Chromatin, structure and function 3rd ed a wolffe (AP, 1998)

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Contents

Preface to the firs t edition
Preface 10 the second edition
Preface to the third edition
1

Ove rview
L1 Introductory comml'nts
1.2 Dcvelopm<-'Tl t o f r~'SCa rch into chroma tin structure ,md
function

2

C hromatin Structu.re

XIII

1
I
2

7

2..1 DNA and hislones
2.1.1 DNA st ructure
2.1.2 The histone>

7
8
15

2.2 The

19

nucl~me

2.2.1 The nucleosome hypothesis
2.2..2 The organization of DNA and hiSionl'S in the
nuclcosome
2.2.3 The s tructu re of DNA in the nudeosome
2.2.4 The po6ition of the core histones in the nuck"O(lOflle
2.2..5 DNA sequenre-dir«I<-'2.3 The organiza tion of nudoosomes into the chromatin fibre
2.3.1 lIistone H I and the compaction of nUdl"D50mal
arrays
2.3.2 The chromatin fibre

19
20

26
30
41
50
50
58


vi

O m/I'II/S

2.4 Chromosomal architecture
2.4.1 The radial loop and helical folding models al
chr()l'n(l6QrTle structure
2.4,2 The nuclear infrastructu re, the ct'ntromere,
telomeres, protein oom~ts and their function
2.43 Lampbrnsh and polytene chromosomes
2.5 Modulation of chrom0l!K)fl1.11 structure
2.5.1 Histone variants
2.5.2 rOSHr'H\slalion.a1 modification of core histones
2.5.3 Unkl'r histone phosphorylation
2.5.4 ActiYators and represson that malo! use of
chromatin modiflCatiom 10 n'gulate tr'H\5Cription
2.5.5 Remodelling al chromatin during s permatogt'T1eSis
2..5.6 Heterochromatin, po6ition effect, locus control

regions and insulatOB
2..5.1 ONA methylation and chromatin
2.5.8 The HMCs and reJ~ted proteins
2.5.9 Functional compartmentalization althe nucleus

3 Chromatin and Nudear Assembly
3.1 Interactions between nuclear ~tructUT1' and cytoplasm
3.1.1 Nuclear transplant .. tion, remodelling and assembly
3,1.2 Heterokaryons
3.2 Chromatin assembly
3.2. 1 Chromatin assembly on n.'Plica ting mdogCllOUli
chromosomal DNA i,r vit'll
3.2.2 Chromatin aSS"rlLbly 111 VItro
3.3 Experimental approocne towards the n.'OOruItitution of
transcriptionally acth'e and silent states
3.3.1 Purified systems
3.3.2 Chromatin assembly and transcription in Xmopus
egw; and oocytes
3.3.3 Chromatin assembly on DNA introduced into
somatk cells
3.3A Yeast minichromosomes
3.4 Modulation of the chromosomal environmen t du ring
dt.'Velopment
3,4.1 Tttruhymtnll

3.4,2 Xrnopli!! and sea urchin
3.4.3 The mouse
3.4.4 Developmental regulation allong-range chromatin
structure

66
61
11
SI
81

88
97
108
I II

126
128
141
151
151

173

In

114
187
189
190

195

200
203

206
215

220
224
224
226
232


Contellls

4

How do Nuclear Processes Occur in Chromatin?
4.1 Overview of nuclear PH)('C5sro'l
4.1.1 The problem of specificity
4.1.2 Action at a distance
4.1.3 The traMCriptiooal machiJl(.'I')'
4.1.4 Stable and ullStable transcription complexes
4.1.5 Regulation of gene activity

4.\ .6 Sequence sptdfic DNA-bindin8 protein!l
4.1.7 PToblems for nuclear prtJCeSSeS in chromatin
4.2. Interaction of Imlls-acting factors with chromatin
4.2.1 Non-specific in te ractiortll
4.2.2 5p<.'1:ific IrQ/Is-acting factors and non-speciflC
chromatin
4.2.3 Specific InlIIs-acting factors and specific chroma tin
4.2.4 Tnrns-acting fact~. DNase I .5eIlSitivity. DNase I

hyper.;ensitive sites and chl"()<ll(l5(Xllil architectun>
4.2.5 Tnln$-acting fa(:tors and the Jocal organization of
chl'Ofl1.illin structun>
<3 1'lOc" ' sive enzyme complexes and chl'Ofl1.illin structun>
4.3.1 Replication and the iKCl'!SS of tral\SCription factors
to DNA
4.3.2 The fate of nucleosomes and transcription
complexes during replication
4.3.3 Chromatin structun> and DNA replication
4.3.4 Tr~ nscription and chromatin ink'grity 111 [)itv
Transcription and chromatin integrity III 1I.lro
Chroma tin structu n> and DNA repair
4.4.1 Influence of chromatin structun> on DNA damage
4.4.2 Repairing DNA and chromatin

."
•••

5

Future Prospects
5.1 Loca l chromatin 5tructUn>
5.' Long·range chroma tin and chrom06OfTl.a! structun>

Referencell
Index

240

24.

