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A genomic code for nucleosome positioning
DNA double helix
Nucleosomes
ChromosomeFelsenfeld & Groudine, Nature (2003)
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GC
Deciphering the nucleosome positioning code
•In vitro selection of nucleosome-favoring DNAs
•Isolation of natural nucleosome DNAs
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Random sequence DNA synthesis(1 each of 5 x 1012 different DNA sequences)
Make many copies by PCR
Equilibrium selection of highest affinity 10%
Extract DNA
Clone, sequence, analyze individuals
Physical selection for DNAsthat attract nucleosomes
Lowary & Widom, 1998
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•Differing DNA sequences exhibit a > 5,000-fold range of affinities for nucleosome formation
Lowary & Widom, 1998Thåström et al., 1999Widom, 2001Thåström et al., 2004
Summary
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AATTTA
AATTTA
AA
TTTA
AATTTA
AATT
TA
AA TT TA
AA
TT
TA
GC
GC
GC
GC
GC
GC
GC
DNA sequence motifs that stabilize nucleosomes and facilitate spontaneous sharp looping
Thåström et al., 2004Cloutier & Widom 2004Segal et al., 2006
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Digest unwrapped DNA
Extract protected DNA
Isolation of natural nucleosome DNAs
Clone, sequence, analyze individuals
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The nucleosome signature in living yeast cells
0.22
0.25
0.28
0.31
0.34
0 20 40 60 80 100 120 140
Position on nucleosome (bp)
AA/TT/TA (fraction)
Position on nucleosome (bp)
Fra
ctio
n(A
A/T
T/T
A)
• ~10 bp periodicity of AA/TT/TA
• Same period for GC, out of phase with AA/TT/TA
• Same signals from the in vitro nucleosome selection
• NO signal from randomly chosen genomic regions
Segal et al., 2006
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0.15
0.2
0.25
0.3
0.35
0.4
0 50 100 150
Position in nucleosome (bp)
Wang & Widom, 2005
Two alignments of nucleosome DNAs
Center alignment
Location mixture model alignment
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The nucleosome signatureis common to yeast and chickens
Chicken (in vivo)
Yeast (in vivo)
0.16
0.2
0.24
0.28
0.32
0.36
0 20 40 60 80 100 120 140
Position on nucleosome (bp)
AA/TT/TA (fraction)
0.16
0.2
0.24
0.28
0.32
0 20 40 60 80 100 120 140
Position on nucleosome (bp)
AA/TT/TA (fraction)
0.22
0.25
0.28
0.31
0.34
0 20 40 60 80 100 120 140
Position on nucleosome (bp)
AA/TT/TA (fraction)
Chicken + Yeast merge
Segal et al., 2006
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The nucleosome signature in vitro and in vivo
0.2
0.25
0.3
0.35
-70 -50 -30 -10 10 30 50 70
AA/TT/TA (fraction)
0.22
0.25
0.28
0.31
0.34
-70 -50 -30 -10 10 30 50 70
Position on nucleosome (bp)
AA/TT/TA (fraction)
0.16
0.2
0.24
0.28
0.32
-70 -50 -30 -10 10 30 50 70
AA/TT/TA (fraction)
0.14
0.19
0.24
0.29
-70 -50 -30 -10 10 30 50 70
AA/TT/TA (fraction)
0
0.1
0.2
0.3
0.4
0.5
-70 -50 -30 -10 10 30 50 70
AA/TT/TA (fraction)
Chicken (in vivo)
Yeast (in vivo)
Yeast (in vitro)
Mouse (in vitro)
Random DNA (in vitro)
Segal et al., 2006
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Summary
Differing DNA sequences exhibit a > 5,000-fold range of affinities for nucleosome formation
We have a predictive understanding of the DNA sequence motifs that are responsible
Sequences matching these motifs are abundant in eukaryotic genomes, and are occupied by nucleosomes in vivo
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Log
likel
ihoo
d
Genomic Location (bp)
Placing nucleosomes on the genome
A free energy landscape, not just scores and a threshold !!
