Nucleosomes

1
Chapter 19

• Nucleosomes

2
19.1 Introduction19.2 The nucleosome is the
subunit of all chromatin19.3 DNA is coiled in
arrays of nucleosomes19.4 Nucleosomes have a
common structure 19.5 DNA structure varies on
the nucleosomal surface19.6 Supercoiling and the
periodicity of DNA19.7 The path of nucleosomes
in the chromatin fiber19.8 Organization of the
histone octamer19.9 Histones are modified19.10
Reproduction of chromatin requires assembly of
nucleosomes19.11 Do nucleosomes lie at specific
positions?19.12 Are transcribed genes organized
in nucleosomes? 19.13 Histone octamers are
displaced by transcription19.14 DNAase
hypersensitive sites change chromatin
structure19.15 Domains define regions that
contain active genes19.16 Heterochromatin
propagates from a nucleation event19.17
Heterochromatin depends on interactions with
histones19.18 X chromosomes undergo global
changes19.19 Chromosome condensation is caused
by condensins19.20 Methylation is perpetuated by
a maintenance methylase19.21 Methylation is
responsible for imprinting19.22 Epigenetic
effects can be inherited19.23 Yeast prions show
unusual inheritance 19.24 Prions cause diseases
in mammals
3
Histones are conserved DNA-binding proteins of
eukaryotes that form the nucleosome, the basic
subunit of chromatin.Nucleosome is the basic
structural subunit of chromatin, consisting of
200 bp of DNA and an octamer of histone proteins.
19.1 Introduction
4
Figure 18.9 The sister chromatids of a mitotic
pair each consist of a fiber (30 nm in diameter)
compactly folded into the chromosome. Photograph
kindly provided by E. J. DuPraw.
19.1 Introduction
5
Micrococcal nuclease is an endonuclease that
cleaves DNA in chromatin, DNA is cleaved
preferentially between nucleosomes.
19.2 The nucleosome is the subunit of all
chromatin
6
Figure 19.1 Chromatin spilling out of lysed
nuclei consists of a compactly organized series
of particles. The bar is 100 nm. Photograph
kindly provided by Pierre Chambon.
19.2 The nucleosome is the subunit of all
chromatin
7
Figure 19.2 Individual nucleosomes are released
by digestion of chromatin with micrococcal
nuclease. The bar is 100 nm. Photograph kindly
provided by Pierre Chambon.
19.2 The nucleosome is the subunit of all
chromatin
8
Figure 19.3 The nucleosome consists of
approximately equal masses of DNA and histones
(including H1). The predicted mass of the
nucleosome is 262 kD.
19.2 The nucleosome is the subunit of all
chromatin
9
Figure 19.4 The nucleosome may be a cylinder with
DNA organized into two turns around the surface.
19.2 The nucleosome is the subunit of all
chromatin
10
Figure 19.5 The two turns of DNA on the
nucleosome lie close together.
19.2 The nucleosome is the subunit of all
chromatin
11
Figure 19.6 Sequences on the DNA that lie on
different turns around the nucleosome may be
close together.
19.2 The nucleosome is the subunit of all
chromatin
12
Core DNA is the 146 bp of DNA contained on a core
particle.Core particle is a digestion product of
the nucleosome that retains the histone octamer
and has 146 bp of DNA its structure appears
similar to that of the nucleosome itself.Linker
DNA is all DNA contained on a nucleosome in
excess of the 146 bp core DNA.
19.3 DNA is coiled in arrays of nucleosomes
13
Figure 19.7 Micrococcal nuclease digests
chromatin in nuclei into a multimeric series of
DNA bands that can be separated by gel
electrophoresis. Photograph kindly provided by
Markus Noll.
19.3 DNA is coiled in arrays of nucleosomes
14
Figure 19.8 Each multimer of nucleosomes contains
the appropriate number of unit lengths of DNA.
Photograph kindly provided by John Finch.
19.3 DNA is coiled in arrays of nucleosomes
15
Figure 19.9 Micrococcal nuclease reduces the
length of nucleosome monomers in discrete steps.
Photograph kindly provided by Roger Kornberg.
