biophysicists are emerging. Rapid developments in super-resolution imaging technologies and dense Hi-C data arestarting to overlap; these advances are expected to elucidatethree-dimensional chromatin organization and chromatindynamics at the single-cell level.
The seminal contributions of Jorg Langowski to thebiophysics and biochemistry of nucleic-acid-protein com-plexes have had a major impact on our understanding ofthe dynamics of DNA, the nucleosome, and chromatin.His elegant work, published in more than 200 articles, hasrevealed important insights into DNA flexibility (109) andlooping (110), nucleosome unwrapping (111), histone taildynamics (112), and the structure of the 30 nm fiber(113). We dedicate this article to the memory of ProfessorJorg Langowski.
ACKNOWLEDGMENTS
We thank Josefina Ocampo for unpublished data and helpful discussions.
This work was supported by the Intramural Research Program of the
National Institute of Child Health and Human Development, National Insti-
tutes of Health.
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