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Paris 12-12-2005

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 collaboration with: Paolo Milani’s group and Laura Finzi’s Group in Milano. The chromatin remodeling proteins are

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Paris 12-12-2005

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  1. 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 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. ~3mm Eiffel tower ~300m Human genome (1.1m) fly

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

  8. DNA compaction and chromatin remodeling histones

  9. Langst, G. et al. J Cell Sci 2001;114:2561-2568 DNA compaction and chromatin remodeling Histones-DNA interaction

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

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

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

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

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

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

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

  17. i ii iii DNA compaction and chromatin remodeling chromatin remodeling

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

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

  20. NURF CH”I”RAC ACF p301 ACF1 ACF1 CHRAC14 NURF55 NURF38 ISWI ISWI ISWI CHRAC15 ISWI DNA compaction and chromatin remodeling ISWI

  21. DNA compaction and chromatin remodeling ISWI activities

  22. DNA compaction and chromatin remodeling RSC

  23. DNA compaction and chromatin remodeling Different contacts during the remodeling activity Langst, G. et al. J Cell Sci 2001;114:2561-2568

  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

  26. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + + + + + + + + + + + + + + + + + + + + + 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. ISWI ISWI ISWI Results AFM w/o ISWI

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

  30. ISWI w ISWI w/o ATP ISWI ISWI Results AFM

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

  32. ISWI ISWI ISWI Results AFM Contour Length with ISWI

  33. ISWI ISWI ISWI Results AFM Diameter distribution ISWI on mica surface = 7.83 ± 0.13nm ISWI on DNA = 11.15 ± 0.26nm

  34. ISWI a = 120.6 ° +/- 2.5° Angle 60 50 a 40 Counts 30 ISWI 20 10 0 0 20 40 60 80 100 120 140 160 180 200 Angle° ISWI Results AFM

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

  36. Results AFM w RSC w/o ATP

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

  38. RSC RSC RSC Results AFM RSC diameter

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

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

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

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

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

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

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

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

  48. 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

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