Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


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

1 Etude du mécanisme daction des facteurs de remodelage de la chromatine par micromanipulation et visualisation de lADN Paris LIA Giuseppe In collaboration with: Paolo Milanis group and Laura Finzis 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 remodelingDNA compaction and chromatin remodeling Experimental setups: AFMExperimental setups: AFM Results: AFMResults: AFM Model of DNA translocationModel of DNA translocation Experimental setups: Magnetic TweezersExperimental setups: Magnetic Tweezers Results: Magnetic TweezersResults: Magnetic Tweezers

4 DNA compaction and chromatin remodelingDNA compaction and chromatin remodeling Experimental setups: AFMExperimental setups: AFM Results: AFMResults: AFM Model of DNA translocationModel of DNA translocation Experimental setups: Magnetic TweezersExperimental setups: Magnetic Tweezers Results: Magnetic TweezersResults: Magnetic Tweezers

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

6 DNA compaction and chromatin remodeling Human genome 3.3*10 9 bp = ~1.1m radius 33.2x nucleus human genome Cell nucleus m

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

8 DNA compaction and chromatin remodeling histones

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

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

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

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

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

14 DNA compaction and chromatin remodeling Family ATP-chromatin remodeling factors SWI/SNFISWI RSFRSF WCRF/hACFWCRF/hACF NURFNURF CHRACCHRAC ACFACF ISWI1ISWI1 ISWI2ISWI2 man hSWI/SNF(BRG1)hSWI/SNF(BRG1) hSWI/SNF(BRM)hSWI/SNF(BRM) fly BrahmaBrahma yeast SWI/SNFSWI/SNF RSCRSC

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

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

17 DNA compaction and chromatin remodeling chromatin remodeling i ii iii

18 DNA compaction and chromatin remodeling dsDNA (bp) % ATP Hydrolysis 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

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

20 DNA compaction and chromatin remodeling ISWI p301 ISWI NURF38 NURF55NURFACF1 ISWI CHRAC14 CHRAC15CHIRACACF1 ISWIACF ISWI

21 ISWI activities

22 DNA compaction and chromatin remodeling RSC

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

24 DNA compaction and chromatin remodelingDNA compaction and chromatin remodeling Experimental setups: AFMExperimental setups: AFM Results: AFMResults: AFM Model of DNA translocationModel of DNA translocation Experimental setups: Magnetic TweezersExperimental setups: Magnetic Tweezers Results: Magnetic TweezersResults: Magnetic Tweezers

25 Experimental setup AFM Tip scanning direction

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

27 DNA compaction and chromatin remodelingDNA compaction and chromatin remodeling Experimental setups: AFMExperimental setups: AFM Results: AFMResults: AFM Model of DNA translocationModel of DNA translocation Experimental setups: Magnetic TweezersExperimental setups: Magnetic Tweezers Results: Magnetic TweezersResults: Magnetic Tweezers

28 w/o ISWI ResultsAFM ISWI

29 Expected length= 307nm l = 306,6 +/- 1,0nm l = 306,6 +/- 1,0nm Probability ,0 0,1 0,2 0,3 0,4 0,5 DNA Length (nm) l ResultsAFM ISWI

30 w ISWI w/o ATP ResultsAFM ISWI

31 Relative position w/o ATP 13.8%86.2% observed expected % % internalextremities ResultsAFM ISWI

32 Contour Length with ISWI ResultsAFM ISWI

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

34 ResultsAFM

35 Model of binding ResultsAFM Lateral viewView on the top ISWI

36 w RSC w/o ATP ResultsAFM

37 Contour Length with RSC ResultsAFM l = 17nm RSC

38 ResultsAFM RSC diameter RSC

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

40 ResultsAFM 6.1%93.9% observed internalextremities expected 4.7%95.3% RSC

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

42 w ISWI w/o 25 M ATP relaxed loops supercoiled loops ResultsAFM ISWI

43 Relative position w ATP 78%22% observed Relative position w/o ATP internalextremities 14%86% ResultsAFM ISWI

44 Contour Length x=38nm x=106nm ResultsAFM ISWI

45 w RSC w/o 2 M ATP ResultsAFM RSC

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

47 RSC Relative position w ATP 64%36% observed Relative position w/o ATP internalextremities 6%94% ResultsAFM RSC

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

49 DNA compaction and chromatin remodelingDNA compaction and chromatin remodeling Experimental setups: AFMExperimental setups: AFM Results: AFMResults: AFM Model of DNA translocationModel of DNA translocation Experimental setups: Magnetic TweezersExperimental setups: Magnetic Tweezers Results: Magnetic TweezersResults: Magnetic Tweezers

