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Paris 12-12-2005 Etude du mécanisme d’action des facteurs de remodelage de la chromatine par micromanipulation et visualisation de l’ADN LIA Giuseppe In.

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Presentation on theme: "Paris 12-12-2005 Etude du mécanisme d’action des facteurs de remodelage de la chromatine par micromanipulation et visualisation de l’ADN LIA Giuseppe In."— Presentation transcript:

1 Paris Etude du mécanisme d’action des facteurs de remodelage de la chromatine par micromanipulation et visualisation de l’ADN LIA Giuseppe In collaboration with: Paolo Milani’s group and Laura Finzi’s Group in Milano

2 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.

3 DNA compaction and chromatin remodeling
Experimental setups: AFM Results: AFM Experimental setups: Magnetic Tweezers Results: Magnetic Tweezers Model of DNA translocation

4 DNA compaction and chromatin remodeling
Experimental setups: AFM Results: AFM Experimental setups: Magnetic Tweezers Results: Magnetic Tweezers Model of DNA translocation

5 Eiffel tower ~300m Human genome (1.1m) fly ~3mm

6 Human genome Cell nucleus radius 33.2x 3.3*109 bp = ~1.1m 10-20 mm
DNA compaction and chromatin remodeling Human genome radius 33.2x nucleus 3.3*109 bp = ~1.1m Cell nucleus 10-20 mm human genome

7 DNA compaction DNA compaction and chromatin remodeling DNA nucleosomes
30nm fiber chromatin loops chromosome compacted inside the nucleus

8 DNA compaction and chromatin remodeling
histones

9 Histones-DNA interaction
DNA compaction and chromatin remodeling Histones-DNA interaction Langst, G. et al. J Cell Sci 2001;114:

10 chromatin “bead on a string” “30nm fiber”
DNA compaction and chromatin remodeling chromatin “bead on a string” “30nm fiber”

11 de-compaction compaction Cellular activities on chromatin
DNA compaction and chromatin remodeling compaction de-compaction Chromatin presents a barrier to transcription factors and other proteins that must access DNA in Eukaryotic cells chromatin remodeling complexes Homologous recombination Cellular activities on chromatin duplication DNA repair transcription

12 DNA compaction and chromatin remodeling
factor transcription factor binding Chromatin compaction Chromatin remodeling factor transcription factor binding

13 chromatin remodeling DNA compaction and chromatin remodeling
chemical mediated remodeling mechanical mediated remodeling histones modifications ATP hydrolyzing enzymes i ) ii ) iii ) iv )

14 Family ATP-chromatin remodeling factors
DNA compaction and chromatin remodeling Family ATP-chromatin remodeling factors SWI/SNF ISWI RSF WCRF/hACF NURF CHRAC ACF ISWI1 ISWI2 man hSWI/SNF(BRG1) hSWI/SNF(BRM) fly Brahma yeast RSC

15 Negative supercoiling diffusion Histones-DNA Destabilization
DNA compaction and chromatin remodeling Mechanical mediated remodeling models Negative supercoiling diffusion nucleosome Loop DNA diffusion torsion nucleosome sliding Histones-DNA Destabilization loop nucleosome sliding exposition nucleosomal DNA Dimer lost dimer lost exposition nucleosomal DNA histone variants variants incorporation

16 i ii iii Comparing the ATPases domain helicase
DNA compaction and chromatin remodeling Comparing the ATPases domain helicase i ii iii Flaus A and Owen-Hughes. Curr Opin Genet Dev. vol.11 pag.148 (2001)

17 DNA compaction and chromatin remodeling
ii iii

18 in vitro evidences of a possible translocation
DNA compaction and chromatin remodeling in vitro evidences of a possible translocation DNA length dependence in ATP hydrolysis Triplex DNA displacement dsDNA (bp) % ATP Hydrolysis Iswi Whitehouse I. et al. Mol Cell Biol. vol.23 pag.1935 (2003)

19 Proteins that I studied
DNA compaction and chromatin remodeling Proteins that I studied ISWI family the ATPase subunit ISWI (fly) SWI/SNF family complex RSC (yeast)

20 ISWI NURF CH”I”RAC ACF DNA compaction and chromatin remodeling p301
CHRAC14 CHRAC15 CH”I”RAC ACF ISWI

21 DNA compaction and chromatin remodeling
ISWI activities

22 DNA compaction and chromatin remodeling
RSC

23 Different contacts during the remodeling activity
DNA compaction and chromatin remodeling Different contacts during the remodeling activity Langst, G. et al. J Cell Sci 2001;114:

24 DNA compaction and chromatin remodeling
Experimental setups: AFM Results: AFM Experimental setups: Magnetic Tweezers Results: Magnetic Tweezers Model of DNA translocation

25 Tip scanning direction
Experimental setup AFM Tip scanning direction

26 tapping mode in dry condition
Experimental setup AFM scanning mode tapping mode in dry condition surfaces : MICA - DNA + + + poly-ornithin - - - - - - - - - - - - - - - - - - mica