19.3 DNA is coiled in arrays of nucleosomes
16
Figure 19.10 Microccocal nuclease initially
cleaves between nucleosomes. Mononucleosomes
typically have 200 bp DNA. End-trimming reduces
the length of DNA first to 165 bp, and then
generates core particles with 146 bp.
19.3 DNA is coiled in arrays of nucleosomes
17
Figure 19.4 The nucleosome may be a cylinder with
DNA organized into two turns around the surface.
19.3 DNA is coiled in arrays of nucleosomes
18
Figure 19.11 Nicks in double-stranded DNA are
revealed by fragments when the DNA is denatured
to give single strands. If the DNA is labeled at
(say) 5? ends, only the 5? fragments are visible
by autoradiography. The size of the fragment
identifies the distance of the nick from the
labeled end.
19.4 DNA structure varies on the nucleosomal
surface
19
Figure 2.4 When restriction fragments are
identified by their possession of a labeled end,
each fragment directly shows the distance of a
cutting site from the end. Successive fragments
increase in length by the distance between
adjacent restriction sites.
19.4 DNA structure varies on the nucleosomal
surface
20
Figure 19.12 Sites for nicking lie at regular
intervals along core DNA, as seen in a DNAase I
digest of nuclei. Photograph kindly provided by
Leonard Lutter.
19.4 DNA structure varies on the nucleosomal
surface
21
Figure 19.13 Two numbering schemes divide core
particle DNA into 10 bp segments. Sites may be
numbered S1 to S13 from one end or taking S7 to
identify coordinate 0 of the dyad symmetry, they
may be numbered -7 to 7.
19.4 DNA structure varies on the nucleosomal
surface
22
Figure 19.4 The nucleosome may be a cylinder with
DNA organized into two turns around the surface.
19.4 DNA structure varies on the nucleosomal
surface
23
Figure 19.14 The most exposed positions on DNA
recur with a periodicity that reflects the
structure of the double helix. (For clarity,
sites are shown for only one strand.)
19.4 DNA structure varies on the nucleosomal
surface
24
Figure 19.15 High resolution analysis shows that
each site for DNAase I consists of several
adjacent susceptible phosphodiester bonds as seen
in this example of sites S4 and S5 analyzed in
end-labeled core particles. Photograph kindly
provided by Leonard Lutter.
19.4 DNA structure varies on the nucleosomal
surface
25
Linking number paradox describes the discrepancy
between the existence of -2 supercoils in the
path of DNA on the nucleosome compared with the
measurement of -1 supercoil released when
histones are removed.Minichromosome of SV40 or
polyoma is the nucleosomal form of the viral
circular DNA.
19.5 Supercoiling and the periodicity of DNA
26
Figure 19.16 The supercoils of the SV40
minichromosome can be relaxed to generate a
circular structure, whose loss of histones then
generates supercoils in the free DNA.
19.5 Supercoiling and the periodicity of DNA
27
Figure 19.4 The nucleosome may be a cylinder with
DNA organized into two turns around the surface.
19.5 Supercoiling and the periodicity of DNA
28
Figure 19.17 The 10 nm fiber in partially unwound
state can be seen to consist of a string of
nucleosomes. Photograph kindly provided by
Barbara Hamkalo.
19.6 The path of nucleosomes in the chromatin
fiber
29
Figure 19.18 The 10 nm fiber is a continuous
strong of nucleosomes.
19.6 The path of nucleosomes in the chromatin
fiber
30
Figure 19.19 The 30 nm fiber has a coiled
structure. Photograph kindly provided by Barbara
Hamkalo.
19.6 The path of nucleosomes in the chromatin
fiber
31
Figure 19.20 The 30 nm fiber may have a helical
coil of 6 nucleosomes per turn, organized
radially.
19.6 The path of nucleosomes in the chromatin
fiber
32
Figure 19.21 In a symmetrical model for the
nucleosome, the H32- H42 tetramer provides a
kernel for the shape. One H2A-H2B dimer can be
seen in the top view the other is underneath.
