Etude du mécanisme daction des facteurs de remodelage de la chromatine par micromanipulation et visualisation de lADN Paris 12-12-2005 LIA Giuseppe In

Etude du mécanisme d’action des facteurs de Etude du mécanisme d’action des facteurs de remodelage de la chromatine par remodelage de la chromatine par micromanipulation et visualisation de l’ADNmicromanipulation et visualisation de l’ADN

Paris 12-12-2005Paris 12-12-2005

LIA GiuseppeLIA Giuseppe

In collaboration with:In collaboration with:Paolo Milani’s group and Paolo Milani’s group and Laura Finzi’s Group in MilanoLaura Finzi’s Group in Milano

The chromatin remodeling proteins are some molecular motors.

They remodel locally a structure called chromatin,

Guarantying to the cell to access the genetic information kept inside the

nucleus.

•DNA compaction and chromatin remodelingDNA compaction and chromatin remodeling

•Experimental setups: AFMExperimental setups: AFM

•Results: AFMResults: AFM

•Model of DNA translocation Model of DNA translocation

•Experimental setups: Magnetic TweezersExperimental setups: Magnetic Tweezers

•Results: Magnetic TweezersResults: Magnetic Tweezers







Hum

an g

enom

e (1

.1m

)

~300

m

fly

Eiffel tower

~3mm

DNA compaction and chromatin remodelingDNA compaction and chromatin remodeling

Human genomeHuman genome3.3*103.3*109 9 bp = ~1.1mbp = ~1.1m

radius 33.2xradius 33.2x nucleusnucleus

human genomehuman genome

Cell nucleusCell nucleus10-20 10-20 mm


DNADNA

nucleosomesnucleosomes

30nm fiber30nm fiber

chromatinchromatin loopsloops

chromosome compactedchromosome compacted inside the nucleusinside the nucleus

DNA compactionDNA compaction


histoneshistones


Histones-DNA interactionHistones-DNA interaction

Langst, G. et al. J Cell Sci 2001;114:2561-2568


““bead on a string”bead on a string” ““30nm fiber”30nm fiber”

chromatinchromatin

DNA repairDNA repair

transcriptiontranscription

duplicationduplication


Cellular activities on chromatinCellular activities on chromatinHomologous Homologous recombinationrecombination

compactioncompaction

de-compactionde-compaction

Chromatin presents a barrier to transcription factors and Chromatin presents a barrier to transcription factors and other proteins that must access DNA in Eukaryotic cellsother proteins that must access DNA in Eukaryotic cells

chromatinchromatinremodelingremodelingcomplexescomplexes


Chromatin remodeling factor

transcription factor

binding

Chromatin remodeling factor

transcription factor

binding

Chromatin compaction


chromatin remodelingchromatin remodelingchemical mediatedchemical mediated

remodelingremodelinghistones modificationshistones modifications

mechanical mediatedmechanical mediatedremodelingremodeling

ATP hydrolyzing enzymesATP hydrolyzing enzymes

i )i )

ii )ii )

iii )iii )

iv )iv )


Family ATP-chromatin remodeling factorsFamily ATP-chromatin remodeling factors

SWI/SNFSWI/SNFISWIISWI

•RSFRSF•WCRF/hACFWCRF/hACF

•NURFNURF•CHRACCHRAC•ACFACF

•ISWI1ISWI1•ISWI2ISWI2

manman •hSWI/SNF(BRG1)hSWI/SNF(BRG1)•hSWI/SNF(BRM)hSWI/SNF(BRM)

flyfly •BrahmaBrahma

yeastyeast •SWI/SNFSWI/SNF•RSCRSC


Mechanical mediated remodeling modelsMechanical mediated remodeling models

Negative supercoiling diffusionNegative supercoiling diffusionnucleosomenucleosome

torsiontorsion

nucleosome slidingnucleosome sliding

looploop

nucleosome slidingnucleosome sliding

Loop DNA diffusionLoop DNA diffusion

Histones-DNA DestabilizationHistones-DNA Destabilization

exposition nucleosomal exposition nucleosomal DNADNA

dimer lostdimer lost

expositionexpositionnucleosomalnucleosomal

DNADNA

histonehistonevariantsvariants

histonehistonevariants incorporationvariants incorporation

Dimer lostDimer lost


Comparing the ATPases domainComparing the ATPases domain

•Flaus A and Owen-Hughes. Curr Opin Genet Dev. vol.11 pag.148 (2001)

i

ii

iii

helicase


chromatin remodeling

i

ii

iii


dsDNA (bp)