•Nucleosomes occupy 147 bp and exclude 157 bp
Segal et al., 2006
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• One of very many possible configurations
P(S)
PB(S) P(S)
P(S)
P(S)
PB(S) PB(S) PB(S)
Chemical potential – apparent concentration
Equilibrium configurations of nucleosomeson the genome
Probability of placing a nucleosome starting at each allowed basepair i of S
Probability of any nucleosome covering position i ( average occupancy)
Locations i with high probability for starting a nucleosome ( stable nucleosomes)
Segal et al., 2006
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Summary
Differing DNA sequences exhibit a > 5,000-fold range of affinities for nucleosome formation
We have a predictive understanding of the DNA sequence motifs that are responsible
Sequences matching these motifs are abundant in eukaryotic genomes, and are occupied by nucleosomes in vivo
A model based only on these DNA sequence motifs and nucleosome-nucleosome exclusion explains ~50% of in vivo nucleosome positions
![Page 15: A genomic code for nucleosome positioning DNA double helix Nucleosomes Chromosome Felsenfeld & Groudine, Nature (2003)](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697c0041a28abf838cc489a/html5/thumbnails/15.jpg)
Distinctive nucleosome occupancy adjacent to TATA elements at yeast promoters
0.83
0.84
0.85
0.86
0.87
0.88
-500 -250 0 250 500 750 1000
Distance from Coding Start (bp)
Average Nucleosome Occupancy
Stable nucleosome
Semi-stable nucleosomes
Semi-stable nucleosomes
Permuted Model
0
0.05
0.1
-500 -250 0
Fraction
TATA Box
Segal et al., 2006
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Segal et al., 2006Fondufe-Mittendorf, Segal, & JW
Predicted nucleosome organization near5’ ends of genes – comparison to experiment
![Page 17: A genomic code for nucleosome positioning DNA double helix Nucleosomes Chromosome Felsenfeld & Groudine, Nature (2003)](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697c0041a28abf838cc489a/html5/thumbnails/17.jpg)
Summary
Differing DNA sequences exhibit a > 5,000-fold range of affinities for nucleosome formation
We have a predictive understanding of the DNA sequence motifs that are responsible
Sequences matching these motifs are abundant in eukaryotic genomes, and are occupied by nucleosomes in vivo
A model based only on these DNA sequence motifs and nucleosome-nucleosome exclusion explains ~50% of in vivo nucleosome positions
These intrinsically encoded nucleosome positions are correlated with, and may facilitate, essential aspects of chromosome structure and function
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Felsenfeld & Groudine, 2003
A genomic code for higher order chromatin structure?
30 nm fiber
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Widom, 1992
Regular 3-d superstructures favor~10 bp quantized linker DNA lengths
End of nucleosome i
Start of nucleosome i+1
Nucleosome iNucleosome i+1
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Stable nucleosomes come in correlated groups
Segal et al., 2006
Pairwise distances histogram
(stable nucleosomes)
Autocorrelations(average
occupancy)Stable nucleosomes (model)
Stable nucleosomes (permuted)
1
10
100
1000
157 357 557 757 957 1157
Distance between centers of proximal nucleosomes (bp)
Frequency
220000
225000
230000
235000
240000
245000
-1000 -500 0 500 1000
Correlation offset (bp)
Correlation
Correlation offset (bp)C
orr
ela
tion
Fre
que
ncy
Center-center distance (bp)
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Clone & sequence Yao et al., 1990;Fondufe-Mittendorf, Wang, & Widom
Digestlinker DNA
Isolatedinucleosomes
Biochemical isolation of dinucleosomes
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Linker DNA length (bp)
Fre
quen
cy
Linker DNA length (bp)
Wang & Widom
Linker lengths in purified dinucleosomes
•Biochemically isolate dinucleosomes
•Predict locations of the two nucleosomes
•Defines the linker DNA length and sequence
Duration HMM Location mixture model