19.7 Organization of the histone octamer
33
Figure 19.22 The crystal structure of the histone
core octamer is represented in a space-filling
model with the H32-H42 tetramer shown in white
and the H2A-H2B dimers shown in blue. Only one of
the H2A-H2B dimers is visible in the top view,
because the other is hidden underneath. The
potential path of the DNA is shown in the top
view as a narrow tube (one quarter the diameter
of DNA), and in the side view by the parallel
lines in a 20 ? wide bundle. Photographs kindly
provided by Evangelos Moudrianakis.
19.7 Organization of the histone octamer
34
Figure 19.4 The nucleosome may be a cylinder with
DNA organized into two turns around the surface.
19.7 Organization of the histone octamer
35
Figure 19.23 Histone positions in a top view show
H3-H4 and H2A-H2B pairs in a half nucleosome the
symmetrical organization can be seen in the
superimposition of both halves.
19.7 Organization of the histone octamer
36
Figure 19.24 The globular bodies of the histones
are localized in the histone octamer of the core
particle, but the locations of the N-terminal
tails, which carry the sites for modification,
are not known, and could be more flexible.
19.7 Organization of the histone octamer
37
Figure 19.25 Acetylation of lysine or
phosphorylation of serine reduces the overall
positive charge of a protein.
19.7 Organization of the histone octamer
38
Figure 19.26 Replicated DNA is immediately
incorporated into nucleosomes. Photograph kindly
provided by S. MacKnight.
19.8 Reproduction of chromatin requires assembly
of nucleosomes
39
Figure 19.27 In vitro, DNA can either interact
directly with an intact (crosslinked) histone
octamer or can assemble with the H32-H42
tetramer, after which two H2A-H2B dimers are
added.
19.8 Reproduction of chromatin requires assembly
of nucleosomes
40
Figure 19.28 If histone octamers were conserved,
old and new octamers would band at different
densities when replication of heavy octamers
occurs in light amino acids (part 1) but
actually the octamers band diffusely between
heavy and light densities, suggesting disassembly
and reassembly (part 2).
19.8 Reproduction of chromatin requires assembly
of nucleosomes
41
Figure 19.29 Nucleosome positioning places
restriction sites at unique positions relative to
the linker sites cleaved by micrococcal nuclease.
19.9 Do nucleosomes lie at specific positions?
42
Figure 19.30 In the absence of nucleosome
positioning, a restriction site lies at all
possible locations in different copies of the
genome. Fragments of all possible sizes are
produced when a restriction enzyme cuts at a
target site (red) and micrococcal nuclease cuts
at the junctions between nucleosomes (green).
19.9 Do nucleosomes lie at specific positions?
43
Figure 19.31 Translational positioning describes
the linear position of DNA relative to the
histone octamer. Displacement of the DNA by 10 bp
changes the sequences that are in the more
exposed linker regions, but does not alter which
face of DNA is protected by the histone surface
and which is exposed to the exterior. DNA is
really coiled around the nucleosomes, and is
shown in linear form only for convenience.
19.9 Do nucleosomes lie at specific positions?
44
Figure 19.32 Rotational positioning describes the
exposure of DNA on the surface of the nucleosome.
Any movement that differs from the helical repeat
(10.2 bp/turn) displaces DNA with reference to
the histone surface. Nucleotides on the inside
are more protected against nucleases than
nucleotides on the outside.
19.9 Do nucleosomes lie at specific positions?
45
Figure 19.33 The extended axis of an rDNA
transcription unit alternates with the only
slightly less extended non-transcribed spacer.
Photograph kindly provided by Charles Laird.
19.10 Are transcribed genes organized in
nucleosomes?
46
Figure 19.34 An SV40 minichromosome can be
transcribed. Photograph kindly provided by Pierre
Chambon.
19.10 Are transcribed genes organized in
nucleosomes?
47
Figure 19.35 RNA polymerase is comparable in size
to the nucleosome and might encounter
difficulties in following the DNA around the
histone octamer.
19.10 Are transcribed genes organized in
nucleosomes?
48
Figure 19.36 A protocol to test the effect of
transcription on nucleosomes shows that the
histone octamer is displaced from DNA and rebinds
at a new position.
19.10 Are transcribed genes organized in
nucleosomes?