% A

TP

Hyd

roly

sis

Iswi

•Whitehouse I. et al. Mol Cell Biol. vol.23 pag.1935 (2003)

in vitro evidences of a possible translocation•Triplex DNA displacement•DNA length dependence in

ATP hydrolysis


SWI/SNF family•complex RSC (yeast)

ISWI family•the ATPase subunit ISWI (fly)

Proteins that I studiedProteins that I studied


ISWIISWI

p301p301

ISWIISWINURF38NURF38NUR

F55NUR

F55

NURFNURF

ACF1ACF1

ISWIISWI

CHRAC14CHRAC14

CHRAC15CHRAC15

CH”I”RACCH”I”RAC

ACF1ACF1

ISWIISWI

ACFACF

ISWIISWI


ISWI activitiesISWI activities


RSCRSC

Langst, G. et al. J Cell Sci 2001;114:2561-2568


Different contacts during the Different contacts during the remodeling activityremodeling activity







Experimental setupExperimental setup

AFMAFM

Tip scanning directionTip scanning direction

AFMAFMExperimental setupExperimental setup

scanning modescanning mode

tapping mode in dry conditiontapping mode in dry condition

surfaces : MICAsurfaces : MICA

-- ---- -- -- ---- -- -- ---- -- -- ---- -- -- --++ ++ ++ ++

++ ++ ++ ++++ ++ ++ ++

++ ++ ++ ++++ ++ ++ ++

++ ++ ++ ++-- -- -- -- -- -- -- -- -- -- -- -- -- ---- -- -- -- -- -- -- -- -- -- -- -- -- ----

mica

DNA

poly-ornithin







w/o ISWIw/o ISWI

ResultsResults AFMAFMIS

WI

ISW

IIS

WI

Expected length= 307nm ll = 306,6 +/- 1,0nm= 306,6 +/- 1,0nm

Pro

bab

ility

Pro

bab

ility

220 260 300 340 3800,0

0,1

0,2

0,3

0,4

0,5

DNA Length (nm)DNA Length (nm)

l


WI

ISW

IIS

WI

w ISWI w/o ATPw ISWI w/o ATP


WI

ISW

IIS

WI

Relative position w/o ATP

13.8%86.2%observed

expected

5.17.3%92.794.9%

internal extremities


WI

ISW

IIS

WI

200 220 240 260 280 300 320 340 360 3800,0

0,1

0,2

0,3

0,4

0,5 without ISWI with ISWI

Pro

bab

ilit

y

DNA Length (nm)

x = 37,9 +/- 3,1nm

Contour Length with ISWI


WI

ISW

IIS

WI

ISWI on mica surface = 7.83 ± 0.13nm

ISWI on DNA = 11.15 ± 0.26nm

Diameter distribution


WI

ISW

IIS

WI

0 20 40 60 80 100 120 140 160 180 2000

10

20

30

40

50

60

Cou

nts

Angle°

Angle = 120.6 ° +/- 2.5°


WI

ISW

IIS

WI

Model of bindingModel of binding

ResultsResults AFMAFM

Lateral view View on the top

ISW

IIS

WI

ISW

I

w RSC w/o ATPw RSC w/o ATP


Contour Length with RSC


180 200 220 240 260 280 300 320 340 3600,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

0,45 w/o RSC

l = 303nm

w RSC w/o ATP

l = 286 +/- 5 nm

Pro

babi

lity

DNA length (nm)

l = 17nm

RS

CR

SC

RS

C


10 20 30 40 50 60 700,00

0,05

0,10

0,15

0,20

0,25

0,30

Pro

babi

lity

DNA Length (nm)

35 +/- 1 nm

RSC diameterRSC diameter

RS

CR

SC

RS

C

No changes has observedNo changes has observed w and w/o DNA!!w and w/o DNA!!

