Download C - IGH

10,000 nm
DNA compaction
in a human nucleus
11 nm
1bp (0.3nm)
compact size
DNA
length
compaction
nucleus (human)
2 x 23 = 46 chromosomes
92 DNA molecules
10 m ball
12,000 Mbp
4 m DNA
400,000 x
mitotic chromosome
2 chromatids, 1 m thick
2 DNA molecules
10 m long X
2x 130 Mbp
2x 43 mm DNA
10,000 x
DNA domain
anchored DNA loop
1 replicon ?
60 nm x 0.5 m
60 kbp
20 m DNA
35 x
chromatin fiber
approx. 6 nucleosomes per ‘turn’ of 11 nm
30 nm diameter
1200 bp
400 nm DNA
35 x
nucleosome
disk 1 ¾ turn of DNA (146 bp) + linker DNA
6 x 11 nm
200 bp
66 nm DNA
6 - 11 x
1 bp
0.33 nm DNA
1x
base pair
0.33 x 1.1 nm
Compaction of DNA by histones
JHW
Compaction by chromosome scaffold / nuclear matrix
3-99
Copyright 1999, J.H.Waterborg, UMKC
30nm
H1
HISTONES
Linker histone
are
highly conserved,
small, basic proteins
H2A
helix
Core histones
variable
H3
H4
conserved
Histone acetylation
is a reversible modification
of lysines in the N-termini
of the core histones.
Result:
• reduced binding to DNA
• destabilization of chromatin
N
JHW
3-99
Copyright 1999, J.H.Waterborg, UMKC
H2B
Core Histones
The basic structure of ALL
core histones is the same:
• 1 long hydrophobic alpha-helix,
bordered by
• 2 short hydrophobic alpha helices
that form pairs
• H2A - H2B and H3 - H4
which interact.
References: Moudrianakis et al. PNAS 88, 10138 (1991);
PNAS 90, 10489 (1993); PNAS 92, 11170 (1995)
JHW
3-99
Copyright 1999, J.H.Waterborg, UMKC
The histone-fold
H3-H4
tetramer
JHW
Histone
octamer
H2A-H2B
dimer
3-99
Copyright 1999, J.H.Waterborg, UMKC
Histone octamer assembly
JHW
3-99
Copyright 1999, J.H.Waterborg, UMKC
The Nucleosome as the fundamental
chromatin unit
• 146-149 bp DNA in a 1.65 turns of a
flat, left-handed superhelix
• one pseudo twofold axis centered at
the “dyad” (reference: 0 helical turns)
• one base-pair precisely at the dyad
• sharp bends at + 1.5 and + 4-5 turns
• Histone-fold domains organize 121
bp of DNA. The DNA is bound at 10 bp
intervals through many contacts,
including penetration of arginines at all
14 minor grooves facing the protein
core
• The grooves from neighboring DNA
turns line up; forming channels
• H3 and H2B N-termini exit one of
these channels every 20bp.
• The H4 tail establishes contacts with
the next core particle.
JHW
H2A
H2B
H3
H4
3-99
Copyright 1999, J.H.Waterborg, UMKC
Nucleosome features
C
Chromatosome
Core histone octamer + 1 Linker Histone + 2 full turns of DNA (168 bp)
N
H1
H3
1 mM
5 mM
Linker Histone and histone termini
control linker DNA entry/exit of
chromatosome in chromatin fiber.
JHW
Zhou, Gerchman, Ramakrishnan, Travers,
Muyldermans Nature 395, 402 (1998)
An, Leuba, van Holde, Zlatanova
PNAS 95, 3396 (1998)
3-99
Copyright 1999, J.H.Waterborg, UMKC
H3
Chromatin
fibers
+ charged N termini
(bind DNA on neigboring
nucleosomes)
11 nm
(beads)
highly acetylated
core histones
(especially H3 and H4)
• HIGH level of histone H1
• Reduced level of histone H1
• NO gene transcription
• Gene transcription possible
JHW
3-99
Copyright 1999, J.H.Waterborg, UMKC
30 nm
chromatin fiber
Alternative chromatin fiber models
Zigzag fiber
Histone modifications
27
Cell (2002) 111, 285-91
Acetylation of conserved lysines
The N-termini of histones H4 and H3, and their acetylation patterns, are absolutely conserved.
