1. 免疫学信息网 http://www.immuneweb.com

2. “Chromatin is the last refuge of scoundrels”

3. 免疫学信息网 http://www.immuneweb.com A Mitotic Human Chromosome DNA length: 4.6 x 10 7 bp = 1.5 cm Chromosome length: 2 µm Compaction ratio = 8000 1

4. 免疫学信息网 http://www.immuneweb.com Chromatin DNA Compaction DNA Accessibility Chromatin Remodeling Machines NIH

5. 免疫学信息网 http://www.immuneweb.com Basic Unit of Chromatin Structure: Polynucleosome 2 Compaction ratio = 6

6. 免疫学信息网 http://www.immuneweb.com Limited Micrococcal Nulcease Digestion of Chromatin Generates a ~200 bp Ladder 3

7. 免疫学信息网 http://www.immuneweb.com H1 Micrococcal Nuclease Digestion Time MononucleosomeChromatosomeCore Particle Linker DNA (variable) Operational Definititions of Nucleosome Particles Chromatin 4

8. 免疫学信息网 http://www.immuneweb.com The 30 nm Chromatin Fiber 5 Compaction ratio = 50

9. 免疫学信息网 http://www.immuneweb.com A Mitotic Human Chromosome DNA length: 4.6 x 10 7 bp = 1.5 cm Chromosome length: 2 µm Compaction ratio = 8000 1 30 nm filament

10. 免疫学信息网 http://www.immuneweb.com Nuclear Scaffold/Matrix DNA Loops (5-200 kb) SAR/MAR 6

11. 免疫学信息网 http://www.immuneweb.com The Nucleosome 146 bp DNA 2X H3, H4, H2A, H2B H4 H2B H2A H3 Not in Handout

12. 免疫学信息网 http://www.immuneweb.com Identities = 74/82 (90%), Positives = 79/82 (96%) Query: 22 ILRDNIQGITKPAIRRLARRGGVKRISGLIYEEVRAVLKSFLESVIRDSVTYTEHAKRKT 81 +LRDNIQGITKPAIRRLARRGGVKRISGLIYEE R VLK FLE+VIRD+VTYTEHAKRKT Sbjct: 21 VLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRDAVTYTEHAKRKT 80 Query: 82 VTSLDVVYALKRQGRTLYGFGG 103 VT++DVVYALKRQGRTLYGFGG Sbjct: 81 VTAMDVVYALKRQGRTLYGFGG 102 Blast Search with S. cerevisiae Histone H4 Query: S. cerevisiae Subject: H. sapiens Not in Handout

13. 免疫学信息网 http://www.immuneweb.com MW Chromosomal Proteins 200,000 36,000 97,000 66,000 55,000 31,000 21,000 14,000 116,000 H4 H2A H2B/H3 Histones are Low MW Chromosomal Proteins 7

14. 免疫学信息网 http://www.immuneweb.com Domain Structure of Histones 11 22 33 L1L2NC Core Domain 8

15. 免疫学信息网 http://www.immuneweb.com H2B H2A H3 H4 22 33 11 22 33 11 22 33 11 22 33 11 9 L1 L2 NN

16. 免疫学信息网 http://www.immuneweb.com H3 H4 10

17. 免疫学信息网 http://www.immuneweb.com 4 helix-bundle H3 H4 H3’ H4’ 11

18. 免疫学信息网 http://www.immuneweb.com 4 helix-bundle H4H2B H2A 12

19. 免疫学信息网 http://www.immuneweb.com H4 H2B H2A H3 Not in Handout

20. 免疫学信息网 http://www.immuneweb.com H4 H2B H2A H3 Diameter = 110 Aº 13

21. 免疫学信息网 http://www.immuneweb.com H4 H2B H2A H3 Width = 45 Aº 14

22. 免疫学信息网 http://www.immuneweb.com H4 H2B H2A H3 15 Axis of dyad symmetry

23. 免疫学信息网 http://www.immuneweb.com Histone Core Tilt +Roll Tilt -Roll 15A Tilt Roll

24. 免疫学信息网 http://www.immuneweb.com H2B H2A L1-L2 Minor groove  1-  1 Minor groove L1-L2 Minor groove 16

