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

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Interference Footprinting

DNA + MMS

+ Saturating [Protein]

Gel Shift

14

Cleave backbone @ modified bases


“Chromatin is the last refuge of scoundrels”


A Mitotic Human Chromosome

DNA length: 4.6 x 107 bp = 1.5 cm Chromosome length: 2 µm

Compaction ratio = 8000 1


Chromatin

DNA Compaction

DNA AccessibilityChromatin

RemodelingMachines

NIH


Basic Unit of Chromatin Structure: Polynucleosome

2Compaction ratio = 6


Limited Micrococcal Nulcease Digestion of Chromatin Generates a ~200 bp Ladder

3


H1 H1

H1 H1

H1

H1

Micrococcal Nuclease Digestion Time

Mononucleosome Chromatosome Core Particle

Linker DNA (variable)

Operational Definititions of Nucleosome Particles

Chromatin

4


The 30 nm Chromatin Fiber



A Mitotic Human Chromosome

DNA length: 4.6 x 107 bp = 1.5 cm Chromosome length: 2 µm

Compaction ratio = 8000 1

30 nm filament


Nuclear Scaffold/Matrix

DNA Loops (5-200 kb)

SAR/MAR

6


The Nucleosome146 bp DNA 2X H3, H4, H2A, H2B

H4

H2B

H2A

H3

Not in Handout


Identities = 74/82 (90%), Positives = 79/82 (96%)

Query: 22 ILRDNIQGITKPAIRRLARRGGVKRISGLIYEEVRAVLKSFLESVIRDSVTYTEHAKRKT 81 +LRDNIQGITKPAIRRLARRGGVKRISGLIYEE R VLK FLE+VIRD+VTYTEHAKRKTSbjct: 21 VLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRDAVTYTEHAKRKT 80

Query: 82 VTSLDVVYALKRQGRTLYGFGG 103 VT++DVVYALKRQGRTLYGFGGSbjct: 81 VTAMDVVYALKRQGRTLYGFGG 102

Blast Search with S. cerevisiae Histone H4

Query: S. cerevisiaeSubject: H. sapiens

Not in Handout


MW Chromosomal Proteins200,000

36,000

97,000

66,000

55,000

31,000

21,000

14,000

116,000

H4H2A

H2B/H3

Histones are Low MW Chromosomal Proteins

7


Domain Structure of Histones

1 2 3L1 L2N C

Core Domain

8


H2B

H2A

H3

H4

23

1

23

12

3

1

23

1

9

L1L2

N


H3

H4

10


4 helix-bundle

H3

H4

H3’

H4’

11


4 helix-bundle H4H2B

H2A

12


H4

H2B

H2A

H3

Not in Handout


H4

H2B

H2A

H3

Diameter = 110 Aº

13


H4

H2B

H2A

H3

Width = 45 Aº

14


H4

H2B

H2A

H3

15

Axis of dyad symmetry


Histone CoreTilt

+Roll

Tilt

-Roll

15A

Tilt

Roll


H2B

H2A

L1-L2Minor groove

1- 1Minor groove

L1-L2Minor groove

16


H2B

H2A

Not in Handout

DNA entry

DNA exit


N-term helix

H2B

H2A

H3

Not in Handout

DNA entry

DNA exit


N-term helix

N-term helix

H2B

H2A

H3

Not in Handout

DNA entry

DNA exit


Energetic and TopologicalConsequences of DNA Wrapping


Basepair: 1 5 10 15

HistoneCore

Translational and Rotational Positioning


Basepair: 1 5 10 15

HistoneCore



Basepair: 1 5 10 15

HistoneCore


ARECOUPLED!

免疫学信息网 http://www.immuneweb.com 19


Are Nucleosomes “Positioned?”

1. Sequence at Unique Position

2. Micrococcal Nuclease Digest

3. Restriction Digest

4. Southern Blot

Discrete Band PositioningSmear Random Orientation

17


Implications of Translational Positioning

18


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

Core

Histones Have Variable Length Tails

20


H4

H2B

H2A

H3

H3 N-terminus

H3 N-terminus

21


H4

H2B

H2A

H3

H2B N-terminus

22


H4

H2B

H2A

+++

_

__

23


N-SGRGKGGKGLGKGGAKRHRKVLRDNIQ -H4

P

AcAc AcAc

N-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTE- H3

Core P P

10 28

Ac Ac Ac Ac AcAc

N-SGRGKQGGKTRAKSKTRSSRAGL-H2A

P

Ac Ac

N-SDPAKSAPAAKKGSKKAVTKTQKKDGKKRRKSRKES-H2B

P

AcAcAcAc Ac Ac23A

Newly-synthesized histones

M M M

M

M

K79

Activeregion

InactiveRegion (heterochromatin, X-inactivation)

Activeregion

Mitosis(Aurora B)

SSN6/TUP1


???

“Beads on a String” 30 nm Filament

24

Low salt High salt

How does chromatin become compacted??


The “Linker” Histone H1/H5

• Largest histone (21 kD), less conserved

• Binds 165 bp: “Chromatosome”

• Facilitates chromatin compaction


Histone H5 reduces salt requirement for chromatin compaction

25


Helix III - Major groove binding

Loop(Secondary DNA binding site)

Globular domain of Histone H5“winged helix-turn-helix domain”

26


NC

C

C

C

C

C

26Zhou et al., 1998 Nature 395, 402


The Solenoid Model for the 30 nm Fiber

26A

Histone H1


The 30 nm Chromatin Fiber



Condensins

Condensins Are Required for Mitotic ChromosomeCondensation

Hirano and Mitchison, Cell 79, 449

Condensin-Depleted

Mock-Depleted

Hirano et al., Cell 89, 511

27

Documents

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