5
Ac
Ac
Ac
Ac
9
Ac
18
Ac
14
Ac
Acetyl-CoA
O
27
Ac or Me
P
O
DNA
P backbone
binding
Histone
Deacetylase
reversible reactions
CoA
O
O
C C g C e
N+
O C a b C d C
HAT (Histone
Acetyl-Transferase)
-
-
-
-
C
C e
C
C
C
N
C
O
no DNA
binding
-
-
-
-
-
-
-
-
-
-
P
C C
O
-
-
N
e-N-Acetyl-Lysine
JHW
23
Ac
A-R-T-K-Q-T-A-R-K-S-T-G-G-K-A-P-R-K-Q-L-A-T-K-A-A-R-K-S-A-P+
+
+ +
+ +
+ +
+
N
Lysine
20
Ac or Me
Ac-S-G-R-G-K-G-G-K-G-L-G-K-G-G-A-K-R-H-R-K-V-L-R-D+
+ + + + +
+
+
+
+
4
Me
H3 N-terminus
16
12
-
-
-
-
3-99
Copyright 1999, J.H.Waterborg, UMKC
H4 N-terminus
8
Acetylation of Chromatin Domains: analysis by the
technique of Chromatin Immunoprecipitation (ChIP)
Example: the chicken b-globin gene domain
a(Ac-Lys) antibody
nucleosome ppt
DNA remaining
domain
boundary
10
30 kb
20
b
bH
bA
be
DNA loop domain
domain
boundary
b = b-globin genes:
DNase I
hyper-sensitive site
Control:
inactive gene
chicken
ovalbumin
General DNase I
sensitivity
DNase I
l High levels of chromatin acetylation, across complete chromatin domains
(DNA loops), induces chromatin changes detected as “general DNase I sensitivity”
l Within these chromatin domains, at functional genes or transcription factors,
the chromatin structure is interrupted by small “DNase I hypersensitive sites”
JHW
Hebbes, Clayton, Thorne, Crane-Robinson EMBO J. 13, 1823 (1994)
0 1 2
DNase I
(U/ml)
3-99
Copyright 1999, J.H.Waterborg, UMKC
0
Acetylation at Promoters leads to
transcriptional activation
Thyroid Hormone Receptor
TH
example of DNA-binding
Transcription Factor
+
hormone
Transcriptional
ACTIVATION
TH
GCN5
HAT
HAT
TAF II250
p300
CPB
HAT
P/CAF
co-activator
Histone
Acetyl
Transferases
pol.II
Hyper-acetylated
decondensed
Chromatin fiber
JHW
3-99
Copyright 1999, J.H.Waterborg, UMKC
ADA2
ADA3
Deacetylation at Promoters leads to
Transcriptional repression
Without Thyroid Hormone
co-repressor
Histone
Deacetylase
HAT
TAF II250
N-CoR
Sin3
RPD3
X
Hypo-acetylated
condensed
Chromatin fiber
JHW
3-99
Copyright 1999, J.H.Waterborg, UMKC
Transcriptional
REPRESSION
Acétylation vs déacetylation des histones
Acétylation
des histones
Chromatine décondensée:
Etat transcriptionnel
actif
Déacetylation
des histones
Chromatine condensée:
Etat transcriptionnel
réprimé
Histone Methylation
Histones can be methylated at lysines or arginines. Example: H3 K4 methylation
N
Lysine
S-adenosylmethyionine
C C g C e
N+
O C a b C d C
HMT (Histone
Methyl-Transferase)
Histone
demethylase
N
e-N-monomethyl-Lysine
C
C C
O
C
C e N+
C
C
S-adenosylmethyionine
HMT (Histone
Methyl-Transferase)
Histone
demethylase
N
e-N-dimethyl-Lysine
S-adenosylmethyionine
DNA backbone
binding may not be
strongly affected, but
specific proteins may
recognize these
modifications
C
C C
O
C
HMT (Histone
Methyl-Transferase)
C
N
e-N-trimethyl-Lysine
C C
O
C
Histone demethylases
found for K4, K9,
K27 and K36
C e N+
C
C
C
C e N+ C
C C
C
Histone
demethylase
Histone methylation : Histone methyltransferases
Enhancer of Zeste
Dm Trx, Ash1
Mammalian MLL1-3
H3 K27
Euchromatin methylation,
PcG-dependent gene silencing
Euchromatin methylation, gene activation
trxG-dependent gene silencing
Histone demethylases
2 classes:
LSD1
JmjC domain proteins
Histone demethylases
LSD1 demethylases target mono or dimethyl K4 and K9 of histone H3, depending
on their interacting cofactor
Histone demethylases
JmjC domain demethylases target many different lysines and are conserved
Histone methylation
Utx
JMJD3
A reversible phenomenon with
many dedicated components
Coffee break…