,..
N'
,""..
'"
'"
241

275
276

''''
2B6
311

315
316
320
326
328
333

337
338
339

342

'"

345

34B

."

vii


Preface to the First
Edition

Research on chromatin structure and function is expanding rapidly.
Technical advances allow us to follow the events regulating gene
expression in the eukaryotic nucleus in molecular detail. Within the
chromosome, alterations in the organization and accessibility of key
regulatory DNA sequences can be documented and interpreted. This
book is intended to introduce scientists to this exciting field, in the
expectation that many more contributions will be required before we
understand completely how the nucleus of a eukaryotic cell functions.
The book has five sections. The first section is a brief overview of
the issues discussed and an historical account of their development.
The second section describes the structure of chromatin and
chromosomes as far as it is known. Concepts concerning chromatin
structure are already very well developed; indeed, many of the
biophysical techniques and paradigms for studying protein-nucleic
acid interactions were pioneered using the basic unit of chromatin, the
nucleosome, as a model. In contrast, large-scale chromosomal
architecture is much less well defined, as is the influence of
modifications of structural proteins on chromatin and chromosome

organization. How these changes may contribute to the various
requirements for correct chromosomal function is a recurring theme.
A complete understanding of the eukaryotic nucleus requires not
only that we know how to take it apart, but also that we can assemble
it from the various component macromolecules. The third section
describes the approaches, results and interpretations of experiments
designed to accomplish this task. The biological constraints of


x

Chromatin: Structure and Function

assembling a chromosome rapidly are discussed with reference to its
final form and properties.
Form and function are intimately related. Once a complete
understanding of a process is achieved, it is impossible to separate
one from the other. The fourth section describes the multitude of
approaches taken towards resolving how DNA can be folded into a
chromosome and still remain accessible to the regulatory proteins, and
allow processive enzymes to move along the length of the DNA
molecules. It is in this field of research that much of the current
progress on the interrelationship of chromatin structure and function
is taking place. The final section offers a perspective on where
prospects for future development might lie.
I would like to thank participants in the NIH chromatin group for
sharing their ideas and results, especially Drs Trevor Archer, David
Clarke and Sharon Roth. I am indebted to Drs Randall Morse,
Genevi6ve Almouzni, Jeffrey Hayes and my Editor Dr Susan King for
their comments on the text. Appreciation and thanks are given to Ms

Thuy Vo and Mr William Mapes for preparing the manuscript and
figures. Finally I thank my wife Elizabeth for her patience and support
during the preparation of this book.
Alan Wolffe


Preface to the Second
Edition

The impact of chromatin structure on gene activity and many other
nuclear events has become increasingly apparent over the past four
years. Tremendous progress has been made concerning the structure
and function of the nucleoprotein structures regulating transcription,
replication and repair within the eukaryotic chromosome. Important
recent advances include the determination of the internal organization
of the nucleosome. The histones are found to have unexpected
structural similarities to known transcription factors. Similar structures point to similar functions and this emphasizes the importance of
considering both the architectural roles of histones and transcription
factors in regulatory complexes. Genetic experiments have introduced
a whole new significance both to the histones and to other proteins
that control long-range chromosomal compaction and regulate
differential gene activity. The current text has been extensively
modified to incorporate such new discoveries into the framework of
established knowledge. The principal aim remains to introduce
interested scientists to chromatin.
I would like to thank my colleagues at NIH for sharing their ideas
and results. I am indebted to Drs Dmitry Pruss, Horace Drew, Jeffrey
Hayes, Stefan Dimitrov, Mary Dasso and Genevi6ve Almouzni for
invaluable discussions. Drs Randall Morse and Jeffrey Hansen read
the text for which I am particularly grateful. The interpretation of data

xii

Chromatin: Structure and Function
and any errors are my own. Appreciation and thanks are given to my
Editor Dr Tessa Picknett and to Ms Thuy Vo for help with preparation
of the text. Finally, I thank Elizabeth and Max for their patience and
support.

