NucleosomeClass3

NucleosomeClass3 - Nucleosomes:
what,
why
and
...

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Unformatted text preview: Nucleosomes:
what,
why
and
 where?
 Rob
Brewster
 What
is
a
nucleosome?
 Outline
 
‐
how
is
DNA
packaged/organized
in
Eukaryotes?
 Why
do
nucleosomes
form?
 
‐
DNA
is
sEff,
how
do
~100
bp
loops
form
so
readily?
 Where
do
nucleosomes
form?
 ‐ 
What
controls
the
spacing
and
structure
of
nucleosomes
on
 the
chromosome?
 Accessibility
of
DNA
in
the
nucleosomes
 
‐
How
is
DNA
inside
a
nucleosomes
accessed?
 DNA
OrganizaEon
in
Eukaryotes
 DNA
is
packaged
and
condensed
into
chromosome
 ‐ Human
genome
is
big,
nucleus
is
small
 ~
2
billion
basepairs
≈
2m
 nucleus
radius
~
6
µm
 ‐ 
Many
different
levels
of
organizaEon
 ‐ 
Compacts
chromosome
 ‐ 
Regulates
transcripEon
by
making
 


porEons
of
the
chromosome
 


more/less
accessible
(up
to
80%
 


is
inaccessible
to
protein
binding)
 (Lee
W
et
al.,
Nat.
GeneEcs

2007)
 ‐ 
We
will
focus
on
nucleosome
 


formaEon
 (Alberts,
EssenEal
Cell
Biology

1998)
 What
is
a
nucleosome?
 Electron
micrograph
of
chromaEn
at
low
ionic
strength
 
‐
Nucleosomes
appear
as
“beads
on
a

 


string
 (Electron
Micrograph
from
Olins
and
Olins)
 Basic
repeaEng
structure

can
be
probed
(protect
and
seq
method)
 
‐
DigesEon
enzyme
cuts
accessible
regions
of
DNA
 
‐
DNA
protected
by
nucleosome
is
not
cut
 Mono
 Dual
 Mono
 Mono
 (Furuyama
and
Biggins
,PNAS)
 EnzymaEc
digests
 ParEcular
enzymes
can
cut
double
stranded
DNA
between
basepairs
 
‐
Most
have
specific
recogniEon
sites
 
‐
Micrococcal
nuclease
cleaves
everything
it
can
(no
specific
seq.)
 DiluEon
of
nuclease
1:3
 (pictures
from
NEB)
 Structure
of
individual
nucleosome
 RepeaEng
structure
contains
5
different
proteins
 






 
‐
Main
body
is
an
octomer
formed
from:
 


two
copies
each
of
H2A,
H2B,
H3,
H4
 
‐
DNA
wraps
147
bp
(1.75
turns)
around
 


the
core
 
‐14
non
specific
adhesive
contacts
with
 


histone
(major
groove
–
histone)
 
‐
H1
agaches
to
the
linker
region
and

 


changes
the
conformaEon;
required
 


for
chromaEn
formaEon
 
‐
H1
covers
30‐50bp
 


 (Peter
J.
Russell,
iGene%cs)
 DNA
rigidity
 DNA
is
sEff…
 
‐
~150bp
persistence
length,
λ,
for
lysed
 


bacterial
genome
 
‐
loops
smaller
than
λ
should
be
rare
 …
but
not
that
sEff
 
‐
single
turn
around
histone
core
~100
bp
 Back
of
the
powerpoint
calculaEon
 Energy
paid
to
bend
loop
of
147
bp
DNA
in
16.5
bp
circle
 Assume
λ=150
bp
then:

 This
cost
must
be
balanced
out
(and
then
some)
by
the
14
contacts
so
 each
contact
must
contribute
~3
KT
 However,
nucleosome
contacts
are
~
1.5
KT
(Polach
and
Widom,
2005;
Schiessel,
2003)
 Reversing
this
and
solving
for
maximum
sEffness
for
stable
 nucleosomes,
λ
<
~
80
bp

 What
gives?
 Bending
of
short
DNA
fragments
 Looping
probability
for
small
fragments
is
 larger
than
expected
 
‐
~100
bp
fragments
form
loops
more
 


readily
than
predicted
 
‐
certain
sequences
are
more
flexible
 



than
others
 
‐
sequences
which
loop
more
readily
 


also
more
readily
form
nucleosomes
 


(asmuch
as
10^3‐fold
difference)
 Does
this
sequence
dependence
control
 where
nucleosomes
are
posiEoned?
 (ClouEer
and
Widom,
Molecular
Cell

2004)
 Where
are
Nucleosomes?
 The
EukaryoEc
genome!
 
