A biophysical approach to predicting intrinsic and extrinsic nucleosome positioning signals

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A biophysical approach to predicting intrinsic
and extrinsic nucleosome positioning signals

• Alexandre V. Morozov
• Department of Physics Astronomy and
• the BioMaPS Institute for Quantitative Biology,
• Rutgers University
• morozov_at_physics.rutgers.edu

IPAM, Nov. 26 2007
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Introduction to chromatin scales
Electron micrograph of D.Melanogaster chromatin
arrays of regularly spaced nucleosomes, each 80
A across.
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Overview of gene regulation
RNA Pol II TAFs
mRNA
Gene
TF1
TF2
TF3
Nucleosomes

• Prediction and design of gene expression levels
  from
• DNA sequence
• Prediction of transcription factor and nucleosome
  occupancies in vitro and in vivo from genomic
  sequence
• Prediction of levels of mRNA production from
  transcription factor and nucleosome occupancies

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Data for modeling eukaryotic gene regulation

• Available data sources
• DNA sequence data for multiple organisms
• Genome-wide transcription factor
• occupancy data (ChIP-chip)
• Structural data for 100s of protein-DNA
  complexes
• Nucleosome positioning data MNase digestion
  sequencing or microarrays

accagtttacgt
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Biophysical picture of gene transcription
Wray, G. A. et al. Mol Biol Evol 2003 201377-1419
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Chromatin Structure Nucleosomes
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Structure of the nucleosome core particle (NCP)
Left-handed super-helix (1.84 turns, 147 bp, R
41.9 A, P 25.9 A) PDB code 1kx5
T.J.Richmond K.Luger et al. Nature 1997 (2.8
?) T.J.Richmond C.A.Davey Nature 2003 (1.9 ?)
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Gene regulation through chromatin structure

• Transcription factor DNA interactions are
  affected by the chromatin
• Chromatin remodeling by ATP-dependent complexes
• Histone variants (H2A.Z)
• Post-translational histone modifications
• (histone code)

H2A
H3
H4
H2B
H3 tail
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Experimental validation of thehistone-DNA
interaction model
Jon Widom

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

dyad
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Histone-DNA interaction model and DNA flexibility

• Nucleosome affinity depends on the presence and
  spacing of key dinucleotide motifs (e.g. TA,CA)
• Nucleosome affinity can be explained by DNA
  flexibility

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Base-pair steps are fundamental units for DNA
mechanics
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Data-driven model for DNA elastic energy (DNABEND)
Geometry distributions for TA steps in
100 non-homologous protein-DNA complexes

• Quadratic sequence-specific
• DNA elastic energy
• mean lt?gt
• width lt(? - lt?gt)2gt-1
• Matrix of force constants F

W.K. Olson et al., PNAS 1998
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Elastic rod model
DNA looping induced by a Lac repressor tetramer
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Elastic energy and geometry of DNA constrained to
follow an arbitrary curve (DNABEND)
?r
Sequence-specific DNA elastic energy
Constraint energy
Minimize to determine energy geometry
System of linear equations ½ x 6Nbs x 6Nbs
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Example of DNA geometry prediction nucleosome
structure
Ideal superhelix
Prediction for NCP (1kx5)
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Predictions of nucleosome binding affinities

• Experimental techniques
• nucleosome dialysis
• A.Thastrom et al., J.Mol.Biol. 1999,2004
• P.T.Lowary J.Widom, J.Mol.Biol. 1998
• nucleosome exchange
• T.E.Shrader D.M.Crothers PNAS 1989
• T.E.Shrader D.M.Crothers J.Mol.Biol. 1990

Alignment model (Segal E. et al. Nature 2006)

Collect nucleosome-bound sequences in yeast
Center align sequences
Construct nucleosome-DNA model using observed
dinucleotide frequencies
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Alignment Model (in vivo selection)
MNase digestion
Extract DNA, clone into plasmids
Sequence and center-align
AGGTTTATAG.. AGGTTAATCG.. AGGTAAATAA.. ..
142-152 bp
Di-nucleotide log score
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From nucleosome energies to probabilities and
occupancies
Nucleosome energy
Chromosomal coordinate
Use dynamic programming to find the partition
function and thus
probabilities and occupancies of each
DNA-binding factor, e.g. nucleosomes
Nucleosome Probability Occupancy
Chromosomal coordinate
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Nucleosome occupancy is dynamic
Nucleosome-free site
TGACGTCA
Nucleosome-occluded site
TGACGTCA
Nucleosome is displaced by the bound TF
TGACGTCA
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Nucleosome occupancy of TATA boxes explains gene
expression levels
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Nucleosome occupancy in the vicinity of genes
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Nucleosome occupancy in the vicinity of TATA
boxes default repression
TATA
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Functional sites by ChIP-chipin vivo
genome-wide measurementsof TF occupancy

