Anda di halaman 1dari 31

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)

JHW

Compaction of DNA by histones

Compaction by chromosome scaffold / nuclear matrix


3-99

Copyright 1999, J.H.Waterborg, UMKC

30nm

Nuclear - chromosome compaction


+ 2M NaCl

histones

JHW

DNA loops
+ 2M NaCl

histones

chromatid

Compaction by chromosome scaffold / nuclear matrix


3-99

Copyright 1999, J.H.Waterborg, UMKC

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

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 Zrich,
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 ?
Zhou et al. Nature 395, 402 (1998)

On the OUTSIDE of the DNA gyres ?


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

Chromatosome

Core histone octamer + 1 Linker Histone + 2 full turns of DNA (168 bp)

H1
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

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.
12

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

Copyright 1999, J.H.Waterborg, UMKC

H4 N-terminus

Gel Electrophoresis of Histones

gel type:

SDS

AU

electrode:

size + charge +
hydrophobicity

size +
charge

Separation by: size

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

Gel scans: alfalfa histones (Waterborg, 90s)

_
3-99

Copyright 1999, J.H.Waterborg, UMKC

+ + 34
H3.2 +1 2

++
H3 +

H3

TT

Almost All Acetylation is Dynamic


Chlamydomonas
reinhardtii

Level of histone acetylation:


Average level of acetylation:
Average AcLys turnover T:

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)

AcLys turnover T (min): 2.8 1.8 1.8 1.6 1.5


25

% 20

protein

Transcriptionally
active fraction
of genome 20 %

Steady-state [*Ac] labeling

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

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 -globin gene domain
(Ac-Lys) antibody
nucleosome ppt

DNA remaining

domain
boundary

10

30 kb

20

DNA loop domain


domain
boundary

= -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

Acetylation at Promoters
TR/RXR

Transcriptional
REPRESSION

Thyroid Hormone Receptor


example of DNA-binding
Transcription Factor

TH

HAT

JHW

co-activator
co-repressor
Histone
Deacetylase

Histone
Acetyl
Transferases

pol.II

TA
TA

TB

TAFII250

HAT
P/CAF

HAT

Adapted from Wolffe Nature 387, 16 (1997)

N-CoR
Sin3

RPD3

HAT

TAFII250

3-99

Copyright 1999, J.H.Waterborg, UMKC

GCN5
HAT

p300
CPB

TACCCG

ADA2
ADA3

TACCCG

TB

ACGGTC

Without Thyroid Hormone

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

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

Sin3

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 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

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
Swi6

Me
K9 K14

H3

Recruitment of Swi6 or HP-1


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