Alan Wolffe
February 1995


Preface to the Third
Edition

Progress in chromatin research in the past three years has been
remarkable. Pre-eminent in recent discoveries is the role of transcriptional coactivators and corepressors as histone modification enzymes.
Scientists investigating transcriptional control and signal transduction
are now faced with the need to consider chromatin structural
modifications as a primary regulatory mechanism. Other advances
concerning the nucleosome include the definition of unusual
chromatin architecture on human disease genes, the expansion of
the families of proteins that resemble chromatin components, and the
solution of the crystal structure of the nucleosome core. The nucleus
itself is also increasingly recognized as having structural and
functional compartmentalization. This organization can contribute to
epigenetic effects that have important roles in gene expression and
development. The reversibility of such compartmentalization has been

dramatically demonstrated through the successful mammalian cloning experiments. New sections and extensive rewriting have integrated these discoveries into the framework of established knowledge.
The principal aim remains to introduce interested scientists to
chromatin.
I would like to recognize the contributions of my colleagues at NIH
and especially the Chromatin Interest Group in sharing their ideas
and results. I am indebted to Drs Dmitry Pruss for many of the
illustrations, and to Drs Mary Dasso, Jeffrey Hansen, Stefan Kass,
Hitoshi Kurumizaka, Nicoletta Landsberger, Guofu Li, John
Strouboulis, Alexander Strunnikov, Paul Wade and Jiemin Wong for


xiv

Chromatin: Structure and Function

invaluable discussions. A special thanks to Ms Thuy Vo for the
preparation of the text. I thank my wife Elizabeth for her patience and
support, Max and Katherine for limiting their destruction of the
manuscript.
I also thank Dr Tessa Picknett and Sign Davies at Academic Press,
for their assistance in bringing this edition to print. Thanks also to
Blackwell Science, the Company of Biologists, Elsevier Science and
Oxford University Press for permission to reproduce previously
published material.
Alan Wolffe
September 1997


CHAPTER ONE

Overview

1.1 INTRODUCTORY COMMENTS
Our knowledge of how the hereditary information within eukaryotic
chromosomes is organized and used by a cell has increased
enormously through the application of molecular biology and
genetics. Technical advances now allow individual DNA sequences
to be isolated and their association with proteins within the cell
nucleus to be determined. Experimental progress has led the biologist
to explore long-standing questions concerning how a particular cell
acquires and maintains its individual identity. Developmental
biologists have used new methodologies to investigate at a molecular
level how an egg differentiates into different cell types. These
questions have led scientists to the realization that growth, development and differentiation are directed by regulated changes in the form
and composition of specific complexes of protein and DNA within the
nucleus. Understanding how these complexes are assembled and
function has become a central theme in modern biology.
Many of the techniques used to probe protein-DNA interactions
were developed by researchers interested in the basic structural matrix
of chromosomes - chromatin. This complex of DNA, histones and
non-histone proteins has been exposed to a multitude of biochemical,
biophysical, molecular biological and genetic manipulations. The
structure of chromatin is by now well understood, but how it is folded
and compacted into a chromosome is not. Knowledge of how


2

Chromatin: Structure and Function

chromatin is constructed preceded the development of methods
capable of exploring function. The purification and cloning of nonhistone proteins required to perform the complex events involved in
DNA transcription, replication, recombination and repair is the focus
of a continuing and intense research effort. Investigators now make
use of their experience with chromatin structure and assembly to
examine the function of the structural proteins and enzymes required
for the maintenance, expression and duplication of the genome in a
true chromosomal environment.
The conclusion from this research effort is that the organization of
DNA into chromatin and chromosomes is essential for regulated
processes within the nucleus. Histones, nucleosomes and the
chromatin structures they assemble function as integral components
of the machinery determining transcriptional activity, cellular identity
and fate. It might be anticipated that a comparable integration of
structure and function will have occurred with the molecular
machines controlling replication, recombination and repair.