‐
FormaEon
on
Yeast
genome
is
more 


probable
than
for
e.
coli
genome
 
‐
Implies
Euk.
Genome
has
sequence
 


preferenEal
for
nucleosomes
 (Zhang
et
al.,
Nature
struct.
&
mol.
bio.,
2009)
 EukaryoEc
genome
shows
specific
pagerns
 Power
Spectrum
of
AA/AT/TT
repeats
 
‐
AA/AT/TT
dinucleoEde
frequency
 
~10
bp
(one
DNA
twist)
 Nucleosome
code
 10bp
Periodicity
is
evident
both
in
vivo
and
in
vitro
 (Kaplan
et
al,
nature
2009)
 
‐
GC
is
5bp
out
of
phase
with
AT
dinucleoEdes
 
‐
due
to
bending
differences
in
GC
and
AT?
 Major
groove
 Wrap
5bp
 Major
groove
 More
examples
 (Segal
et
al.,
Nature
,
2006)
 Nucleosome
vs
random
sequence
 Periodicity
is
missing
from
arbitrary
sequences
 (Segal
et
al.,
Nature
,
2006)
 ProbabilisEc
model
of
nucleosome
occupancy
 Method:
From
in
vivo
occupancy
data,
calculate
the
 probability
of
any
dinucleoEde
pair
at
a
given
nuc.
 posiEon.
 Probability
that
147bp
sequence
S
is
wrapped:
 The
staEsEcal
weight
of
a
longer
sequence
with
mulEple
nucleosomes
is
 just
the
product
of
the
probability
for
every
basepair
to
be
in
that
state,
 The
probability
must
now
be
computed
computaEonally
due
to
the
 enormous
number
of
possible
configuraEons
(C)
 (Segal
et
al.,
Nature
,
2006)
 ProbabilisEc
landscape
for
occupancy
 At
a
parEcular
nucleosomal
coverage
can
predict
where
stable
 nucleosomes
will
be
located
 (Kaplan
et
al,
nature
2009)
 PredicEons
from
thermodynamic
model
 Prob.
of
coverage
by
nuc
 Predicted
stable
nuc
 Exp.
Determined
nuc
 Success
rate
within
32bp
of
predicted
posiEon
 StaEsEcal
PosiEoning
–
A
(semi)compeEng
view
 In
Vitro
 Various
 Transcribed
Genes
 Less
 more
 (Zhang
et
al.,
Nature
struct.
&
mol.
bio.,
2009)
 Nucleosomes
pagern
emerges
from
steric
exclusion
on
a
line
 
‐
Promoter
region
is
always
“nucleosome
free”
 
‐
DNA
sequence
appears
to
play
only
a
small
role
 Random
1D
hard
sphere
gas?
 (Zhang
et
al.,
Nature
struct.
&
mol.
bio.,
2009)
 Occurrence
 Distance
from
TSS
 Phasing
in
vivo
resembles
RDF
of
(for
instance)
LJ
Gas
 Barrier
 Hard
Spheres
 Can
a
simple
model
of
a
 1D
hard
sphere
gas
predict
 nucleosome
posiEoning
 pagern?
 Conclusions
on
posiEoning
 DNA
sequence
magers
for
nucleosomes
posiEons…
 
‐
preferenEally
bind
to
parEcularly
repeaEng
sequences
 
‐
avoid
long
tracks
of
A/T
 …
however,
this
effect
seems
to
be
minimized
in
vivo
 
‐
Simple
1D
gas
model
fits
in
vivo
nucleosome
posiEons
well
 
 
(data
not
shown)
 How
does
one
resolve
the
apparent
conflict
in
these
views?
 Accessing
nucleosomal
DNA
 How
is
protected
DNA
accessed?
 
