e genomic code for nucleosome positioni DNA double helix Nucleosomes Chromosome Felsenfeld & Groudine, Nature (200
Jan 16, 2016
The genomic code for nucleosome positioning
DNA double helix
Nucleosomes
ChromosomeFelsenfeld & Groudine, Nature (2003)
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
Nucleosomes dislike forming on this DNA sequence;
Nucleosomes like forming on this DNA sequence;CCAGCACCACCTGTAACCAATACAATTTTAGAAGTACTTTCACTTTGTAACTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAA
The nucleosome positioning code
ACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCCAATCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGT
Nucleosomes dislike forming on this DNA sequence;
Nucleosomes like forming on this DNA sequence;CCAGCACCACCTGTAACCAATACAATTTTAGAAGTACTTTCACTTTGTAACTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAA
The nucleosome positioning code
CC
AA
TAccess of proteins to target site is hindered
ACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCCAATCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGT
Nucleosomes dislike forming on this DNA sequence;ACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCCAATCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGT
Nucleosomes like forming on this DNA sequence;CCAGCACCACCTGTAACCAATACAATTTTAGAAGTACTTTCACTTTGTAACTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAA
The nucleosome positioning code
CC
AA
TAccess of proteins to target site is hindered
CCAAT
Easy access of proteins to target site in this region
GC
Deciphering the nucleosome positioning code
•In vitro selection of nucleosome-favoring DNAs
•Isolation of natural nucleosome DNAs
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
•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
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
Digest unwrapped DNA
Extract protected DNA
Isolation of natural nucleosome DNAs
Clone, sequence, analyze individuals
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
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
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
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
dyad
c
In vitro experimental validationof histone-DNA interaction model
• Adding key motifs increases nucleosome affinity• Deleting motifs or disrupting their spacing decreases affinity
dyad
Segal et al., 2006
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
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
• 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
Reading the nucleosome code andpredicting the in vivo locations of nucleosomes
GAL10 GAL1Binding sites
for Gal4activator protein
147 bp
Segal et al., 2006
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
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
Segal et al., 2006Fondufe-Mittendorf, Segal, & JW
Predicted nucleosome organization near5’ ends of genes – comparison to experiment
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
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, Fortney, Widom, & Siggia
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
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, Fortney, Widom, & Siggia
€
E = E0 +1
2fij Δθ ˆ
i j =1
6
∑i =1
6
∑ Δθ ˆ j
E0 = Energy at equilibrium conformation 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
E = Eelastic + Edeviation from superhelix
Elastic energy model for nucleosomal DNA
Ideal superhelixCrystal structure
Morozov, Fortney, Widom, & Siggia
Felsenfeld & Groudine, 2003
A genomic code for higher order chromatin structure?
30 nm fiber
Widom, 1992
Regular 3-d superstructures favor~10 bp quantized linker DNA lengths
Stable nucleosomes come in correlated groups
Segal et al., 2006
Pairwise distances histogram
(stable nucleosomes)
Auto-correlations(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)
Wang, Fondufe-Mittendorf, & Widom
Fourier transforms in extended regions
Averaged for extended regions starting i = 11,…20 bp beyond end of mapped nuclesome:
Period with max amplitude = 10.2 bp
Phase offset at max period = 5 bp
Yao et al., 1990;Fondufe-Mittendorf, Wang, & Widom
Clone & sequence
Digestlinker DNA
Isolatedinucleosomes
Biochemical isolation of dinucleosomes
Linker lengths in purified dinucleosomes
Predict locations of the two nucleosomes
•Duration hidden Markov model: L’, N, L, N, L’’
LNL’ L’’N
N: Nucleosome
L: Linker
L’, L’’: Partial linkers
Wang, Fondufe-Mittendorf, & Widom
Felsenfeld & Groudine, 2003
The genomic code for nucleosome positioning
DNA
Nucleosomes
30 nm fiber
Multiplexing
Layering two or more signals on top of each other without cross-interference
•Multiple phone conversations in a single wire or optical fiber
•Stereo broadcast on an FM channel
•Text message hidden in a picture, in a spy novel
How is multiplexing accomplished?
•Nucleosomes not evolved for highest affinity; many ways to have suboptimal affinity over 147 bp length
•Protein coding sequences and gene regulatory sequences are degenerate
•A remarkable feature of DNA mechanics
Evolution of the nucleosome positioning code
Sandman & Reeve,Curr. Op. Microbiol. 2006
+ Nucleosomes– nucleosomes
Acknowledgements
The genomic code for nucleosome positioning
Northwestern University
Yvonne Fondufe-MittendorfIrene MooreLingyi Chen
Karissa FortneyAnnchristine Thåström
Timothy CloutierPeggy Lowary
Jiping Wang (NU Statistics Dept.)
Weizmann Institute
Eran SegalYair Field
Rockefeller University
Eric SiggiaAlexandre Morozov
UCLA
Robijn BruinsmaJoe Rudnick
David Schwab