1.2 DEVELOPMENT OF RESEARCH INTO CHROMATIN
STRUCTURE AND FUNCTION
Towards the end of the nineteenth century numerous investigators
formulated the theory that chromosomes determined inherited
characteristics (see Voeller, 1968). These studies were almost entirely
based on cytological observations with the light microscope. Although
chromosomes are clearly only present in the nucleus, the influence of
components of the cytoplasm on inherited characteristics was
examined by forcing embryonic nuclei into regions of the cytoplasm
in which they would not normally be found (Wilson, 1925). These
experiments and others led Morgan (1934) to propose the theory that
differentiation depended on variation in the activity of genes in
different cell types. The genes were clearly in the chromosomes, but

their biochemical composition remained completely unknown.
The last quarter of the nineteenth century also saw the recognition
of RNA (first identified as yeast nucleic acid), DNA (thymus nucleic
acid) and the discovery of histones. Albrecht Kossel isolated nuclei
from the erythrocytes of geese and examined the basic proteins in his
preparations, which he named the histones (reviewed by Kossel,
1928). The apparent biochemical simplicity of DNA and the obvious
complexity of protein in chromosomes led investigators mistakenly to
regard the latter component as the major constituent of the elusive


Overview

genes (Stedman and Stedman, 1947). Only the gradual acceptance of
experiments on the capacity of DNA alone to change the genetic
characteristics of the cell (Avery et al., 1944) led to the recognition of
nucleic acid as the key structural component of a gene.
The elucidation of the double helical structure of DNA with its
immediate implications for self-duplication, opened up the new
approaches of molecular biology to clarifying the nature of genes
(Watson and Crick, 1953). Although the double helix was now
recognized as containing the requisite information to specify a genetic
function, how this information was controlled was not understood.
The apparent heterogeneity of the histones due to proteolysis and the
various modifications of these proteins suggested that they might be
important in regulating genes. Eventually methodological improvements for isolating and resolving the different histones demonstrated
that they were highly conserved in eukaryotes and that only a few
basic types existed (Fitzsimmons and Wolstenholme, 1976). This lack
of variety implied that histones themselves were unlikely to be the
determinants of gene specific transcription. However, a key role for

histone modification remained central to prevailing ideas of transcriptional regulation (Allfrey et al., 1964).
A major breakthrough came in the 1970s when a combination of
methodologies, including nuclease digestion, protein-protein crosslinking, electron microscopy and sedimentation analysis, determined
that chromatin consisted of a repetitive fundamental nucleoprotein
complex, which came to be called the nucleosome.
Structural studies on the nucleosome continue to the present time.
Current and past research reveals the nucleosome to be a remarkably
complex structure in which DNA is wrapped around the histones. The
integrity of the nucleosome depends on highly specific histonehistone interactions, and the recognition by the histones of DNA
structural features as the nucleosome is assembled. The core histones
are present as an octamer, consisting of two molecules of H2A, H2B,
H3 and H4. Histones H3 and H4 assemble a tetramer ((H3, H4)2) that
wraps DNA such that two dimers of H2A and H2B can stably
associate. Once two turns of DNA are wrapped around the octamer, a
fifth linker histone, such as histone H1, can be stably incorporated to
complete the assembly process. Although all nucleosomes maintain
these architectural features, there are many variations built upon this
common theme.
Nucleosomal structures can contain different forms of particular
core histones or linker histones. These histone variants are the products
of distinct genes which may be differentially expressed during
development (Newrock et al., 1977). The histones can also be post-

3


4

Chromatin: Structure and Function

translationally modified to different extents. Early experiments
associated different types of histone modification with particular
nuclear functions such as transcription (Allfrey et al., 1964). Many early
attempts were made to interrelate general differences in the transcriptional activity of genes to the solubility properties of chromatin
dependent on histone modification or differences in histone content.
Recombinant DNA methodologies facilitated the isolation and
cloning of defined DNA sequences, and DNA sequencing enabled the
cis-acting elements potentially controlling gene expression to be
defined (Brown, 1981). Hybridization analysis allowed the transcriptional activity of specific genes to be related to their accessibility to
nucleases such as DNase I (Weintraub and Groudine, 1976). More
detailed studies revealed that the regulatory DNA, such as promoter
and enhancer sequences, was hypersensitive to DNase I cleavage (Wu
et al., 1979). Chromatin was perceived as having a precise organization
that was certainly modified by the transcription process. It was even
possible to infer that structural features of chromatin might actually
determine the potential for transcription to occur. Nevertheless,
analysis of the nuclease sensitivity of chromatin was primarily
descriptive. The molecules that actually directed the transcription of
specific eukaryotic genes could not be determined through these
approaches.
The enzymatic activities of the eukaryotic RNA polymerases had
been characterized through the early 1970s. An initially disappointing
conclusion from these studies was that these polymerases alone did
not recognize the regulatory elements of eukaryotic genes with any
specificity when the template was naked DNA. Roeder and colleagues
(Parker and Roeder, 1977; Jaehning and Roeder, 1977) made the
seminal discovery that RNA polymerases would accurately transcribe
genes within chromatin, but not as naked DNA. The hunt was now on
for the auxiliary proteins that would determine the specific initiation
of transcription by RNA polymerase.