50 Experimental setup Magnetic Tweezers

51 Experimental setup Magnetic Tweezers

52 Experimental setup Video Tracking Focus plane Magnetic Tweezers

53 Experimental setup Magnetic Tweezers

54 Results DNA topology refresher Force = 0.4pN Lk=Tw+WrLk=Tw+Wr L k Intertwining of DNA strands T w Wrapping of DNA strands W r Global measure of DNA crossing = L k /L k0

55 Magnetic Tweezers Experimental setup

56 DNA compaction and chromatin remodelingDNA compaction and chromatin remodeling Experimental setups: AFMExperimental setups: AFM Results: AFMResults: AFM Model of DNA translocationModel of DNA translocation Experimental setups: Magnetic TweezersExperimental setups: Magnetic Tweezers Results: Magnetic TweezersResults: Magnetic Tweezers

57 Results Magnetic Tweezers ISWI Binding on Nicked DNA w/o ATP 2pN [ ISWI ] 2nM X 0 = -46,5 ± 2,2nm ISWI

58 Results Magnetic Tweezers ISWI binding on: Force = 0.4pN PositivesupercoiledDNA NegativesupercoiledDNA DNA extension increases !! DNA extension decreases !! ISWI

59 Results With positive supercoiled DNA i ii Magnetic Tweezers ISWI

60 Results With negative supercoiled DNA i ii Magnetic Tweezers ISWI

61 Results l nicked = -46nm l negative -149nm l positive 104nm Positive Supercoiling l + Nicked DNA l - Negative Supercoiling nl p lnln lnln l l + =-l n +nl p+ l + =-l n +nl p+ l - =-l n -nl p- l - =-l n -nl p- l=-l n l=-l n l + =-l n +nl p+ l - =-l n -nl p- l =-l n N= 2.3 ± 0.2 turns dl + =104nm dl - =-149nm l n =-46nm l p+ =62.5nm/turns l p- = 49.3nm/turns Magnetic Tweezers ISWI

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

63 ResultsAFM Summary DNA binding w/o ATP w/o ATP ISWI reduces DNA length by a wrapping that introduces 2 turns w/o ATP ISWI reduces DNA length by a wrapping that introduces 2 turns w/o ATP RSC doesnt change or the DNA extension either the DNA linking number w/o ATP RSC doesnt change or the DNA extension either the DNA linking number ISWI RSC

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

65 Results Processivity L max = ± 14.0 bp K M = 4.5 ± 1.0 M Base Pairs ATP M ATP M Translocation from the initial loop Magnetic Tweezers ISWI

66 Results Loop formation and loop deformation are active process Loop formation and loop deformation are active process They show similar V max and K M They show similar V max and K M Magnetic Tweezers on on off off ISWI

67 Results Magnetic Tweezers Stalling Force ISWI

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

69 ResultsAFM Loop size (nm) Probability loop translocated (200µM ATP) Processivity Loop max = ~700bp RSC

70 ResultsAFM 200nm Event aEvent b ON OFF RSC

71 ResultsAFM RSC Stalling Force RSC

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

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

74 Turns added by translocation Results Magnetic Tweezers ISWI

75 Results Effect of topology on translocation Magnetic Tweezers RSC

76 Results Effect of topology on translocation Magnetic Tweezers RSC

77 ResultsAFM ATP ( M) turns (-)sc DNA (+)sc DNA Topology effects Turns introduced RSC

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

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

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

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

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

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

84 Magnetic Tweezers RSC translocation model

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

86 Thanks!!

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

88 Magnetic Tweezers possible in vivo mechanism I nucleosome sliding

89 Magnetic Tweezers possible in vivo mechanism II

90 Magnetic Tweezers Translocation on ssDNA

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

92 bulk tests Digestion with endonuclease w/ochromatin remodeling remodelingfactorswithchromatin factors

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


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

Similar presentations


Ads by Google