27 DNA compaction and chromatin remodeling
Experimental setups: AFM Results: AFM Experimental setups: Magnetic Tweezers Results: Magnetic Tweezers Model of DNA translocation

28 Results AFM ISWI w/o ISWI

29 l l = 306,6 +/- 1,0nm Results AFM ISWI Expected length= 307nm
Probability 220 260 300 340 380 0,0 0,1 0,2 0,3 0,4 0,5 DNA Length (nm) l Expected length= 307nm l = 306,6 +/- 1,0nm

30 Results AFM ISWI w ISWI w/o ATP

31 Relative position w/o ATP
Results AFM ISWI Relative position w/o ATP observed 86.2% 13.8% internal extremities expected 92.794.9% 5.17.3%

32 Results AFM ISWI Contour Length with ISWI

33 Diameter distribution
Results AFM ISWI Diameter distribution ISWI on mica surface = 7.83 ± 0.13nm ISWI on DNA = ± 0.26nm

34 Results AFM ISWI a = 120.6 ° +/- 2.5° a Counts Angle° 20 40 60 80 100
20 40 60 80 100 120 140 160 180 200 10 30 50 Counts Angle° Angle a = ° +/- 2.5° a

35 Results AFM ISWI Model of binding Lateral view View on the top

36 Results AFM w RSC w/o ATP

37 Results AFM RSC Contour Length with RSC Dl = 17nm

38 Results AFM RSC RSC diameter

39 No changes has observed
Results AFM RSC No changes has observed w and w/o DNA!!

40 Results AFM RSC 93.9% 6.1% 95.3% 4.7% observed internal extremities
expected 95.3% 4.7%

41 Summary AFM DATA w/o ATP
Results AFM Summary AFM DATA w/o ATP ISWI wraps the DNA ISWI ISWI position is randomized along DNA molecule RSC binds the DNA w/o changing the DNA extension RSC RSC position is randomized along DNA molecule

42 Results AFM ISWI w ISWI w/o 25mM ATP relaxed loops supercoiled loops

43 Relative position w ATP
Results AFM ISWI Relative position w ATP observed 22% 78% internal extremities Relative position w/o ATP 86% 14%

44 Results AFM ISWI Contour Length Dx=38nm Dx=106nm

45 Results AFM RSC w RSC w/o 2mM ATP

46 Results AFM RSC Contour Length with RSC and ATP l = 17nm l = 106nm

47 RSC Relative position w ATP
Results AFM RSC RSC Relative position w ATP observed 36% 64% internal extremities Relative position w/o ATP 94% 6%

48 Summary AFM DATA w ATP ISWI and RSC extrude DNA loops
Results AFM Summary AFM DATA w ATP ISWI and RSC extrude DNA loops ISWI ISWI and RSC change their position during DNA extrusion RSC

49 DNA compaction and chromatin remodeling
Experimental setups: AFM Results: AFM Experimental setups: Magnetic Tweezers Results: Magnetic Tweezers Model of DNA translocation

50 Experimental setup Magnetic Tweezers

51 Experimental setup Magnetic Tweezers

52 Experimental setup Magnetic Tweezers Video Tracking Focus plane

53 Experimental setup Magnetic Tweezers

54 DNA topology refresher s>0 s<0
Results Magnetic Tweezers DNA topology refresher s>0 s<0 Force = 0.4pN Lk=Tw+Wr Lk Intertwining of DNA strands Tw Wrapping of DNA strands Wr Global measure of DNA crossing s = DLk /Lk0

55 Experimental setup Magnetic Tweezers

56 DNA compaction and chromatin remodeling
Experimental setups: AFM Results: AFM Experimental setups: Magnetic Tweezers Results: Magnetic Tweezers Model of DNA translocation

57 ISWI Binding on Nicked DNA w/o ATP
Results Magnetic Tweezers ISWI ISWI Binding on Nicked DNA w/o ATP X0= -46,5 ± 2,2nm [ ISWI ]  2nM 2pN

58 ISWI binding on: Force = 0.4pN Results Magnetic Tweezers ISWI Positive
supercoiled DNA DNA extension increases !! Negative supercoiled DNA DNA extension decreases !! Force = 0.4pN

59 Results Magnetic Tweezers ISWI With positive supercoiled DNA i ii

60 Results Magnetic Tweezers ISWI With negative supercoiled DNA i ii

61 dl+ dl- N= 2.3 ± 0.2 turns dl+=-ln+nlp+ dl-=-ln-nlp- dl=-ln
Results Magnetic Tweezers ISWI dl+=-ln+nlp+ dl-=-ln-nlp- dl=-ln N= 2.3 ± 0.2 turns dl+=104nm dl-=-149nm ln=-46nm lp+=62.5nm/turns lp-=49.3nm/turns Positive Supercoiling dl+ Nicked DNA dl- Negative Supercoiling nlp ln dl dl+=-ln+nlp+ dl-=-ln-nlp- dl=-ln lnicked = -46nm lnegative  -149nm lpositive  104nm

62 No changes of DNA extension or torsion introduction
Results Magnetic Tweezers RSC RSC w/o ATP w/o RSC w RSC w/o ATP DNA length (µm) Time (s) No changes of DNA extension or torsion introduction was observed w/o ATP