The early searches for these transcription factors were dependent
on the development of in vivo and in vitro assays for transcription.
Microinjection of purified or cloned genes into the nuclei of eukaryotic
cells was an early assay system used to define the cis-acting sequences
recognized by transcription factors (Brown and Gurdon, 1977).
Subsequent assays relied on in vitro transcription extracts (Wu, 1978;
Weil et al., 1979). These assays led to the purification and characterization of the first gene-specific eukaryotic transcription factor in 1980
(Engelke et al., 1980; Pelham and Brown, 1980).
Much of the research effort on transcriptional regulation during the
1980s focused on the further definition of cis-acting elements and


Overview
trans-acting factors involved in the initiation of the transcription
process (Johnson and McKnight, 1989). The in vitro transcription or
transfection assays used to examine the function of transcription
factors did not require templates to be within their normal
chromosomal environment for transcription to occur. In general these
assays examined mechanisms that stimulated gene transcription, but
did not examine the repression of transcription or the regulation of
transcription in a physiological context.
Although far from the mainstream of research on transcription, the
1980s also saw the discovery of nucleosome positioning around
eukaryotic genes (Simpson and Stafford, 1983). Application of
genomic footprinting methodologies established that this phenomenon was a feature of several regulatory DNA sequences (Almer et al.,
1986; Richard-Foy and Hager, 1987). Histones were increasingly
perceived as having the potential for specific effects on the transcription process. Experiments that combined in vitro transcription systems
with natural chromosomal templates revealed a specific role for
histones in transcriptional regulation (Schlissel and Brown, 1984). All
of this work relied upon the detailed analysis of particular promoters

in individual laboratories. The overall relevance of chromatin
structure to the eukaryotic transcription process was difficult to
establish from these studies. Nevertheless, they provided the foundation for the interpretation of genetic experiments that did in fact
determine the general significance for transcription of assembling
DNA into nucleosomes.
In a series of insightful experiments Grunstein, Winston and
colleagues (Han et al., 1987, 1988; Han and Grunstein, 1988; ClarkAdams et al., 1988) determined that changes in nucleosomal packaging
had pleiotropic effects on gene activity. Subsequent work by these
investigators and Mitch Smith established that very specific modifications in histone structure could either activate or repress specific genes
(Megee et al., 1990; Durrin et al., 1991; Mann and Grunstein, 1992). This
led directly to the resurgence of interest towards understanding gene
activity in the natural chromosomal environment that has characterized much of the research effort in eukaryotic transcriptional
regulation over the past few years.
The new-found interest in the role of chromatin in transcriptional
regulation has been fuelled by progress in two specific areas.
Structural studies led to the recognition that the histones were
isomorphous with components of the transcriptional machinery
(Arents and Moudrianakis, 1993; Clark et al., 1993; Ramakrishnan et
al., 1993; Xie et al., 1996; Luger et al., 1997). These observations
provided an architectural foundation for examining the specific roles

5


6

Chromatin: Structure and Function

of histones and transcription factors in the assembly and function of
regulatory nucleoprotein complexes. Specific modifications to nucleosomal architecture through histone acetylation, removal of histones

H2A/H2B or H1 were shown to alleviate the repressive effects of
chromatin assembly (Lee et al., 1993; Bouvet et al., 1994; Ura et al., 1995).
In certain instances chromatin assembly was also shown to stimulate
the transcription process (Schild et al., 1993). Thus the potential roles of
nucleosomal proteins in gene control became more interesting (van
Holde, 1993). Biochemical purification of histone acetyltransferases
and deacetylases (Brownell et al., 1996; Taunton et al., 1996) provided
an even closer link between chromatin and the transcriptional
machinery. Histone acetyltransferases were discovered to be components of large macromolecular complexes known as coactivators,
which are targeted to specific promoters by transcriptional activators.
Therefore a direct link was established between histone acetylation
and transcriptional activation. Histone deacetylases were found
within corepressor complexes that turn genes off. Once again, histone
chemistry became an important variable to consider in transcriptional
control.
It is now recognized that to understand transcriptional control or
any other regulated event in the nucleus it is necessary to define the
chromatin structure within which DNA is utilized. Aside from the
characterization of specific architecture, we must also determine how
structure might change. Chromatin is not static, but dynamic. Targeted
histone modifications within regulatory nucleoprotein complexes
have emerged as a means of modulating the stability of repressive
chromatin structures and the transcription process itself. The
observations made using simple model systems are having an impact
on our understanding of both development and disease. It is now
probable that our increasing knowledge of both chromatin and
chromosome structure and function in the nucleus will provide many
avenues for future advances in biotechnological and medical fields.

