A genomic code for nucleosome positioning DNA double helix Nucleosomes Chromosome Felsenfeld & Groudine, Nature (2003)

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 different DNA sequences) Make many copies by PCR Equilibrium selection of highest affinity 10% Extract DNA Clone, sequence, analyze individuals Physical selection for DNAs that attract nucleosomes Lowary & Widom, 1998

Differing DNA sequences exhibit a > 5,000-fold range of affinities for nucleosome formation Lowary & Widom, 1998 Thåström et al., 1999 Widom, 2001 Thåström et al., 2004 Summary

AA TT TA GC DNA sequence motifs that stabilize nucleosomes and facilitate spontaneous sharp looping Thåström et al., 2004 Cloutier & Widom 2004 Segal 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 Position on nucleosome (bp) Fraction (AA/TT/TA) ~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

Wang & Widom, 2005 Two alignments of nucleosome DNAs Center alignment Location mixture model alignment

The nucleosome signature is common to yeast and chickens Chicken (in vivo) Yeast (in vivo) Chicken + Yeast merge Segal et al., 2006

The nucleosome signature in vitro and in vivo 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 likelihood 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) P B (S)   P(S) P B (S) Chemical potential – apparent concentration Equilibrium configurations of nucleosomes on 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 Stable nucleosome Semi-stable nucleosomes Permuted Model TATA Box Segal et al., 2006

Fondufe-Mittendorf, Segal, & JW Predicted nucleosome organization near 5’ 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 i Nucleosome 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) Correlation offset (bp) Correlation Frequency Center-center distance (bp)

Clone & sequence Yao et al., 1990; Fondufe-Mittendorf, Wang, & Widom Digest linker DNA Isolate dinucleosomes Biochemical isolation of dinucleosomes

Linker DNA length (bp) Frequency 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 HMMLocation 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 Nucleosomes – nucleosomes

An elastic energy model for the sequence-dependent cost of DNA wrapping AA TT TA 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 AA TT TA GC Morozov, Segal, Widom, & Siggia

E 0 = minimum energy for step f ij = elastic constants impeding deformation; calculated from dispersion of parameters in X-ray crystal structures, assuming harmonic potential  i =  i –  i 0, = 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 Zhurkin Olson

Structural basis of sharp DNA bending in 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 = E elastic + E deviation from superhelix Morozov, Segal, Widom, & Siggia Elastic energy model for nucleosomal DNA Ideal superhelixCrystal structure

AA TT TA GC DNA sequence motifs that stabilize nucleosomes and facilitate spontaneous sharp looping Thåström et al., 2004 Cloutier & Widom 2004 Segal et al., 2006

p<10 –8 Lowary & Widom, 1998 Beyond dinucleotides Highly enriched tetranucleotides

Acknowledgements Yvonne Fondufe-Mittendorf Irene Moore Lingyi Chen Karissa Fortney Annchristine 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