‐
DNA
replicaEon,
repair
and
transcripEon
all
require
access
to
 



occluded
DNA
 
‐
Specialized
motors
called
“remodeling
factors”
disassemble
and
 


perturb
nucleosomes
to
allow
access 

 
‐However,
even
without
these
motors,
wrapped
DNA
has
some
 

accessibility
 
‐
DigesEon
assays
probe
equilibrium
accessibility
 
‐
Fret
probes
dynamic
accessibility
 In
vitro
accessibility
mechanism
 What
is
the
mechanism
to
access
a
site
buried
deep
in
the
 nucleosome?
 ?
 TranslaEon
along
dna
 ?
 ?
 Bulged
structures
 Transient
unwrapping
 In
vitro
accessibility
mechanism
 What
is
the
mechanism
to
access
a
site
buried
deep
in
the
 nucleosome?
 ?
 TranslaEon
along
dna
 In
vitro
accessibility
 does
not
show
 length
dependence
 beyond
~100
bp
 ?
 Bulged
structures
 Transient
unwrapping
 (Anderson
et
al,
2002)
 Fret
on
nucleosomal
complex
 One
fret
dye
is
put
on
the
nucleosome,
one
is
put
on
the
end
of
 the
DNA
strand
 Excite
at
low
wavelengths:
 Dyes
are
close
–
signal
~680nm
is
high
 Dyes
are
far
–
signal
~680nm
is
low
 (Li
and
Widom,

2004)
 In
vitro
accessibility
mechanism
 What
is
the
mechanism
to
access
a
site
buried
deep
in
the
 nucleosome?
 !
 TranslaEon
along
dna
 In
vitro
accessability
 does
not
show
 length
dependence
 beyond
~100
bp
 Transient
unwrapping
 FRET
experiments
 by
Li
and
Widom
 Bulged
structures
 Probing
accessibility
 Specific
restricEon
enzyme
binding
site
 DNA
is
cleaved
when
site
becomes
 accessible

from
unwrapping
 Measure
probability
for
being
cut
as
a
 funcEon
of
burial
depth
 Accessibility
is
reduced
(compared
to
naked
dna)
 sites
buried

shallow
10‐100x,

deep
~105x
 (Polach
and
Widom,
2000)
 Measuring
unwrapping
 The
more
flexible
sequence
 is
less
accessible!
 Where
ΔG
is
the
change
in
free
energy
associated
with
opening
the
binding
site
 Theory
Interlude
 Nucleosome
preference
is
enErely
bending?!
 Fizng
bending
energy
 More
rigid
sequence
(5S)
 More
flexible
sequence
(601TA)
 γ
=‐
0.16
kT/bp
 γ
=
‐0.24
kT/bp
 b
 With
R
=13.5bp,
a=10.5bp,
L=147bp

 However,
for
ClouEer
and
Widom
data
 bp
 RNAP
transcripEon
through
the
nucleosome

 Bead
agached
to
end
of
DNA
and
RNAP
 
‐RNAP
movement
through
the
 


nucleosome
can
be
measured
 
‐
Distance
between
beads
is
a
 



readout
for
RNAP
depth
in
 



nucleosome 


 (Hodges
et
al,
2009)
 Model
for
RNAP
transcripEon
through
 nucleosome
 Conclusions
 The
End
 DNA
organizaEon
 E.
Coli
genome

~
4

million
bp
≈
1.2
mm
 E.
Coli
dimensions
~
2
µm^3
 Human
genome

~
6
billion
bp
≈
2000
mm
 Nucleus
dimensions
~
750
µm^3
 (Maghews,
DNA Structure Prerequisite Informa%on.
1997)
 Sequence
preference
of
nucleosomes
 Nucleosome
code
 
‐
AA/TT/TA
dinucleoEde
repeats
 


periodicity
 
‐GC
repeats
5bp
out
of
phase
 Due
to
difference
in
bending
of
 
dinucleoEdes?
 
‐
Helicity
of
DNA
means
bending
 



at
major
and
minor
groove
is
 

 


appear
with
10bp
 

 



opposite
 (Segal
et
al.,
Nature
,
2006)
 
‐
Nucleosomes
may
posiEon
AA/TA/TT
at
minor/major,
GC
at
 


major/minor
groove
due
to
difference
in
bending
 What
is
a
Nucleosome
(how
do
we
know)?
 DigesEon
with
 ‐ Human
genome
is
big,
nucleus
is
small
 ~
2
billion
basepairs
≈
2m
 nucleus
radius
~
6
µm
 ‐ 
Many
different
levels
of
organizaEon
 ‐ 
We
are
interesEng
in
Nucleosome
 


formaEon
 DNA
organizaEon
inEukaryotes
 Olins
and
Olins
 DNA
organizaEon
in
Eukaryotes
 Topics:
 
‐General
introducEon
to
Nucleosomes
 
 
‐compacEon
computaEon?
 
‐Protect
and
Seq:
Widom
hgp://www.wisdom.weizmann.ac.il/~eran/NucleosomeModel.pdf
 
 
weissman
hgp://www.nature.com/nmeth/journal/v6/n4/full/nmeth0409‐244b.html
 
‐Lacra
stuff,
pulling
exp
 
‐Widom
Accessibility
 ...
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