CHAPTER

TWO

Chromatin Structure

Chromosomes represent the largest and most visible physical
structures involved in the transfer of genetic information. Surprisingly,
our understanding of chromosome organization is most complete for
the smallest and most fundamental structural units. These units are
the nucleosomes which contain both DNA and histones. Long folded
arrays of nucleosomes comprise the vast majority of chromatin. In this
section I discuss the structural features of DNA and histones, how
they assemble into nucleosomes and how nucleosomes fold into
chromatin fibres. Finally, I describe what we know about the
organization of the chromatin fibre into a chromosome and how this
can be modified in various ways.

2.1 DNA AND HISTONES
The most striking property of a chromosome is the length of each
molecule of DNA incorporated and folded into it. The human genome
of 3 x 109 bp would extend over a metre if unravelled; however, this
is compacted into a nucleus of only 10-5 m in diameter. It is an
astonishing feat of engineering to organize the long linear DNA
molecule within ordered structures that can reversibly fold and unfold
within the chromosome. Not surprisingly, many aspects of chromosome structure reflect the impediments and constraints imposed by
having to bend and distort DNA.


8

Chromatin: Structure and Function
2.1.1 D N A

structure

DNA has an elegant and simple structure around which the
chromosome is assembled. The DNA molecule exists as a long
unbranched double helix consisting of two antiparallel polynucleotide
chains. DNA always contains an equivalent amount of the deoxyribonucleotide containing the base adenine (A) to that with the base
thymine (T), and likewise of the deoxyribonucleotide containing the
base guanine (G) to that with the base cytosine (C) (Fig. 2.1). Each base
is linked to the pentose sugar ring (2-deoxyribose) and a phosphate
group. The 5' position of one pentose ring is connected to the 3'
position of the next pentose ring via the phosphate group (a 5'-3'
linkage) to create the polynucleotide chain (Fig. 2.2). The two
antiparallel polynucleotide chains are attached to each other by
hydrogen bonding between the bases. G is always base paired to C,
and A is always base paired to T. In addition to the stability imparted
by hydrogen bonding, hydrophobic base stacking interactions occur
along the middle of the double helix (Fig. 2.3) (see Calladine and
Drew, 1997 or Sinden, 1994 for details).
Physical studies using X-ray diffraction indicate that under
conditions of physiological ionic strength, DNA is a regular helix,
making a complete turn every 3.4 nm with a diameter of 2 nm. This
particular DNA structure is known as B-DNA and has approximately
10.5 b p / t u r n of the helix. This means that every base pair is rotated
Thymine (T)

Adenine (A)

H\

CH3
\

// 0

H--C

/ H
Ni
c --H

N--H

'N---C /

i

Sugar

H/ C % 7 c ~ N /

\\

\

0

Sugar

Cytosine (C)
H

\

H
I
N-H

/
C---C
H-C//
X~N
/
Sugar

\\
0

Guanine (G)
0

H

II
\ N/C~cIN\
J
JJ ~H--C

H

Figure 2.1. The four bases found in DNA.

\
Sugar


Chromatin Structure
5' End
OI
O= P--O--CH2

Base

H ~,,~ H

OH H
3' End
I ~ I L

J

Phosphate 2-Deoxyribose
Group
Sugar Ring
OI

O= P--O--CH 2
'

...

Base

O3' Linkage
Phosphodiester
Bond

mO H
i
O= P--O-CH 2

5' Linkage y
Phosphodi
Bond ester

[
Base
~ / d H'H~
H ~"lr H
O H
I

O=P--O--CH2

o'- I
OH H

Figure 2.2. A nucleotide and a polynucleotide chain.

approximately 34 ~ around the axis of the helix relative to the next
base pair. This results in a twisting of the two polynucleotide strands
around each other. A double helix is formed that has a minor groove
(approximately 1.2 nm across) and a major groove (approximately 2.2
nm across). The geometry of the major and minor grooves of DNA
will be seen later to be crucial in determining the interaction of
proteins with the DNA backbone. The double helix is right handed
(Fig. 2.4).
Beyond this basic description, DNA structure is exceedingly plastic.
Crystallization of various oligonucleotides indicates that a variety of
DNA sequences will yield recognizable B-form DNA structures (Priv6
et al., 1991; Yanagi et al., 1991). More severe alterations in the
conditions under which DNA is examined do, however, generate

9


10

Chromatin: Structure and Function
5' Phosphate

i

0!

