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免疫学信息网 “Chromatin is the last refuge of scoundrels”

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Presentation on theme: "免疫学信息网 “Chromatin is the last refuge of scoundrels”"— Presentation transcript:

1 免疫学信息网

2 “Chromatin is the last refuge of scoundrels”

3 免疫学信息网 A Mitotic Human Chromosome DNA length: 4.6 x 10 7 bp = 1.5 cm Chromosome length: 2 µm Compaction ratio =

4 免疫学信息网 Chromatin DNA Compaction DNA Accessibility Chromatin Remodeling Machines NIH

5 免疫学信息网 Basic Unit of Chromatin Structure: Polynucleosome 2 Compaction ratio = 6

6 免疫学信息网 Limited Micrococcal Nulcease Digestion of Chromatin Generates a ~200 bp Ladder 3

7 免疫学信息网 H1 Micrococcal Nuclease Digestion Time MononucleosomeChromatosomeCore Particle Linker DNA (variable) Operational Definititions of Nucleosome Particles Chromatin 4

8 免疫学信息网 The 30 nm Chromatin Fiber 5 Compaction ratio = 50

9 免疫学信息网 A Mitotic Human Chromosome DNA length: 4.6 x 10 7 bp = 1.5 cm Chromosome length: 2 µm Compaction ratio = nm filament

10 免疫学信息网 Nuclear Scaffold/Matrix DNA Loops (5-200 kb) SAR/MAR 6

11 免疫学信息网 The Nucleosome 146 bp DNA 2X H3, H4, H2A, H2B H4 H2B H2A H3 Not in Handout

12 免疫学信息网 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 MW Chromosomal Proteins 200,000 36,000 97,000 66,000 55,000 31,000 21,000 14, ,000 H4 H2A H2B/H3 Histones are Low MW Chromosomal Proteins 7

14 免疫学信息网 Domain Structure of Histones 11 22 33 L1L2NC Core Domain 8

15 免疫学信息网 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 H3 H4 10

17 免疫学信息网 4 helix-bundle H3 H4 H3’ H4’ 11

18 免疫学信息网 4 helix-bundle H4H2B H2A 12

19 免疫学信息网 H4 H2B H2A H3 Not in Handout

20 免疫学信息网 H4 H2B H2A H3 Diameter = 110 Aº 13

21 免疫学信息网 H4 H2B H2A H3 Width = 45 Aº 14

22 免疫学信息网 H4 H2B H2A H3 15 Axis of dyad symmetry

23 免疫学信息网 Histone Core Tilt +Roll Tilt -Roll 15A Tilt Roll

24 免疫学信息网 H2B H2A L1-L2 Minor groove  1-  1 Minor groove L1-L2 Minor groove 16

25 免疫学信息网 H2B H2A Not in Handout DNA entry DNA exit

26 免疫学信息网 N-term  helix H2B H2A H3 Not in Handout DNA entry DNA exit

27 免疫学信息网 N-term  helix N-term  helix H2B H2A H3 Not in Handout DNA entry DNA exit

28 免疫学信息网 Energetic and Topological Consequences of DNA Wrapping

29 免疫学信息网 Basepair: Histone Core Translational and Rotational Positioning

30 免疫学信息网 Basepair: Histone Core Translational and Rotational Positioning

31 免疫学信息网 Basepair: Histone Core Translational and Rotational Positioning ARE COUPLED!

32 免疫学信息网 19

33 免疫学信息网 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 Implications of Translational Positioning 18

35 免疫学信息网 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 H4 H2B H2A H3 H3 N-terminus 21

37 免疫学信息网 H4 H2B H2A H3 H2B N-terminus 22

38 免疫学信息网 H4 H2B H2A _ _ _ 23

39 免疫学信息网 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 ??? “Beads on a String”30 nm Filament 24 Low salt High salt How does chromatin become compacted??

41 免疫学信息网 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 Histone H5 reduces salt requirement for chromatin compaction 25

43 免疫学信息网 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 N C C C C C C 26 Zhou et al., 1998 Nature 395, 402

45 免疫学信息网 The Solenoid Model for the 30 nm Fiber 26A Histone H1

46 免疫学信息网 The 30 nm Chromatin Fiber 5 Compaction ratio = 50

47 免疫学信息网 Condensins Condensins Are Required for Mitotic Chromosome Condensation Hirano and Mitchison, Cell 79, 449 Condensin- Depleted Mock- Depleted Hirano et al., Cell 89,


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