Molecular Cell

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Nucleosome Positioning: MultipleMechanisms toward a Unifying Goal

Arnob Dutta1 and Jerry L. Workman1,*1Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA*Correspondence: jlw@stowers.orghttp://dx.doi.org/10.1016/j.molcel.2012.09.015

In this issue, Hughes et al. (2012) show that nucleosome positioning is determined not by any singlemechanism, but by the coordinated action of multiple factors, including the underlying DNA sequence,species-specific DNA binding proteins, chromatin remodelers, and the transcription machinery.

DNA in eukaryotic organisms is organized

into nucleosome arrays, resembling the

classic structure of ‘‘beads on a string.’’

But what determines where nucleosomes

are positioned across the genome?

Studies from various groups have sug-

gested that a number of factors, including

the DNA sequence, DNA binding factors,

chromatin remodelers, and the transcrip-

tion machinery, either on their own or in

consort, regulate nucleosome positioning

(Iyer, 2012). The new study by Hughes

et al. in this issue provides a model for

genome-wide nucleosome positioning

that is not determined by any single

factor, but is dependent on all the afore-

mentioned mechanisms working together

(Hughes et al., 2012).

In order to address the hotly debated

question of what factors determine

nucleosome positioning, Hughes and

colleagues took an interestingly different

approach. Instead of comparing nucleo-

some organization across multiple

species, the group took a functional

evolutionary approach. To tease out how

cis- and trans- acting factors affect nucle-

osome positioning, studies were carried

out by transforming large segments of

heterologous DNA from evolutionarily

divergent yeast species, K. lactis or

D. hansenii, into S. cerevisiae, followed

by analyses of nucleosome occupancy

and preinitiation complex recruitment

using chromatin immunoprecipitation. If

cis-acting factors such as the underlying

DNA sequence were the major determi-

nants of nucleosome positioning, results

would be similar in both the S. cerevisiae

host and the native species. On the other

hand, if species-specific trans-acting

factors such as DNA binding proteins

or the transcription machinery were

involved, nucleosome positioning would

differ.

A longstanding question has been the

role of DNA sequence in determining the

positioning of nucleosomes. Studies by

Kaplan et al. (2009) and others suggested

that A/T-rich sequences are a character-

istic of nucleosome depleted regions

(NDRs), specifically in yeast, whereas

GC-rich sequences, in higher eukaryotes,

are enriched for nucleosomes (Kaplan

et al., 2009; Segal et al., 2006). This

suggested the existence of a genomic

code for nucleosome positioning. By

comparing nucleosome positioning on

DNA from K. lactis or D. hansenii present

inS. cerevisiae to that in native organisms,

the authors found that many NDRs were

maintained in both host and native

species. Nucleosome depletion from

NDRs correlated well with the length of

A/T-rich sequences. This was particularly

evident on DNA sequences from K. lactis,

where NDRs are characterized by AT

stretches. However, D. hansenii NDRs

are largely deficient in AT sequences

and failed to show nucleosome depletion

when present in S. cerevisiae. This result,

along with studies by other groups, rein-

forces the fact that the underlying inflexi-

bility of A/T-rich DNA sequences plays

a partial role in determining nucleosome

positioning (Figure 1). Alternatively, NDR

formation and positioning of adjacent

nucleosomes has been attributed to

DNA binding factors such as Reb1,

Rap1, and Abf1 in S. cerevisiae (Hartley

and Madhani, 2009). These factors bind

promoters of genes and have been sug-

gested to direct positioning of the +1

nucleosome (Figure 1). Another example

is the NDRs in D. hansenii, which lack

A/T sequences and can be formed as

Molecular Cell

a consequence of the binding of the

factor Cbf1. As S. cerevisiae does not

contain Cbf1, these NDRs are lost on

D. hansenii DNA that is transformed into

S. cerevisiae. This result is consistent

with observations that DNA binding

factors such as CTCF in higher eukary-

otes can help determine nucleosome

positioning (Iyer, 2012).