/H

.~ ,, ,-,

-u= ,r-u

CH3
\

/

(T)

//

c--c
//

o

O

y:_

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Base

Stacking

Figure 2.3. The interactions stabilizing the two antiparallel polynucleotide chains in DNA.

distinct conformations. Dehydrating the fibre will cause the double
helix to take up a structure known as A-DNA (11 bp/turn); or placing
DNA with a defined sequence of alternating G and C bases in
solutions of high ionic strength will lead to the formation of a lefthanded helix known as Z-DNA (12 bp/turn). The existence of either of
these extreme structures in the eukaryotic nucleus under normal
physiological conditions is controversial. However, their formation
indicates the gross morphological changes that DNA can be forced to
undergo (Drew et al., 1988; Calladine and Drew, 1997).
How do we know what structure populations of DNA molecules
have in solution? Two experimental methodologies have been
commonly used. The first employs DNA cleavage reagents and a flat
crystal surface (Rhodes and Klug, 1980). When DNA is absorbed from
solution on to a flat calcium phosphate surface and cut with DNase I,
the enzyme cuts DNA most readily where it is exposed away from the
surface. The average spacing between the sites of cleavage gives the
approximate number of base pairs per turn of DNA (Fig. 2.5). This is


Chromatin Structure

2.2 nm Major Groove
One Turn
1.2nm Minor Groove

3.4nm
10.5 bp

L~

2nm

Figure 2.4. The dimensions of DNA.
Base pairs are shown as horizontal lines for one turn of the double helix.

determined by the electrophoresis of denatured molecules through a
polyacrylamide gel. A better reagent for this purpose is the hydroxyl
radical. Hydroxyl radicals are generated by the Fenton reaction in
which an Fe(II) EDTA complex reduces hydrogen peroxide to a
hydroxide anion and a hydroxyl radical.
[Fe(EDTA)] 2-

+ H202

~

[Fe(EDTA)] 1- + OH- + .OH

The radical is about the size of a water molecule and has little
sequence specificity in cleaving DNA. This it does by breaking the
pentose sugar rings of individual deoxyribonucleotides. In contrast,
DNase I is a large enzyme which has considerable sequence
preferences. In both instances, the number of base pairs per turn of
a large population of different DNA sequences bound to a crystal
surface is found to be 10.5 (Tullius and Dombroski, 1985). This result is
consistent with DNA having a B-form configuration as determined by
X-ray studies.
The second method to examine DNA structure in solution reaches
the similar conclusion that DNA has a B-form conformation at
physiological ionic strength; however, a completely different strategy

is used. It is generally found that a population of closed circular DNA

11


12

Chromatin: Structure and Function

Experiment

Theory

L

Cleavage
Reagent

Crystal
Surface

Number of
base pairs
tum of DNA

Ibp

I

Origin

Cleavage
Products

[-

Migration
Preferred Cleavage Site

Figure 2.5. Determining the helical periodicity of DNA in 'solution' through binding to a flat crystal surface and cleavage with an
enzyme or a chemical reagent.
In theory the most exposed region of the double helix will be cut
preferentially, experimentally this is reflected in a larger population of
DNA fragments cut at this site after resolution on a polyacrylamide gel
(darker bands). The distance between darker bands in base pairs is the
helical periodicity (number of base pairs per turn) of DNA.

molecules, identical in length and sequence, contains different
numbers of superhelical turns. Superhelical turns can be simply
defined by the following description: a single superhelical turn is
introduced into a closed circular DNA molecule if the molecule is
broken, one end of the molecule is then fixed, the other is rotated once
and the two ends then rejoined. Supercoils can be positive or negative
depending on which way the free DNA end is rotated. Closed circular
molecules of the same length and sequence with different numbers of
superhelical turns are known as topoisomers. Each population of
small closed circular DNA molecules that differ in length by a few
base pairs will exist as a distribution of topoisomers. These can be
resolved by electrophoresis through an agarose gel matrix. A molecule
which has a length corresponding to an integral number of helical

