Posicionament de nucleosomes en cromatina mitjançant tècniques

Posicionament de nucleosomes en cromatina mitjançant tècniques

Effect of cytosine methylation on DNA structural
properties and nucleosome binding affinity
Federica Battistini
[email protected]
Nucleosome Workshop, May 13th 2012, Haifa
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Nucleosome and Positioning
GC
Nucleosome Positioning well defined at TSS and TTS
Nucleosome Positioning encoded in DNA sequence
Segal E. et all, A genomic code for nucleosome positioning. Nature, 2006; 442.
Jiang C. and Pugh B.F., Nucleosome positioning and gene regulation: advances through genomics. Nature Reviews
Genetics, 2009;10.
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Trifonov E.N., Cracking the chromatin code: Precise rule of nucleosome positioning. Physics of life Reviews, 2011; 8.
Epigenetics: Cytosine Methylation
 One of the most important
epigenetic modification/mark
 Biological processes, gene
silencing, tumor suppressor…
 In mammals 70-80% of CpG
content is methylated
 CpG islands non methylated,
hypermethylation in cancer
cell.
 Controversy on methylation
location and effect
Chodavarapu RK,. Relationship between nucleosome positioning and DNA methylation. Nature 2010;466:388-392.
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Felle M., Nucleosomes protect DNA from DNA methylation in vivo and in vitro. Nucleic Acid Researc. 2011
Methylated Cytosine
Pointing to
the major
groove
Steric hindrance and hydrophobicity
of methyl group REDUCE FLEXIBILITY DNA.
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Methylation Process
DNA methyltransferases (DNMTs)
Dnmt 1:
Maintenance methyltransferase,
preferentially methylates one DNA strand.
Hemi-methylation.
Dnmt 3a and 3b:
De novo methyltransferases, in germ cells or
the early embryo,
Methylation of both DNA strands.
Homo-methylation.
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Methylation and gene repression
 Direct effect : promoter methylation prevents the binding of transcription
factors.
TF
TF
 Direct effect : DNA methylation may recruit proteins that have competitive
binding/effect (Repressor or MBD_proteins)
MBD
prot
TF
repressor
 Indirect effect: change in nucleosome structure or nucleosome positioning at
TF binding site
TF
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Workflow
Nucleosome Positioning
DNA physical properties
MD simulations, Nucleosome
conformational and
energetical analysis
Methylated CpG
Yeast Nucleosome Maps
Nucleosome Reconstitution
DNA Circularization Assay
CpG in CpG islands
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DNA Physical Properties
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Elastic/Deformation Energy
ΔE
xeq (i+1)‫‏‬
ki+1
ΔE
x (i+1)‫‏‬
xeq (i)‫‏‬
ki
x(i)‫‏‬
xeq (i-1)‫‏‬
ki-1
x(i-1)‫‏‬
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Mesoscopic Method
2
E

k
(
x

x
)
Hooke’s Law: 
 o
( x  xo )
k

w k
wr k
wt k
ws k
wl k
wf


k
k
k
k
k
k
wr r rt rs rl rf
k k k k k k 
st
tl
tf

2

1
 wt rt t


 kBT


E
X

k
k
ws k
rs k
st k
s
ls k
lf
k k k k k k 
lf
 wl rl tl ls l



k
k
wf k
rf k
tf k
lf k
lf
f

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Deformation Energy at TSS
Deformation energy reproduces coverage profile at TSS (and TTS) and describes accurately
nucleosome-depleted region, nucleosome +1 and -1 at the border.
Deniz O et all, Physical properties of naked DNA influence nucleosome positioning and correlate with transcription start
Battistini 2011;12:489
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and termination sites in yeast. BMCF.Genomics,
DNA Physical Properties
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CpG Physical Properties
MD simulations
 BP in Tetramers,
importance of the
neighboring steps
XAAY, XACY…..
AA
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AC AG AT
CA CC CG GA GC TA MG
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CpG
Physical
Properties
MD simulations
 FORCE CONSTANT
VALUES:
CpG and GpC steps in
CpG island environment
are STIFFER when
methylated
AA
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AC AG AT
CA CC CG GA GC TA MG
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CpG Physical Properties
Roll CG
Roll GC
GC
Roll MG
G
C
G
C
C
G
C
M
G
M
G
C
G
G
M
G
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CpG islands not as poli
(CG) or poli(GC), and even more when methylated!
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CpG Physical Properties
Bending stiffness
 Studied 18-mer duplexes.
 (CpG)9 segment is not distinguishable from
the others in stiffness or
properties.
 Methylation increases stiffness
 Dramatic effect on CpG islands
bending
Total Curvature
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CpG methylation and curvature
 Estimation of DNA circularization
with Monte Carlo calculation : 120180 bp long met oligo with
methylation every 21 bp.
 Methylation disfavour circle
formation
 (J factor 0.05 to 0.2 for not met)
Circularization experiments with a 21mer oligo with any or a single CpG
methylation.
Methylation decreases circularization
efficiency
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Elastic/Deformation Energy
ΔE
xeq (i+1)‫‏‬
ki+1
ΔE
x (i+1)‫‏‬
xeq (i)‫‏‬
ki
x(i)‫‏‬
xeq (i-1)‫‏‬
ki-1
x(i-1)‫‏‬
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Deformation Energy
Reduction in flexibility is an important responsible of the lower affinity of methylated DNA for
the histones.
CpG methylation disfavours F.CpG
containing sequence to wrap around histones
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Nucleosome reconstitution
Methylation disfavour
nucleosome formation.
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Nucleosome re-positioning
 Methylation disfavours nucleosome formation:
 Global effect: translational repositioning
NFR
TF
NFR
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Perez A., Castellazzi C. et all, The impact
of methylation on the physical properties of DNA, The Biophysical Journal,22
2012
Conclusions
 CpG are quite curve and flexible
 Methylation increases the curvature of CpG and CpG islands, and makes the CpG
stiffer
 The effect of CpG methylation propagates in the sequence
 Methylation reduces DNA affinity for nucleosomes, probably due to the increased
rigidity and average conformation.
 Methylation might reduce/repress gene activity by reorganizing nucleosome
positioning around the TSS
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Acknowledgments
Prof. Modesto Orozco
Dr. Guillem Portella
Oscar Flores
Chiara Castellazzi
Özgen Deniz
Dr. Alberto Perez
EBL and Orozco group
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THANK YOU for the ATTENTION
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DTI Results: single/multiple mutations
DDG kcal/mol
BAD
GOOD
Major groove
Minor groove
Multiple mutations
Cumulative effect
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DDG kJ/mol
Elastic Energy vs Free Energy
Outliers mutations in
very kinked and open
positions…
not explain only by the
elastic deformation…
something else is going
on….
Sequence/position
dependent?
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Conclusions
 Methylation disfavours nucleosome formation:
 Local effect ---> Rotational repositioning
histones
negative
positive roll
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