![Page 23: A genomic code for nucleosome positioning DNA double helix Nucleosomes Chromosome Felsenfeld & Groudine, Nature (2003)](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697c0041a28abf838cc489a/html5/thumbnails/23.jpg)
Felsenfeld & Groudine, 2003
A genomic code for nucleosome positioning
DNA
Nucleosomes
30 nm fiber
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Evolution of the nucleosome positioning code
Sandman & Reeve,Curr. Op. Microbiol. 2006
+ Nucleosomes– nucleosomes
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An elastic energy model for the sequence-dependent cost of DNA wrapping
AATT
TA
AATTTA
AA
TTTA
AATT
TA
AATT
TA
AA TT TA
AA
TT
TA
GC
GC
GC
GC
GC
GC
GC
Morozov, Segal, Widom, & Siggia
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Luger et al., Nature (1997)
Side view(Space filling representation)
Top view(Ribbon representation)
DNA in nucleosomes is extremely sharply bent
~80 bp per superhelical turn
![Page 27: A genomic code for nucleosome positioning DNA double helix Nucleosomes Chromosome Felsenfeld & Groudine, Nature (2003)](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697c0041a28abf838cc489a/html5/thumbnails/27.jpg)
An elastic energy model for the sequence-dependent cost of DNA wrapping
AATT
TA
AATTTA
AA
TTTA
AATT
TA
AATT
TA
AA TT TA
AA
TT
TA
GC
GC
GC
GC
GC
GC
GC
Morozov, Segal, Widom, & Siggia
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€
E = E0 +1
2fij Δθ ˆ
i j =1
6
∑i =1
6
∑ Δθ ˆ j
E0 = minimum energy for step
fij = elastic constants impeding deformation; calculated from dispersion of parameters in X-ray crystal structures, assuming harmonic potential
i = i – i0,
= fluctuation of step parameter from equilibrium
Olson et al., (1998)
Elastic energy of dinucleotide step
•Knowledge-based harmonic potential
![Page 29: A genomic code for nucleosome positioning DNA double helix Nucleosomes Chromosome Felsenfeld & Groudine, Nature (2003)](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697c0041a28abf838cc489a/html5/thumbnails/29.jpg)
Basepair steps as fundamental units of DNA mechanics
ZhurkinOlson
![Page 30: A genomic code for nucleosome positioning DNA double helix Nucleosomes Chromosome Felsenfeld & Groudine, Nature (2003)](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697c0041a28abf838cc489a/html5/thumbnails/30.jpg)
Structural basis of sharp DNA bendingin nucleosomes
Richmond & Davey, 2003
•Small distortions, and localized larger distortions, along the full wrapped DNA length
Middle of DNA(bp #74) DNA end
![Page 31: A genomic code for nucleosome positioning DNA double helix Nucleosomes Chromosome Felsenfeld & Groudine, Nature (2003)](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697c0041a28abf838cc489a/html5/thumbnails/31.jpg)
Richmond & Davey, 2003
Correlated deformations for sharp DNA wrapping
Roll
Tilt
Shift
Slide
Twist
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E = Eelastic + Edeviation from superhelix
Morozov, Segal, Widom, & Siggia
Elastic energy model for nucleosomal DNA
Ideal superhelixCrystal structure
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AATTTA
AATTTA
AA
TTTA
AATTTA
AATT
TA
AA TT TA
AA
TT
TA
GC
GC
GC
GC
GC
GC
GC
DNA sequence motifs that stabilize nucleosomes and facilitate spontaneous sharp looping
Thåström et al., 2004Cloutier & Widom 2004Segal et al., 2006
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Tetranucleotide Actual #
Occur.
Expecte d#
(actual -expected) std. dev.
ctag 152 65 ± 9 10.0
taga 124 57 ± 9 7.8
tcta 124 58 ± 9 7.8
agag 104 67 ± 8 4.6
p<10–8
Lowary & Widom, 1998
Beyond dinucleotides
•Highly enriched tetranucleotides
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Acknowledgements
Yvonne Fondufe-MittendorfIrene MooreLingyi Chen
Karissa FortneyAnnchristine Thåström
Peggy Lowary
Jiping Wang (Northwestern U. Statistics)
Eran Segal (Weizmann Inst.)Yair Field (Weizmann Inst.)
Eric Siggia (Rockefeller U.)Alexandre Morozov (Rockefeller U.)
The genomic code for nucleosome positioning