63 Summary DNA binding w/o ATP
Results AFM Summary DNA binding w/o ATP w/o ATP ISWI reduces DNA length by a wrapping that introduces 2 turns ISWI w/o ATP RSC doesn’t change or the DNA extension either the DNA linking number RSC

64 ISWI translocation on nicked DNA with ATP
Results Magnetic Tweezers ISWI ISWI translocation on nicked DNA with ATP w/o ISWI and ATP ISWI w/o ATP ISWI and ATP Force =0.4pN

65 Base Pairs ATP [mM] Processivity m L max = 293.4 14.0 bp K M = 4.5 1.0
Results Magnetic Tweezers ISWI Processivity 1 10 100 50 150 200 250 300 350 L max = 293.4 14.0 bp K M = 4.5 1.0 m Base Pairs ATP [mM] Translocation from the initial loop

66 ton toff Speed on Speed off Results Magnetic Tweezers ISWI
Michaelis-Menten Fit V max = 436,6 33.5 bp/s K M = 16,1 4.3 m Speed on 75 150 225 300 375 450 525 Base Pairs/s 50 100 200 ATP [ mM ] Speed off = 429,4 25,3 bp/s = 17,3 3.5 Loop formation and loop deformation are active process They show similar Vmax and KM

67 Results Magnetic Tweezers ISWI Stalling Force

68 RSC w 200µM ATP Results Magnetic Tweezers RSC w/o RSC w RSC w/o ATP
DNA length (µm) Time (s)

69 Loopmax = ~700bp Results AFM RSC loop translocated Processivity
(200µM ATP) Processivity Probability Loop size (nm) Loopmax = ~700bp

70 Results AFM RSC Event a Event b ON OFF 200nm 200nm

71 Results AFM RSC RSC Stalling Force

72 Summary DNA translocation
Results AFM Summary DNA translocation w ATP Translocate DNA by looping ISWI Processivity follows a Michaelis Menten law RSC Both can reverse translocation direction

73 F=0.4pN Effect of topology on translocation Results Magnetic Tweezers
ISWI Effect of topology on translocation F=0.4pN

74 Turns added by translocation
Results Magnetic Tweezers ISWI Turns added by translocation

75 Effect of topology on translocation
Results Magnetic Tweezers RSC Effect of topology on translocation

76 Effect of topology on translocation
Results Magnetic Tweezers RSC Effect of topology on translocation

77 Turns introduced Topology effects (-)sc DNA turns (+)sc DNA ATP (mM)
Results AFM RSC Turns introduced Topology effects 8 (-)sc DNA 6 turns 4 2 (+)sc DNA 10 100 1000 ATP (mM)

78 Enzyme step Results Magnetic Tweezers lateral view side view
Helical pitch

79 Results Magnetic Tweezers ISWI ISWI Enzyme step lateral view side view

80 Results Magnetic Tweezers RSC RSC Enzyme step lateral view side view

81 Summary DNA topology in translocation
Results AFM Summary DNA topology in translocation w ATP Translocate DNA by looping ISWI Processivity follows a Michaelis Menten law RSC ISWI can reverse translocation direction

82 Translocation model DNA compaction and chromatin remodeling Experimental setups Results: AFM Magnetic Tweezers Model of DNA translocation

83 ISWI translocation model
Magnetic Tweezers ISWI translocation model D.J. Fitzgerald et al EMBO J., 23(19), 2004.

84 RSC translocation model
Magnetic Tweezers RSC translocation model

85 Summary Magnetic Tweezers DATA
Results AFM Summary Magnetic Tweezers DATA w/o ATP ISWI reduces DNA length by a wrapping that introduces 2 turns w ATP ISWI forms a DNA loop Loop formation and deformation are both active process Force reduces exponentially the size of extruded loop Translocation introduces some turns on DNA Rate of turns introduction depends of the DNA supercoiling degree

86 Thanks!!

87 bulk tests + DNA compaction and chromatin remodeling remodeled
di-nucleosomes change histone octamers + nucleosome sliding

88 possible in vivo mechanism I
Magnetic Tweezers possible in vivo mechanism I nucleosome sliding

89 possible in vivo mechanism II
Magnetic Tweezers possible in vivo mechanism II nucleosomes regularly spaced

90 Translocation on ssDNA
Magnetic Tweezers Translocation on ssDNA 50 100 150 200 dsDNA ssDNA Loop size (nm) 300 600 900 1200 1500 Base Pairs ATP [ m M ]

91 in vitro evidences of a possible translocation
DNA compaction and chromatin remodeling in vitro evidences of a possible translocation Triplex DNA displacement time dna + triplex free triplex

92 bulk tests DNA compaction and chromatin remodeling Digestion
w/o chromatin remodeling factors with Digestion with endonuclease

93 ISWI Binding on Nicked DNA w/o ATP
Results Magnetic Tweezers ISWI Binding on Nicked DNA w/o ATP F = 0.4pN [ ISWI ]  20-25nM With ISWI Without ISWI F=0.4pN


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