ResultsResults AFMAFMR

SC

RS

CR

SC


6.1%93.9%observed


expected

4.7%95.3%

RS

CR

SC

RS

C


Summary AFM DATA w/o ATPSummary AFM DATA w/o ATP

ISWI wraps the DNAISWI wraps the DNAISWI position is randomized along ISWI position is randomized along DNA molecule DNA molecule

RSC binds the DNA w/o changing RSC binds the DNA w/o changing the DNA extensionthe DNA extensionRSC position is randomized along RSC position is randomized along DNA moleculeDNA molecule

ISWIISWI

RSC

w ISWI w/o 25M ATP

relaxed loops supercoiled loops


WI

ISW

IIS

WI

Relative position w ATP

78%22%observed



14%86%


WI

ISW

IIS

WI

50 100 150 200 250 300 350 4000,0

0,1

0,2

0,3

0,4

0,5 without ISWI with ISWI w/o ATP with ISWI and ATP

Pro

bab

ility

DNA Length (nm)

Contour Length

x=38nm

x=106nm


WI

ISW

IIS

WI

w RSC w/o 2M ATP


SC

RS

CR

SC


Contour Length with RSC and ATP

l = 106nml = 106nm

l = 17nml = 17nm

RS

CR

SC

RS

C

RSC Relative position w ATP

64%36% observed



6%94%


SC

RS

CR

SC


Summary AFM DATA w ATPSummary AFM DATA w ATP

ISWI and RSC ISWI and RSC extrude DNA loopsextrude DNA loops

ISWI and RSC ISWI and RSC change their position change their position during DNA extrusionduring DNA extrusion

ISWIISWI

RSC







Experimental setupExperimental setup Magnetic TweezersMagnetic Tweezers

Experimental setupExperimental setup

Video TrackingVideo Tracking

Focus plane

Magnetic TweezersMagnetic Tweezers

Experimental setupExperimental setup Magnetic TweezersMagnetic Tweezers

ResultsResults Magnetic TweezersMagnetic Tweezers

DNA topology refresher

Force = 0.4pN

Lk=Tw+Wr

Lk Intertwining of DNA strands

Tw Wrapping of DNA strands

Wr Global measure of DNA crossing

= Lk /Lk0

Magnetic TweezersMagnetic TweezersExperimental setupExperimental setup








ISWI Binding on Nicked DNA w/o ATPISWI Binding on Nicked DNA w/o ATP

2pN[ [ ISWI ISWI ] ] 2nM 2nM

XX00= -46,5 ± 2,2nm= -46,5 ± 2,2nm

ISW

IIS

WI

ISW

I


ISWI binding on:ISWI binding on:

Force = 0.4pN

Positive Positive supercoiled supercoiled

DNADNA

Negative Negative supercoiled supercoiled

DNADNA

DNA extensionDNA extensionincreases !!increases !!

DNA extensionDNA extensiondecreases !!decreases !!

ISW

IIS

WI

ISW

I

ResultsResults

With positive supercoiled DNAWith positive supercoiled DNA

iii

Magnetic TweezersMagnetic TweezersIS

WI

ISW

IIS

WI

ResultsResults

With negative supercoiled DNAWith negative supercoiled DNA

iii


WI

ISW

IIS

WI

ResultsResults

lnicked = -46nm

lnegative -149nm

lpositive 104nm

Positive Supercoiling

l+

Nicked DNA

l-

Negative Supercoiling

nlp

nlp

lnln l

ll++=-l=-lnn+nl+nlp+p+ ll--=-l=-lnn-nl-nlp-p- ll=-l=-lnn

l+=-ln+nlp+

l-=-ln-nlp-

l=-ln

N= 2.3 ± 0.2 turns

dl+=104nmdl-=-149nm

ln=-46nm

lp+=62.5nm/turns

lp-=49.3nm/turns


WI

ISW

IIS

WI


No changes of DNA extension or torsion introduction was observed w/o ATP

RSC w/o ATPD

NA

len

gth

(µ

m)

DN

A le

ngt

h (

µm

)

Time (s)Time (s)

w/o RSCw/o RSC w RSC w/o ATPw RSC w/o ATP

RS

CR

SC

RS

C


Summary DNA binding w/o ATPSummary DNA binding w/o ATP

w/o ATP ISWI reduces DNA length by a wrapping w/o ATP ISWI reduces DNA length by a wrapping that introduces 2 turns that introduces 2 turns

w/o ATP RSC doesn’t change or the DNA w/o ATP RSC doesn’t change or the DNA extension either the DNA linking number extension either the DNA linking number

ISWI

RSC

ResultsResults

Force =0.4pN

ISWI translocation on nicked DNA with ATPISWI translocation on nicked DNA with ATP

w/o ISWI and ATP

ISWI w/o ATP

ISWI and ATP


WI

ISW

IIS

WI

ResultsResults

ProcessivityProcessivity

1 10 1000

50

100

150

200

250

300

350

Lmax = 293.4 ± 14.0 bp

KM = 4.5 ± 1.0 M

Bas

e P

airs

Bas

e P

airs

ATP ATP MM

Translocation from the initial loopTranslocation from the initial loop


WI

ISW

IIS

WI

ResultsResults

Michaelis-Menten FitVmax = 436,6 ± 33.5 bp/s

KM = 16,1 ± 4.3 M

Speed onSpeed on

75

150

225

300

375

450

525

Bas

e P

airs

/s

0 50 100 150 2000

ATP [ M ]B

ase

Pai

rs/s

Speed offSpeed off

0 50 100 150 2000

75

150

225

300

375

450

525

ATP [ M ]