Previous studies have shown that a

hallmark of the yeast genome is strongly

positioned +1 and�1 nucleosomes flank-

ing NDRs at promoters and well-phased

nucleosome arrays in the body of genes

(Jiang and Pugh, 2009). Based on these

observations, the barrier model was

suggested, wherein strong positioning of

the first nucleosome determined the

phasing of nucleosomes downstream

(Mavrich et al., 2008). However, subse-

quent studies showed that such ordered

arrays of nucleosomes were lost when

nucleosomes were reconstituted in vitro

by salt dialysis, but could be restored by

the addition of yeast extracts and ATP,

implicating roles for chromatin remodel-

ers (Zhang et al., 2009). Moreover, recent

studies by the Owen-Hughes lab have

shown that phased nucleosomes are lost

when remodelers Chd1, Isw1, and Isw2

are deleted in yeast (Gkikopoulos et al.,

2011) (Figure 1). To address if phasing

of nucleosomes is species specific,

Hughes et al. compared spacing of nucle-

osomes on heterologous DNA present in

S. cerevisiae to that in native species.

Interestingly, the average internucleo-

somal distances were shortened from

�178 bp as observed in K. lactis to

�165 bp when present in S. cerevisiae,

resembling spacing on S. cerevisiae

genes. These studies indicate that phased

nucleosomal arrays are determined not

48, October 12, 2012 ª2012 Elsevier Inc. 1

TF

NDR

AAAA

NDR

AAAA

NDR Promoter+1-1

Chromatin remodelers (Chd1, Isw1, Isw2)

TF

Transcription initiationcomplex

AARemodelers

(Reb1, Abf1, Rsc, CTCF)

Remodelers

A

B

TFRNAPII

Figure 1. Multiple Mechanisms Define Nucleosome Positioning(A) Nucleosome depleted regions (NDRs) can be generated either by the presence of A/T-rich sequencesthat occlude nucleosome formation and/or by transcription factor (TF) binding, along with the action ofchromatin remodelers such as Swi/Snf and RSC, which can displace nucleosomes.(B) Binding of transcription factors and the assembly of the preinitiation complex, along with RNApolymerase II (RNAPII), helps define the well-positioned +1 nucleosome adjacent to the transcriptionstart site. The ordered phasing of nucleosomes downstream is brought about by the coordinated actionof ATP-dependent chromatin remodelers Chd1, Isw1, and Isw2.

Molecular Cell

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by DNA sequences, but by the action of

chromatin remodeling factors inherent to

individual species.

In yeast the +1 nucleosome is posi-

tioned immediately downstream of the

transcription start site (TSS). Its position

can be influenced by DNAbinding factors,

RNA polymerase, and the preinitiation

complex (Weiner et al., 2010; Zhang

et al., 2009). Hughes et al. found that for

�50% of K. lactis or D. hansenii genes,

the position of the +1 nucleosome was

shifted by 20 bp or more when the DNA

was transformed into S. cerevisiae.

Moreover, they observed that the TSS of

K. lactis and D. hansenii genes expressed

in S. cerevisiae were similarly shifted.

These changes in the TSS and the +1

nucleosome closely matched that for

2 Molecular Cell 48, October 12, 2012 ª2012

S. cerevisiae genes. Interestingly, a

number of NDRs absent in D. hansenii

genome were observed in the coding

regions of D. hansenii DNA present in

S. cerevisiae. These NDRs were due to

fortuitous recruitment of the S. cerevisiae

transcription machinery, which conse-

quently positioned adjacent nucleo-

somes. These results support previous

studies (Zhang et al., 2009; Weiner et al.,

2010) that the +1 nucleosome position

is linked to the TSS and regulated by

the transcription initiation machinery

(Figure 1).

While A/T sequences can be instru-

mental in occluding nucleosomes and

forming NDRs, DNA binding factors can

also influence nucleosome occupancy,

resulting in nucleosome-deleted regions.

Elsevier Inc.

Positioning of the nucleosomes flanking

the NDRs is determined by the transcrip-

tion initiation machinery and RNA poly-

merase along with DNA binding factors.

The phased ordering of nucleosomes, on

the other hand, is a consequence of chro-

matin remodeling factors. Thus, in trying

to answer the question—what determines

nucleosome positioning?—the study by

Hughes et al. supports an ‘‘all of the

above’’ model (Figure 1). A question for

the field now becomes if and how nucleo-

some positions are maintained during

cell division and passed on to daughter

cells. For example, are all nucleosome

positions retained, or must they be re-

established from the genetically coded

NDR sequences during each cell division?

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