49
Figure 19.37 RNA polymerase displaces DNA from
the histone octamer as it advances. The DNA loops
back and attaches (to polymerase or to the
octamer) to form a closed loop. As the polymerase
proceeds, it generates positive supercoiling
ahead. This displaces the octamer, which keeps
contact with DNA and/or polymerase, and is
inserted behind the RNA polymerase.
19.10 Are transcribed genes organized in
nucleosomes?
50
Figure 19.38 The URA3 gene has translationally
positioned nucleosomes before transcription. When
transcription is induced, nucleosome positions
are randomized. When transcription is repressed,
the nucleosomes resume their particular
positions. Photograph kindly provided by Fritz
Thoma.
19.10 Are transcribed genes organized in
nucleosomes?
51
Figure 19.39 Indirect end-labeling identifies the
distance of a DNAase hypersensitive site from a
restriction cleavage site. The existence of a
particular cutting site for DNAase I generates a
discrete fragment, whose size indicates the
distance of the DNAase I hypersensitive site from
the restriction site.
19.11 DNAase hypersensitive sites change
chromatin structure
52
Figure 19.40 The SV40 minichromosome has a
nucleosome gap. Photograph kindly provided by
Moshe Yaniv.
19.11 DNAase hypersensitive sites change
chromatin structure
53
Figure 19.41 The SV40 gap includes hypersensitive
sites, sensitive regions, and a protected region
of DNA. The hypersensitive site of a chicken
b-globin gene comprises a region that is
susceptible to several nucleases.
19.11 DNAase hypersensitive sites change
chromatin structure
54
Domain of a chromosome may refer either to a
discrete structural entity defined as a region
within which supercoiling is independent of other
domains or to an extensive region including an
expressed gene that has heightened sensitivity to
degradation by the enzyme DNAase I.
19.12 Domains define regions that contain active
genes
55
Figure 19.42 Sensitivity to DNAase I can be
measured by determining the rate of disappearance
of the material hybridizing with a particular
probe.
19.12 Domains define regions that contain active
genes
56
Figure 19.43 In adult erythroid cells, the adult
b-globin gene is highly sensitive to DNAase I
digestion, the embryonic b-globin gene is
partially sensitive (probably due tIn adult
erythroid cells, the adult b-globin gene is
highly sensitive to DNAase I digestion, the
embryonic b-globin gene is partially sensitive
(probably due to spreading effects), but
ovalbumin is not sensitive. Data kindly provided
by Harold Weintraub.
19.12 Domains define regions that contain active
genes
57
Epigenetic changes influence the phenotype
without altering the genotype. They consist of
changes in the properties of a cell that are
inherited but that do not represent a change in
genetic information.
19.13 Heterochromatin depends on interactions
with histones
58
Figure 19.44 Position effect variegation in eye
color results when the white gene is integrated
near heterochromatin. Cells in which white is
inactive give patches of white eye, while cells
in which white is active give red paPosition
effect variegation in eye color results when the
white gene is integrated near heterochromatin.
Cells in which white is inactive give patches of
white eye, while cells in which white is active
give red patches. The severity of the effect is
determined by the closeness of the integrated
gene to heterochromatin. Photograph kindly
provided by Steve Henikoff.
19.13 Heterochromatin depends on interactions
with histones
59
Figure 19.45 Extension of heterochromatin
inactivates genes. The probability that a gene
will be inactivated depends on its distance from
the heterochromatin region.
19.13 Heterochromatin depends on interactions
with histones
60
Figure 19.46 Formation of heterochromatin is
initiated when RAP1 binds to DNA. SIR3/4 bind to
RAP1 and also to histones H3/H4. The complex
polymerizes along chromatin and may connect
telomeres to the nuclear matrix.
19.13 Heterochromatin depends on interactions
with histones
61
Constitutive heterochromatin describes the inert
state of permanently nonexpressed sequences,
usually satellite DNA.Dosage compensation
describes mechanisms employed to compensate for
the discrepancy between the presence of two X
chromosomes in one sex but only one X chromosome
in the other sex.Facultative heterochromatin
describes the inert state of sequences that also
exist in active copies-for example, one mammalian
X chromosome in females.Single X hypothesis
describes the inactivation of one X chromosome in
female mammals.
