For further reading:
Nucleic Acids,Structures,Properties,and Functions
V.A,Bloomfield
D.M,Crothers
I,Tinoco
Announcements:
Power Point Lecture Notes Available on BCMP200 Website
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
AccessibilityChromatinRemodeling
Machines
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
5Compaction ratio = 50
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 Nucleosome
146 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+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
MW Chromosomal Proteins200,000
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
Domain Structure of Histones
?1 ?2 ?3L1 L2N C
Core Domain
8
H2B
H2A
H3
H4
?2
?3
?1
?2?3
?1
?2
?3
?1
?2
?3
?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-L2
Minor groove
?1- ? 1
Minor groove
L1-L2
Minor 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 Topological
Consequences of DNA Wrapping
Basepair,1 5 10 15
Histone
Core
Translational and Rotational Positioning
Basepair,1 5 10 15
Histone
Core
Translational and Rotational Positioning
Basepair,1 5 10 15
Histone
Core
Translational and Rotational Positioning
ARE
COUPLED!
19
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
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 Ac 23A
Newly-synthesized histones
M M M
M
M
K79
Active
region
Inactive
Region (heterochromatin,X-inactivation)
Active
region
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
N
C
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
5Compaction ratio = 50
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
Nucleic Acids,Structures,Properties,and Functions
V.A,Bloomfield
D.M,Crothers
I,Tinoco
Announcements:
Power Point Lecture Notes Available on BCMP200 Website
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
AccessibilityChromatinRemodeling
Machines
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
5Compaction ratio = 50
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 Nucleosome
146 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+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
MW Chromosomal Proteins200,000
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
Domain Structure of Histones
?1 ?2 ?3L1 L2N C
Core Domain
8
H2B
H2A
H3
H4
?2
?3
?1
?2?3
?1
?2
?3
?1
?2
?3
?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-L2
Minor groove
?1- ? 1
Minor groove
L1-L2
Minor 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 Topological
Consequences of DNA Wrapping
Basepair,1 5 10 15
Histone
Core
Translational and Rotational Positioning
Basepair,1 5 10 15
Histone
Core
Translational and Rotational Positioning
Basepair,1 5 10 15
Histone
Core
Translational and Rotational Positioning
ARE
COUPLED!
19
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
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 Ac 23A
Newly-synthesized histones
M M M
M
M
K79
Active
region
Inactive
Region (heterochromatin,X-inactivation)
Active
region
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
N
C
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
5Compaction ratio = 50
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
