A genomic code for nucleosome positioning

DNA double helix

Nucleosomes

ChromosomeFelsenfeld & Groudine, Nature (2003)

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

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

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

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

End of nucleosome i

Start of nucleosome i+1

Nucleosome iNucleosome i+1

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)

Clone & sequence Yao et al., 1990;Fondufe-Mittendorf, Wang, & Widom

Digestlinker DNA

Isolatedinucleosomes

Biochemical isolation of dinucleosomes

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

Felsenfeld & Groudine, 2003

A genomic code for nucleosome positioning

DNA

Nucleosomes

30 nm fiber

Evolution of the nucleosome positioning code

Sandman & Reeve,Curr. Op. Microbiol. 2006

+ Nucleosomes– nucleosomes

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

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, Segal, Widom, & Siggia

€

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

Basepair steps as fundamental units of DNA mechanics

ZhurkinOlson

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

Richmond & Davey, 2003

Correlated deformations for sharp DNA wrapping

Roll

Tilt

Shift

Slide

Twist

E = Eelastic + Edeviation from superhelix

Morozov, Segal, Widom, & Siggia

Elastic energy model for nucleosomal DNA

Ideal superhelixCrystal structure

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

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

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