The genomic code for nucleosome positioning

1
The genomic code for nucleosome positioning
Felsenfeld Groudine, Nature (2003)
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DNA in nucleosomes is extremely sharply bent
Side view (Space filling representation)
Top view (Ribbon representation)
80 bp per superhelical turn
Luger et al., Nature (1997)
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The nucleosome positioning code
Nucleosomes like forming on this DNA sequence
CCAGCACCACCTGTAACCAATACAATTTTAGAAGTACTTTCACTTTGTAA
CTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGG
ATGGACGCAA
Nucleosomes dislike forming on this DNA sequence
ACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAG
ATGTGCCTCCAATCGCACTGCTCCGAACAATAAAGATTCTACAATACTAG
CTTTTATGGT
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The nucleosome positioning code
CCAAT
Nucleosomes like forming on this DNA sequence
CCAGCACCACCTGTAACCAATACAATTTTAGAAGTACTTTCACTTTGTAA
CTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGG
ATGGACGCAA
Access of proteins to target site is hindered
Nucleosomes dislike forming on this DNA sequence
ACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAG
ATGTGCCTCCAATCGCACTGCTCCGAACAATAAAGATTCTACAATACTAG
CTTTTATGGT
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The nucleosome positioning code
CCAAT
Nucleosomes like forming on this DNA sequence
CCAGCACCACCTGTAACCAATACAATTTTAGAAGTACTTTCACTTTGTAA
CTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGG
ATGGACGCAA
Access of proteins to target site is hindered
CCAAT
Nucleosomes dislike forming on this DNA sequence
ACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAG
ATGTGCCTCCAATCGCACTGCTCCGAACAATAAAGATTCTACAATACTAG
CTTTTATGGT
Easy access of proteins to target site in this
region
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Deciphering the nucleosome positioning code

• In vitro selection of nucleosome-favoring DNAs

• Isolation of natural nucleosome DNAs

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Physical selection for DNAs that attract
nucleosomes
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
Lowary Widom, 1998
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Summary

• Differing DNA sequences exhibit a gt 5,000-fold
  range of affinities for nucleosome formation

Lowary Widom, 1998 Thåström et al., 1999 Widom,
2001 Thåström et al., 2004
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DNA sequence motifs that stabilize nucleosomes
and facilitate spontaneous sharp looping
Thåström et al., 2004 Cloutier Widom 2004 Segal
et al., 2006
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Isolation of natural nucleosome DNAs
Digest unwrapped DNA
Extract protected DNA
Clone, sequence, analyze individuals
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The nucleosome signature in living yeast cells

• 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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Two alignments of nucleosome DNAs
Wang Widom, 2005
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The nucleosome signature is common to yeast and
chickens
Chicken Yeast merge
Chicken (in vivo)
Yeast (in vivo)
Segal et al., 2006
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The nucleosome signature in vitro and in vivo
Segal et al., 2006
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In vitro experimental validationof histone-DNA
interaction model

• Adding key motifs increases nucleosome affinity
• Deleting motifs or disrupting their spacing
  decreases affinity

Segal et al., 2006
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Summary
Differing DNA sequences exhibit a gt 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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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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Equilibrium configurations of nucleosomeson the
genome

• 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
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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Reading the nucleosome code and predicting the in
vivo locations of nucleosomes
Segal et al., 2006
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Summary
Differing DNA sequences exhibit a gt 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
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Distinctive nucleosome occupancy adjacent to TATA
elements at yeast promoters
Segal et al., 2006
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Predicted nucleosome organization near 5 ends of
genes comparison to experiment
Segal et al., 2006 Fondufe-Mittendorf, Segal, JW
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Summary
Differing DNA sequences exhibit a gt 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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An elastic energy model for the
sequence-dependent cost of DNA wrapping
Morozov, Fortney, Widom, Siggia
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DNA in nucleosomes is extremely sharply bent
Side view (Space filling representation)
Top view (Ribbon representation)
80 bp per superhelical turn
Luger et al., Nature (1997)
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An elastic energy model for the
sequence-dependent cost of DNA wrapping
Morozov, Fortney, Widom, Siggia
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Elastic energy of dinucleotide step

• Knowledge-based harmonic potential

Olson et al., (1998)
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Elastic energy model for nucleosomal DNA
E Eelastic Edeviation from superhelix
Ideal superhelix
Crystal structure
Morozov, Fortney, Widom, Siggia
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A genomic code for higher order chromatin
structure?
30 nm fiber
Felsenfeld Groudine, 2003
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Regular 3-d superstructures favor 10 bp
quantized linker DNA lengths
Widom, 1992
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Stable nucleosomes come in correlated groups
Segal et al., 2006
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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
Wang, Fondufe-Mittendorf, Widom
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Biochemical isolation of dinucleosomes
Yao et al., 1990 Fondufe-Mittendorf, Wang,
Widom
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Linker lengths in purified dinucleosomes
Predict locations of the two nucleosomes

• Duration hidden Markov model L, N, L, N, L

L Linker
N Nucleosome
L, L Partial linkers
Wang, Fondufe-Mittendorf, Widom
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The genomic code for nucleosome positioning
DNA
Nucleosomes
30 nm fiber
Felsenfeld Groudine, 2003
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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

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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

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Evolution of the nucleosome positioning code
Sandman Reeve, Curr. Op. Microbiol. 2006
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Acknowledgements
The genomic code for nucleosome positioning
Northwestern University Yvonne
Fondufe-Mittendorf Irene Moore Lingyi
Chen Karissa Fortney Annchristine
Thåström Timothy Cloutier Peggy Lowary Jiping
Wang (NU Statistics Dept.)
Weizmann Institute Eran Segal Yair
Field Rockefeller University Eric
Siggia Alexandre Morozov UCLA Robijn
Bruinsma Joe Rudnick David Schwab