This slide series has been used for teaching to students of the Montpellier University in 2004.
It explains basic features of the folding of DNA in nucleosomes and chromatin fibers, and
discusses the regulatory roles of post-translational histone modificatons
The material displayed is inspired from several sources. The literature used is cited within
the slides. Moreover, several slides (indicated by the JHW logo) have been copied or modified
from Jacob Waterborg, University of Missouri, Kansas City To see his chromatin material in
full, see the website: http://sbs.umkc.edu/waterborg/
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
Nuclear - chromosome compaction
+ 2M NaCl
histones
radial loop
chrosomosome model
Mitosis
+ 2M NaCl
histones
chromatid
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
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
nucleosome
JHW
base pair
Compaction by chromosome scaffold / nuclear matrix
0.33 x 1.1 nm
3-99
Copyright 1999, J.H.Waterborg, UMKC
chromatid 1m
mitotic chromosome
10 m
DNA loops
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
Figure, courtesy from Timothy
Richmond, ETH Zürich,
Switzerland
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-fod 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
<
JHW
6 nm
• Each core histone dimer
has 6 DNA binding surfaces
that organize 3 DNA turns;
• The histone octamer
organizes 145 bp of DNA
in 1 3/4 helical turn of DNA:
48 nm of DNA packaged in a disc of 6 x 11nm
>
3-99
Copyright 1999, J.H.Waterborg, UMKC
<
11 nm
>
Histone octamer organizes 145 bp of DNA
Luger, Mader,
Richmond, Sargent & Richmond
Nature 389, 251-260 (1997)
JHW
Question 1: What is the function of histone N-termini ?
Question 2: Are all N-termini functionally equivalent ?
3-99
Copyright 1999, J.H.Waterborg, UMKC
Where are the N-termini of the core histones ?
H1/H5 globular protein domain
Asymmetric between the DNA gyres ?
On the OUTSIDE of the DNA gyres ?
Zhou et al. Nature 395, 402 (1998)
Wolffe et al. Science 274, 614 (1996)
Linker histone H1 organizes exiting DNA (up to 168 - 200 bp).
H1 stabilizes interaction between nucleosomes in compacted chromatin.
JHW
3-99
Copyright 1999, J.H.Waterborg, UMKC
Linker histone H1
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
Histone modifications
27
Turner (2002). Cell 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
Gel Electrophoresis of Histones
gel type:
SDS
AU
_
electrode:
size + charge +
hydrophobicity
size +
charge
Separation by: size
AUT
++
+
+
H3.1 +
TTTT
135
TT
T
T
+ + 34
H3.2 +1 2
++
H3 +
H3.1
0
135
H3.2
T TT
T
T
4
T
T
3
4
3
2
1
0
2
H3
135 1 0
H3
H4
H4
H3
4
3
2
+
+
H4 +
4
3
TT 2
1
0
T
135 aa
++
H4 +
1
H4
0
102
H4
102
102 aa
electrode:
JHW
+
_
Gel scans: alfalfa histones (Waterborg, 90’s)
_
3-99
Copyright 1999, J.H.Waterborg, UMKC
T
Almost All Acetylation is Dynamic
Chlamydomonas
reinhardtii
Level of histone acetylation:
32 % of H3
Average level of acetylation:
2.4 AcLys/H3
Average AcLys turnover T½: 1.9 0.4 min (H3)
AcLys turnover T½ (min): 2.8 1.8 1.8 1.6 1.5
16 % of H4
1.6 AcLys/H4
3.5 1.1 min (H4)
25
% 20
25
protein
Transcriptionally
active fraction
of genome  20 %
Steady-state [*Ac] labeling
15
H3
H4
protein
20
15
10
10
5
5
0
5
4
LEVEL of
AcLys
turnover
0
LEVEL of
AcLys
turnover
5
4
3
3
2
2
1
1
0
0
1
2
3
4
5
1
2
%
3
4
5
#AcLys/histone
Fraction of acetylated histone 100 92 104 106 82 39 58 81 100 76 %
subject to AcLys turnover: 100 12 % of H3
55 4 % of H4
JHW
Waterborg J. Biol. Chem. 273, 27602 (1998)
(67% of multi-acetylated H4)
3-99
Copyright 1999, J.H.Waterborg, UMKC
Example: alga
Acetylation of Chromatin Domains
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
TR/RXR
example of DNA-binding
Transcription Factor
TH
ADA2
ADA3
GCN5
HAT
HAT
TAFII250
JHW
p300
CPB
HAT
P/CAF
Transcriptional
REPRESSION
Thyroid Hormone Receptor
Without Thyroid Hormone
co-activator
co-repressor
Histone
Acetyl
Transferases
pol.II
Adapted from Wolffe Nature 387, 16 (1997)
Histone
Deacetylase
N-CoR
Sin3
RPD3
HAT
TAFII250
3-99
Copyright 1999, J.H.Waterborg, UMKC
Transcriptional
ACTIVATION
+
hormone
TH
Deacetylation of Chromatin
Transcriptional Silencing — X-chromosome Inactivation


3’..pGpCp..5’
5
me
5’..pCpGp..3’
MeCP2
transcriptional repressor
co-repressor
Sin3
Histone Deacetylase
RPD3
+
l 5meC CpG-methylation is maintained after DNA
replication by Maintenance Methylase action on
hemi-methylated DNA
l 5meC binds transcriptional repressor MeCP2
(MethylC-binding Protein-2)
l MeCP2 binds Sin3 with RPD3 histone deacetylase
+
CpG methylation
JHW
l 5meC CpG DNA modification is observed in
repressed genes and inactivated X chromosomes
Hypo-acetylated repressed chromatin fiber
Nan et al. Mol.Cell.Biol. 16, 414 (1996); Cell 88, 1 (1997); Jones et al. Nat.Genet. 19, 187 (1998)
3-99
Copyright 1999, J.H.Waterborg, UMKC
5
me
Histone Methylation
Histones can be methylated at lysines or arginines. Example: H3 K9 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)
N
e-N-dimethyl-Lysine
S-adenosylmethyionine
C
C C
O
C
HMT (Histone
Methyl-Transferase)
C
N
e-N-trimethyl-Lysine
C C
O
C
C e N+
C
C
C
C e N+ C
C C
C
DNA backbone
binding may not be
strongly affected, but
specific proteins may
recognize these
modifications
Histone methylation : Histone methyltransferases
Enhancer of Zeste
& K27
Silencing of euchromatic genes
Modified from:
Lachner and Jenuwein
(2002). Current Opin.