25. 免疫学信息网 http://www.immuneweb.com H2B H2A Not in Handout DNA entry DNA exit

26. 免疫学信息网 http://www.immuneweb.com N-term  helix H2B H2A H3 Not in Handout DNA entry DNA exit

27. 免疫学信息网 http://www.immuneweb.com N-term  helix N-term  helix H2B H2A H3 Not in Handout DNA entry DNA exit

28. 免疫学信息网 http://www.immuneweb.com Energetic and Topological Consequences of DNA Wrapping

29. 免疫学信息网 http://www.immuneweb.com Basepair:151015 Histone Core Translational and Rotational Positioning

30. 免疫学信息网 http://www.immuneweb.com Basepair:151015 Histone Core Translational and Rotational Positioning

31. 免疫学信息网 http://www.immuneweb.com Basepair:151015 Histone Core Translational and Rotational Positioning ARE COUPLED!

32. 免疫学信息网 http://www.immuneweb.com 19

33. 免疫学信息网 http://www.immuneweb.com Are Nucleosomes “Positioned?” 1. Sequence at Unique Position 2. Micrococcal Nuclease Digest 3. Restriction Digest 4. Southern Blot Discrete Band Positioning Smear Random Orientation 17

34. 免疫学信息网 http://www.immuneweb.com Implications of Translational Positioning 18

35. 免疫学信息网 http://www.immuneweb.com N -ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTE- H3 N -SGRGKGGKGLGKGGAKRHRKVLRDNIQ - H4 N -SGRGKQGGKTRAKSKTRSSRAGL - H2A N -SDPAKSAPAAKKGSKKAVTKTQKKDGKKRRKSRKES - H2B H3 -ERA -C H4 -RTLYGFGG- C H2A - ELNKLLGGVTIAQGGVLPNIQSVLLPKKTESSKSTKSK- C H2B - PGELAKHAVSEGTKAVTKYTSAK- C Core Histones Have Variable Length Tails 20

36. 免疫学信息网 http://www.immuneweb.com H4 H2B H2A H3 H3 N-terminus 21

37. 免疫学信息网 http://www.immuneweb.com H4 H2B H2A H3 H2B N-terminus 22

38. 免疫学信息网 http://www.immuneweb.com H4 H2B H2A + + + _ _ _ 23

39. 免疫学信息网 http://www.immuneweb.com N - S GRG K GG K GLG K GGA K RHRKVLRDNIQ - H4 P Ac N -ART K QTAR KS TGG K APR K QLAT K AAR KS APATGGVKKPHRYRPGTVALREIRRYQKSTE- H3 Core PP 1028 Ac N - S GRG K QGG K TRAKSKTRSSRAGL - H2A P Ac N -SDPA K SAPAAK K GS K KAVT K TQ K KDG K KRRK S RKES - H2B P Ac 23A Newly-synthesized histones MMM M M K79 Active region Inactive Region (heterochromatin, X-inactivation) Active region Mitosis (Aurora B) SSN6/TUP1

40. 免疫学信息网 http://www.immuneweb.com ??? “Beads on a String”30 nm Filament 24 Low salt High salt How does chromatin become compacted??

41. 免疫学信息网 http://www.immuneweb.com The “Linker” Histone H1/H5 Largest histone (21 kD), less conserved Binds 165 bp: “Chromatosome” Facilitates chromatin compaction

42. 免疫学信息网 http://www.immuneweb.com Histone H5 reduces salt requirement for chromatin compaction 25

43. 免疫学信息网 http://www.immuneweb.com Helix III - Major groove binding Loop (Secondary DNA binding site) Globular domain of Histone H5 “winged helix-turn-helix domain” 26

44. 免疫学信息网 http://www.immuneweb.com N C C C C C C 26 Zhou et al., 1998 Nature 395, 402

45. 免疫学信息网 http://www.immuneweb.com The Solenoid Model for the 30 nm Fiber 26A Histone H1

46. 免疫学信息网 http://www.immuneweb.com The 30 nm Chromatin Fiber 5 Compaction ratio = 50

47. 免疫学信息网 http://www.immuneweb.com Condensins Condensins Are Required for Mitotic Chromosome Condensation Hirano and Mitchison, Cell 79, 449 Condensin- Depleted Mock- Depleted Hirano et al., Cell 89, 511 27