Michaelis-Menten FitVmax = 429,4 ± 25,3 bp/s

KM = 17,3 ± 3.5 M

Loop formation and loop deformation are active processLoop formation and loop deformation are active process

They show similar VThey show similar Vmaxmax and K and KMM


onon offoff

ISW

IIS

WI

ISW

I


Stalling ForceStalling Force

ISW

IIS

WI

ISW

I


DN

A le

ngt

h (

µm

)D

NA

len

gth

(µ

m)

Time (s)Time (s)

w/o RSCw/o RSC w RSC w/o ATPw RSC w/o ATP

RSC w 200µM ATP

RS

CR

SC

RS

C


Loop size (nm)Loop size (nm)

Pro

babi

lity

Pro

babi

lity

loop translocated (200µM ATP) Processivity

Loopmax = ~700bp

RS

CR

SC

RS

C


200n

m

200n

mEvent a Event b

ON OFF

RS

CR

SC

RS

C


RSC Stalling ForceRSC Stalling Force

RS

CR

SC

RS

C


Summary DNA translocationSummary DNA translocation

w ATP Translocate DNA by loopingw ATP Translocate DNA by loopingISWIRSC

Processivity follows a Michaelis Menten lawProcessivity follows a Michaelis Menten lawBoth can reverse translocation directionBoth can reverse translocation direction

ResultsResults

Effect of topology on translocationEffect of topology on translocation

1 10 100-200

-150

-100

-50

0

50

100

negative

nicked

l (

nm

)

ATP (M)

positive

F=0.4pN


WI

ISW

IIS

WI

Turns added by translocationTurns added by translocation

ResultsResults Magnetic TweezersMagnetic TweezersIS

WI

ISW

IIS

WI

ResultsResults


Magnetic TweezersMagnetic TweezersR

SC

RS

CR

SC

ResultsResults


Magnetic TweezersMagnetic TweezersR

SC

RS

CR

SC


10 100 1000

ATP (M)

0

2

4

6

8

turn

s

(-)sc DNA

(+)sc DNA

Topology effects Turns introduced

RS

CR

SC

RS

C


Enzyme stepEnzyme steplateral viewlateral view

Helical pitchHelical pitchside viewside view


ISWI Enzyme stepISWI Enzyme stepside viewside viewlateral viewlateral view

ISW

IIS

WI

ISW

I


RSC Enzyme stepRSC Enzyme stepside viewside viewlateral viewlateral view

RS

CR

SC

RS

C


Summary DNA topology in translocationSummary DNA topology in translocation

w ATP Translocate DNA by loopingw ATP Translocate DNA by loopingISWIRSC

Processivity follows a Michaelis Menten lawProcessivity follows a Michaelis Menten lawISWI can reverse translocation directionISWI can reverse translocation direction


•Experimental setupsExperimental setups

•Results:Results: AFMAFMMagnetic TweezersMagnetic Tweezers


Translocation modelTranslocation model


D.J. Fitzgerald et al EMBO J., 23(19), 2004.D.J. Fitzgerald et al EMBO J., 23(19), 2004.

ISWI translocation modelISWI translocation model


RSC translocation modelRSC translocation model


Summary Magnetic Tweezers DATASummary Magnetic Tweezers DATA

w/o ATP ISWI reduces DNA length by a wrapping w/o ATP ISWI reduces DNA length by a wrapping that introduces 2 turns that introduces 2 turns

w ATP ISWI forms a DNA loop w ATP ISWI forms a DNA loop Loop formation and deformation are both active Loop formation and deformation are both active processprocessForce reduces exponentially the size of extruded Force reduces exponentially the size of extruded looploopTranslocation introduces some turns on DNATranslocation introduces some turns on DNA

Rate of turns introduction depends of the DNA Rate of turns introduction depends of the DNA supercoiling degree supercoiling degree

Thanks!!Thanks!!

Documents

Etude du mécanisme daction des facteurs de remodelage de la chromatine par micromanipulation et visualisation de lADN Paris 12-12-2005 LIA Giuseppe In