Cell Biol. 14, 286-98
Multiple roles of H3 K9 methylation
Tri-; di-; mono-HMTases?
S. pombe to man
(Tri-met K27)
and tri- met K9
N. crassa
Modified from:
Lachner and Jenuwein
(2002). Current Opin. Cell
Biol. 14, 286-98
Enhancer of
zeste
In contrast to histone acetylation, histone methylation is STABLE. This makes of this
mark a candidate for inheritance of chromatin states.
Heterochromatin
First discovered in Drosophila in genetic studies of chromosome rearrangements
Heterochromatin induces gene silencing
Singh, P. B. (1994). Molecular mechanisms of cellular determination: their relation to chromatin structure and parental imprinting. J Cell Sci 107, 2653-2668.
Genes involved in heterochromatin
Singh, P. B. (1994). Molecular mechanisms of cellular determination: their relation to chromatin structure and parental imprinting. J Cell Sci 107, 2653-2668.
Heterochromatin formation involves histone H3 lysine 9 methylation by
Su(var)3-9 and recruitment of HP-1
Ac
Ac
K9
K14
H3
Open chromatin
HDAC
K9
K14
H3
Methylation of K9 (Clr4; Suvar39)
Me
K9
K14
Recruitment of Swi6 or HP-1
Swi6
Me
K9
H3
K14
H3
Recruitment of more Clr4 (Suvar39) molecules
by Swi6 (HP-1)
Condensed chromatin
Epigenetic regulation of centromere function and RNAi
Outer repeats
Central domain
Outer repeats
S. Pombe Centromere
dsRNAs
Nucleosomes
K9 Me
Swi6
Nucleosomes
K9 Me
Swi6
This structure requires proteins that are responsible for the phenomenon of RNA interference
-> RNA interference is gene silencing mediated by short RNA molecules produced from
double stranded RNA (dsRNA).
-> Short RNAs are produced by cleavage of dsRNA by the nuclease called Dicer. Dicer
produces short dsRNA called siRNA duplexes. Then, a complex of proteins called RISC
unwinds the duplex and produces siRNA. siRNA hybridize to the target mRNA which is
degraded.
…But RNAi also acts on chromatin
-> Centromeres produce dsRNA, and Dicer as well as the RISC are required for centromere
silencing. Transposons induce gene silencing in a similar manner.
Mechanism for coupling of RNAi and chromatin regulation?
Model:
Nascent RNA
si RNA
K9 methylation, Swi 6 recruitment!
Ago (RISC), Clr4 Dcr complex
See also: Grewal and Moazed (2003). Science 301, 798-802
Opposing functions of H3 K9 versus K4 methylation
Lachner and Jenuwein. (2002) Current Opin. Cell Biol. 14, 286-98
A functional link between histone H3 methylation and
DNA cytosine methylation in Arabidopsis thaliana
HP1?
Modified from: Lachner and Jenuwein. (2002) Current Opin. Cell Biol. 14, 286-98
Multiple biological phenomena involving histone methylation
E(z)
H3-K27 methylation and
and H3-K27 methylation
Modified from: Lachner and Jenuwein. (2002) Current Opin. Cell Biol. 14, 286-98
Summary and perspectives
Chromatin is packaged in a hierarchy of structures. Each of these
levels of packaging has regulatory roles in the genome
The level of packaging we know best, also thanks to formidable
tools & technology development in the recent years, is the
nucleosome.
In particular, post-translational histone modifications play key roles
in regulation of genome function, and the combinatorial power of
these modifications is only beginning to be unraveled.
Future challenges will be to decrypt the detailed meaning of these
modifications, and to understand the roles of the higher order levels
of chromatin and chromosome folding