19.14 Global changes in X chromosomes
62
Figure 19.47 Different means of dosage
compensation are used to equalize X chromosome
expression in male and female.
19.14 Global changes in X chromosomes
63
Figure 19.48 X-linked variegation is caused by
the random inactivation of one X chromosome in
each precursor cell. Cells in which the allele
is on the active chromosome have wild phenotype
but cells in which the - allele is on the active
chromosome have mutant phenotype.
19.14 Global changes in X chromosomes
64
Figure 19.49 X-inactivation involves
stabilization of XIST RNA, which coats the
inactive chromosome.
19.14 Global changes in X chromosomes
65
Imprinting describes a change in a gene that
occurs during passage through the sperm or egg
with the result that the paternal and maternal
alleles have different properties in the very
early embryo. May be caused by methylation of DNA.
19.15 Methylation is responsible for imprinting
66
Figure 19.50 The state of methylated sites could
be perpetuated by an enzyme that recognizes only
hemimethylated sites as substrates.
19.15 Methylation is responsible for imprinting
67
Figure 19.51 The state of methylation is
controlled by three enzymes.
19.15 Methylation is responsible for imprinting
68
Figure 19.52 The parental alleles of Igf2 are
differentially methylated in the early embryo,
but the patterns of methylation are reset when
gametes are formed by the adult.
19.15 Methylation is responsible for imprinting
69
Prion is a proteinaceous infectious agent, which
behaves as an inheritable trait, although it
contains no nucleic acid. Examples are PrPSc, the
agent of scrapie in sheep and bovine spongiform
encephalopathy, and Psi, which confers an
inherited state in yeast.
19.16 Epigenetic effects can be inherited
70
Figure 19.50 The state of methylated sites could
be perpetuated by an enzyme that recognizes only
hemimethylated sites as substrates.
19.16 Epigenetic effects can be inherited
71
Figure 19.53 What happens to protein complexes on
chromatin during replication?
19.16 Epigenetic effects can be inherited
72
Figure 19.45 Extension of heterochromatin
inactivates genes. The probability that a gene
will be inactivated depends on its distance from
the heterochromatin region.
19.16 Epigenetic effects can be inherited
73
Figure 19.54 Acetylated cores are conserved and
distributed at random to the daughter chromatin
fibers at replication. Each daughter fiber has a
mixture of old (acetylated) cores and new
(unacetylated) cores.
19.16 Epigenetic effects can be inherited
74
Figure 19.55 The state of the Sup35 protein
determines whether termination of translation
occurs.
19.17 Yeast prions show unusual inheritance
75
19.17 Yeast prions show unusual inheritance
Figure 19.56 Newly synthesized Sup35 protein is
converted into the PSI state by the presence
of pre-existing PSI protein.
76
Figure 19.57 Purified protein can convert
thepsi- state of yeast to PSI.
19.17 Yeast prions show unusual inheritance
77
Scrapie is a infective agent made of protein.
19.18 Prions cause diseases in mammals
78
Figure 19.58 A PrpSc protein can only infect an
animal that has the same type of endogenous PrPC
protein.
19.18 Prions cause diseases in mammals
79
1. All eukaryotic chromatin consists of
nucleosomes.2. The path of DNA around the
histone octamer creates 3. Nucleosomes are
organized into a fiber of 30 nm diameter which
has 6 nucleosomes per turn and a packing ratio of
40. 4. RNA polymerase displaces histone octamers
during transcription. 5. Two types of changes in
sensitivity to nucleases are associated with gene
activity. 6. Formation of heterochromatin may be
initiated at certain sites and then propagated
for a distance that is not precisely determined.
19.19 Summary
80
7. Inactive chromatin at yeast telomeres and
silent mating type loci appears to have a common
cause, and involves the interaction of certain
proteins with the N-terminal tails of histones H3
and H4.8. Inactivation of one X chromosome in
female (eutherian) mammals occurs at random. 9.
Methylation of DNA is inherited epigenetically.
10. Prions are proteinaceous infectious agents
that are responsible for the disease ofscrapie in
sheep and for related diseases in man.
19.19 Summary