turns will exist predominantly as a single topoisomer whereas a


Chromatin Structure

molecule which deviates from this by half a helical turn will be equally
likely to exist with the superhelical turn in a positive or negative sense.
The number of DNA molecules with a particular mobility in the
agarose gel will be reduced by half since the molecules exist as an
equal mixture of topoisomers. Examining the relationship between
DNA length and the distribution of topoisomers allows the number of
base pairs per turn of DNA to be calculated. The result of 10.5 b p / t u r n
is close to that derived from crystal binding studies (Horowitz and
Wang, 1984). Finally, theoretical calculations of the most stable
configuration of DNA, which actually preceded much of the
experimental work, suggested a value of 10.6bp/turn (Levitt, 1978).
The range of values around 10.5bp/turn, obtained both experimentally and theoretically, provides a sound basis for considering
alterations in this structure based on DNA sequence content and
histone-DNA interaction.
Aside from the dramatic changes in DNA structure seen on
formation of A- or Z-DNA, local variations in DNA sequence can
significantly influence DNA conformation and properties of the helix.
Our most extensive knowledge of the local changes in B-form DNA
structure due to sequence content comes from studying AT-rich
DNAs. For example, oligo(dA).oligo(dT) tracts are found experimentally, using both spectroscopic techniques and DNA cleavage reagents
such as the hydroxyl radical, to be straight and rigid with a constant
narrow minor groove width (Nelson et al., 1987; Hayes et al., 1991a).
This is believed to be a consequence of maximizing the hydrophobic
base stacking interactions between adjacent A.T base pairs in the
DNA helix (Fig. 2.3). This stabilization process requires the bases to

be more twisted relative to each other than would normally be found
in typical B-form DNA. Chains of these base pairs have the correct
geometry to allow at least two water molecules per base pair to
become highly ordered along the DNA backbone. This creates a
'spine of hydration' which contributes to the rigidity of oligo(dA)
.oligo(dT) tracts (Berman, 1991). Changes in sequence that affect these
structural features lead to widening of the minor groove; for example,
a G.C base pair will disrupt the straight path and rigidity of an
oligo(dA).oligo(dT) tract. In contrast to oligo(dA).oligo(dT), oligo
[d(AT)] tracts are conformationally flexible. This flexibility is a
consequence of not being able to achieve efficient hydrophobic base
stacking interactions between consecutive T.A and A.T base pairs
without severely distorting the DNA helix (Travers and Klug, 1987;
Travers, 1989). Finally, short oligo(dA).oligo(dT) tracts (4-6 bp in
length) that are phased with a periodicity similar to that of the DNA
helix itself will cause the molecule to be curved. This is due to a

13


14

Chromatin: Structure and Function

narrowing of the minor groove every turn of DNA caused by the
phased oligo(dA).oligo(dT) tract (Koo et al., 1986). Periodicities that
are greater or smaller than 10-11 bp will cause the normally straight
DNA to take on a 'corkscrew-like' path. In spite of this wide variation
in 'B-form' DNA structure, all of these DNA sequences can be
assembled into chromatin (Section 2.2.5).

DNA structure is thought to have an important role in certain
human genetic diseases characterized by the presence of repeats of
particular trinucleotide sequences. These trinucleotide repeats are
found in the gene whose aberrant expression leads to the disease
phenotype (Bates and Lehrach, 1994; Sutherland and Richards,
1995). The segments of DNA containing trinucleotide repeats are
unstable with the potential to expand from generation to generation.
Two trinucleotide repeat sequences are of particular interest: (CTG)n
is associated with many diseases including Huntington's disease,
myotonic dystrophy, spinocerebellar ata•
type 1, and hereditary
dentatorubral-pallidoluysian atrophy; (CCG)n is associated with
fragile X mental retardation. Normal individuals have relatively few
copies of these repeat sequences whereas diseased individuals have
many copies (> 50). The number of repeats influences both the
expansion process and the disease. How this influence is exerted
has been the focus of a great deal of attention (see also Section
2.2.5).
Of all the many potential trinucleotides present in the genome only
reiterated CTG and CCG sequences show the special properties of
instability and tendency towards expansion (Han et al., 1994). These
sequences have the capacity to form stable hairpin structures when
they reach a certain threshold length of 40 to 50 repeats (Gacy et al.,
1995). It has been suggested that the ability to form stable hairpins
might explain both the dependence on particular trinucleotides and
the length of sequence for repeat expansion. The favoured model for
expansion predicts that DNA polymerase might 'slip' on reiterated
sequences during replication leading to small increases in trinucleotide repeat copy number. Once the copy number becomes large
enough, the single-stranded DNA at the replication fork might form a
stable hairpin looping out intervening DNA and leading a small 'slip'

to generate a large expansion of the trinucleotide repeat sequence
(Gacy et al., 1995). Under these special circumstances the capacity to
form unusual hairpin DNA structures might contribute to the
generation of a disease phenotype.
The (CTG)n and (CCG)n sequences appear to have no reason to
adopt unusual structural features when present as duplex DNA,
however there is evidence that these sequences might differ from