• Genome-wide occupancies for 203 transcription
  factors in yeast by ChIP-chip (Harbison et al.,
  Nature 2004 Transcriptional regulatory code)
• MacIsaac et al., BMC Bioinformatics 2006 An
  improved map of phylogenetically conserved
  regulatory sites
• (98 factor specificities 26 more from the
  literature)

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Nucleosome occupancy of transcription factor
binding sites default repression

• ltOcc(functional sites)gt - ltOcc(non-functional
  sites)gt
• In vitro nucleosomes compete for DNA sequence
  only with each other

DNABEND Nucleosomes
p lt 0.05
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Nucleosome occupancy of transcription factor
binding sites

• ltOcc(functional sites)gt - ltOcc(non-functional
  sites)gt
• In vivo nucleosomes compete for DNA sequence
  with TFs

DNABEND Nucleosomes TFs
p lt 0.05
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Functional transcription factor sites are
clustered
DNABEND Nucleosomes TFs, randomized functional
sites
p lt 0.05
functional sites
non-functional sites
Clustering!
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Functional transcription factor sites are not
occupied by nucleosomes in vivo
Yuan et al. microarray experiment DNABEND
Transcription Factors DNABEND Alignment model
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Nucleosome-induced cooperativity
Nucleosome-occluded TF sites no separate binding
TGACGTCA
TAAGGCCT
Nucleosome-occluded TF sites cooperative binding
TAAGGCCT
TGACGTCA
Miller and Widom, Mol.Cell.Biol. 2003
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Nucleosome occupancy of TF sites in a model system
TF sites
pCYC1
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Nucleosome-induced cooperativityexample
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Nucleosome position predictionsGAL1-10 locus
GAL1
GAL10
Nucleosomes in vitro Nucleosomes in vivo TBP GAL4
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Nucleosome position predictionsHIS3-PET56 locus
Nucleosomes in vitro Nucleosomes in vivo TBP GCN4
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Conclusions

• Predicted histone-DNA binding affinities and
  genome-wide nucleosome occupancies using a DNA
  mechanics model a thermodynamic model of
  nucleosomes competing with other factors for
  genomic sequence
• Chromatin structure around ORF starts is
  consistent with microarray-based measurements of
  nucleosome positions, and can be explained with a
  simple model of nucleosomes phasing off bound
  TBPs
• Nucleosome-induced cooperativity (brought about
  by clustering of functional transcription factor
  binding sites) is responsible for the increased
  accessibility of functional sites

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Future Directions

• Lots of nucleosome positioning sequences soon
  to become available can a better model of
  dinucleotide (base stacking) energies be built?
  Anirvan Sengupta, Rutgers
• Can such a model be used to inform a better DNA
  mechanics model? Conversely, can a DNA mechanics
  model be compressed, i.e. encapsulated in a
  simple set of dinucleotide energies? Anirvan
  Sengupta, Rutgers
• DNABEND extensions to non-nucleosome systems,
  i.e. nucleoid proteins, DNA loops etc.? John
  Marko, Jon Widom, Northwestern
• Prediction of in vivo nucleosome positions in
  gene expression libraries Ligr et al., Genetics
  2006 random libraries of yeast promoters Lu Bai
  et al., unpublished

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Acknowledgements

• PEOPLE
• Eric Siggia (Rockefeller University)
• Jon Widom (Northwestern University)
• Harmen Bussemaker (Columbia University)
• FUNDING
• Leukemia Lymphoma Society Fellowship
• BioMaPS Institute, Rutgers University

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Nucleosome occupancy of chromosomal regions
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Induced periodicity of stable nucleosomes
stable
stable
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Nucleosome position predictionssummary