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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)
JHW
30nm
histones
JHW
DNA loops
+ 2M NaCl
histones
chromatid
chromatid 1m
Mitosis
mitotic chromosome
10 m
radial loop
chrosomosome model
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
JHW
3-99
H2B
Core Histones
3-99
The histone-fold
H3-H4
tetramer
JHW
Histone
octamer
H2A-H2B
dimer
3-99
JHW
JHW
H2A
H2B
H3
H4
3-99
Nucleosome features
<
JHW
6 nm
>
3-99
<
11 nm
>
Luger, Mader,
Richmond, Sargent & Richmond
Nature 389, 251-260 (1997)
JHW
3-99
Linker histone H1
Chromatosome
Core histone octamer + 1 Linker Histone + 2 full turns of DNA (168 bp)
H1
1 mM
5 mM
3-99
H3
H3
Chromatin
fibers
+ charged N termini
(bind DNA on neigboring
nucleosomes)
11 nm
(beads)
highly acetylated
core histones
(especially H3 and H4)
NO gene transcription
JHW
3-99
30 nm
chromatin fiber
Histone modifications
27
16
Ac
Ac
Ac
Ac
9
Ac
18
Ac
14
Ac
Acetyl-CoA
23
Ac
27
Ac or Me
P
O
DNA
P backbone
binding
Histone
Deacetylase
reversible reactions
CoA
C C C
N+
O C C C
HAT (Histone
Acetyl-Transferase)
C C
O
C
C
C
N
C
O
no DNA
binding
-N-Acetyl-Lysine
JHW
Ac or Me
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-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
3-99
H4 N-terminus
gel type:
SDS
AU
electrode:
size + charge +
hydrophobicity
size +
charge
AUT
++
H3.1 +
TTTT
135
4
2
H4
4
3
1
102
H3
+
H4 +
4
3
TT 2
1
0
T
H3
H4
135 aa
H3.2
T TT
T
135 1 0
++
H4 +
H3.1
135
3
4
3
2
1
0
H4
H4
102
102 aa
electrode:
JHW
_
3-99
+ + 34
H3.2 +1 2
++
H3 +
H3
TT
32 % of H3
2.4 AcLys/H3
1.9 0.4 min (H3)
16 % of H4
1.6 AcLys/H4
3.5 1.1 min (H4)
% 20
protein
Transcriptionally
active fraction
of genome 20 %
15
H3
H4
protein
25
20
15
10
10
0
5
4
LEVEL of
AcLys
turnover
LEVEL of
AcLys
turnover
5
4
5 #AcLys/histone
Example: alga
DNA remaining
domain
boundary
10
30 kb
20
= -globin genes:
DNase I
hyper-sensitive site
Control:
inactive gene
chicken
ovalbumin
General DNase I
sensitivity
DNase I
0 1 2
DNase I
(U/ml)
3-99
Acetylation at Promoters
TR/RXR
Transcriptional
REPRESSION
TH
HAT
JHW
co-activator
co-repressor
Histone
Deacetylase
Histone
Acetyl
Transferases
pol.II
TA
TA
TB
TAFII250
HAT
P/CAF
HAT
N-CoR
Sin3
RPD3
HAT
TAFII250
3-99
GCN5
HAT
p300
CPB
TACCCG
ADA2
ADA3
TACCCG
TB
ACGGTC
TA
TA
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
Histone Deacetylase
RPD3
CpG methylation
JHW
Sin3
Nan et al. Mol.Cell.Biol. 16, 414 (1996); Cell 88, 1 (1997); Jones et al. Nat.Genet. 19, 187 (1998)
3-99
5
me
Histone Methylation
Histones can be methylated at lysines or arginines. Example: H3 K9 methylation
N
Lysine
S-adenosylmethyionine
C C C
N+
O C C C
HMT (Histone
Methyl-Transferase)
Histone
demethylase?
N
-N-monomethyl-Lysine
C
C C
C N+
C
C
S-adenosylmethyionine
HMT (Histone
Methyl-Transferase)
N
-N-dimethyl-Lysine
S-adenosylmethyionine
C
C C
HMT (Histone
Methyl-Transferase)
-N-trimethyl-Lysine
C C
O
C N+
C
C
C
C N+ C
C C
C
DNA backbone
binding may not be
strongly affected, but
specific proteins may
recognize these
modifications
Enhancer of Zeste
& K27
Modified from:
Lachner and Jenuwein
(2002). Current Opin.
Cell Biol. 14, 286-98
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
Heterochromatin
First discovered in Drosophila in genetic studies of chromosome rearrangements
Singh, P. B. (1994). Molecular mechanisms of cellular determination: their relation to chromatin structure and parental imprinting. J Cell Sci 107, 2653-2668.
Ac
K9 K14
H3
Open chromatin
HDAC
K9 K14
H3
Me
K9 K14
H3
Condensed chromatin
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
Lachner and Jenuwein. (2002) Current Opin. Cell Biol. 14, 286-98
HP1?
Modified from: Lachner and Jenuwein. (2002) Current Opin. Cell Biol. 14, 286-98
E(z)
Modified from: Lachner and Jenuwein. (2002) Current Opin. Cell Biol. 14, 286-98