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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

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This slide series has been used for teaching to students of the montpellier university in 2004

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/


Dna compaction in a human nucleus

Compaction by chromosome scaffold / nuclear matrix

10,000 nm

DNA compactioninahuman nucleus

11 nm

30nm

1bp (0.3nm)

Compaction of DNA by histones

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

+ 2M NaCl

histones

+ 2M NaCl

mitotic chromosome

histones

1mm

chromatid

Compaction by chromosome scaffold / nuclear matrix

Nuclear - chromosome compaction

radial loop

chrosomosome model

Mitosis

DNA loops

10 mm

chromatid

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

H1

HISTONESare highly conserved,small, basic proteins

Linker histone

H2A

H2B

helix

  • Histone acetylation

  • is a reversible modification

  • of lysines in the N-termini

  • of the core histones.

  • Result:

  • reduced binding to DNA

  • destabilization of chromatin

Core histones

variable

H3

H4

conserved

N

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

Core Histones

The histone-fold

  • 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


This slide series has been used for teaching to students of the montpellier university in 2004

Histone octamer assembly

Histone

octamer

H3-H4

tetramer

H2A-H2B

dimer

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

The Nucleosome as the fundamental chromatin unit

Figure, courtesy from Timothy Richmond, ETH Zürich, Switzerland

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

H4

H3

H2B

H2A

Nucleosome features

  • 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


This slide series has been used for teaching to students of the montpellier university in 2004

Histone octamer organizes 145 bp of DNA

< 11 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

< 6 nm >

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

Where are the N-termini of the core histones ?

Luger, Mader,

Richmond, Sargent & Richmond

Nature389, 251-260 (1997)

Question 1: What is the function of histone N-termini ?

Question 2: Are all N-termini functionally equivalent ?

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

Linker histone H1

H1/H5 globular protein domain

On the OUTSIDE of the DNA gyres ?

Asymmetric between the DNA gyres ?

Zhou et al. Nature 395, 402 (1998)

Wolffe et al. Science274, 614 (1996)

Linker histone H1 organizes exiting DNA (up to 168 - 200 bp).

H1 stabilizes interaction between nucleosomes in compacted chromatin.

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

1 mM

5 mM

Zhou, Gerchman, Ramakrishnan, Travers,

Muyldermans Nature 395, 402 (1998)

An, Leuba, van Holde, Zlatanova

PNAS 95, 3396 (1998)

Chromatosome

C

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

N

H1

H3

H3

Linker Histone and histone termini

control linker DNA entry/exit of

chromatosome in chromatin fiber.

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

Chromatinfibers

11 nm

(beads)

30 nm

chromatin fiber

highly acetylated

core histones

(especially H3 and H4)

+ charged N termini

(bind DNA on neigboring

nucleosomes)

  • HIGH level of histone H1

  • Reduced level of histone H1

  • Gene transcription possible

  • NO gene transcription

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

Histone modifications

27

Turner (2002). Cell 111, 285-91


This slide series has been used for teaching to students of the montpellier university in 2004

5

16

20

8

12

Ac or Me

Ac

Ac

Ac

Ac

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-

+

+

+

+

+

+

+

+

+

+

27

4

18

9

23

14

Ac or Me

Me

Ac

Ac

Ac

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

-

O

O

-

-

C

C

C

g

e

-

N+

P

P

-

a

d

b

C

C

C

-

O

O

O

-

-

-

-

-

-

-

N

P

-

-

C

C

N

C

C

e

-

-

-

C

C

C

C

-

O

-

-

O

-

Acetylation of conserved lysines

The N-termini of histones H4 and H3, and their acetylation patterns, are absolutely conserved.

H4 N-terminus

H3 N-terminus

DNA

backbone

binding

Lysine

Acetyl-CoA

HAT (Histone

Acetyl-Transferase)

Histone

Deacetylase

reversible reactions

CoA

e-N-Acetyl-Lysine

no DNA

binding

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

gel type:

SDS

_

+

+

+

+

electrode:

+

H3.1

135

T

T

T

T

T

T

T

T

4

T

3

2

1

0

4

+

+

3

+

H3.2

2

1

0

+

+

135

+

H3

T

T

T

T

T

T

T

4

3

H3

2

1

135

0

+

+

+

+

+

135 aa

H4

4

4

+

H4

3

3

H3

T

2

2

T

T

H4

1

1

H4

0

0

102

102

102 aa

_

_

+

electrode:

Gel Electrophoresis of Histones

size + charge +

hydrophobicity

size +

charge

Separation by: size

AU

AUT

H3.1

H3.2

H3

H4

H4

Gel scans: alfalfa histones (Waterborg, 90’s)

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

Example: alga

Chlamydomonas

reinhardtii

Level of histone acetylation: 32 % of H316 % of H4

Average level of acetylation: 2.4 AcLys/H31.6 AcLys/H4

Average AcLys turnover T½: 1.9 0.4 min (H3) 3.5 1.1 min (H4)

AcLys turnover T½ (min): 2.8 1.8 1.8 1.6 1.5

Transcriptionally

active fraction

of genome  20 %

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 H355 4 % of H4

(67% of multi-acetylated H4)

AlmostAll Acetylation is Dynamic

WaterborgJ. Biol. Chem. 273, 27602 (1998)

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

(Ac-Lys) antibody

nucleosome ppt

DNA loop domain



H

A



 = -globin genes:

domain

boundary

domain

boundary

0

10

30

kb

20

Control:

inactive gene

DNase I

hyper-sensitive site

chicken

ovalbumin

General DNase I

sensitivity

DNA remaining

0 1 2

DNase I

DNase I

(U/ml)

Acetylation of Chromatin Domains

Example: the chicken -globin gene domain

  • High levels of chromatin acetylation, across complete chromatin domains

    (DNA loops), induces chromatin changes detected as “general DNase I sensitivity”

  • Within these chromatin domains, at functional genes or transcription factors,

  • the chromatin structure is interrupted by small “DNase I hypersensitive sites”

Hebbes, Clayton, Thorne, Crane-RobinsonEMBO J. 13, 1823 (1994)

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

+

TR/RXR

TH

Transcriptional

ACTIVATION

hormone

TH

Without Thyroid Hormone

TACCCG

ACGGTC

TACCCG

co-activator

ADA2

ADA3

p300

CPB

HAT

co-repressor

N-CoR

Sin3

GCN5

HAT

Histone

Deacetylase

HAT

P/CAF

Histone

Acetyl

Transferases

RPD3

HAT

HAT

TAF 250

II

TAF 250

pol.II

II

TATA

TBP

TATA

TBP

Acetylation at Promoters

Transcriptional

REPRESSION

Thyroid Hormone Receptor

example of DNA-binding

Transcription Factor

Adapted from WolffeNature 387, 16 (1997)

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

transcriptional repressor

co-repressor

5

Histone Deacetylase

me

3’..pGpCp..5’

RPD3

5

me

5’..pCpGp..3’

MeCP2

Sin3

+

+

Deacetylation of ChromatinTranscriptional Silencing — X-chromosome Inactivation

  • 5meC CpG DNA modification is observed in repressed genes and inactivated X chromosomes

  • 5meC CpG-methylation is maintained after DNA replication by Maintenance Methylase action on hemi-methylated DNA

  • 5meC binds transcriptional repressor MeCP2 (MethylC-binding Protein-2)

  • MeCP2 binds Sin3 with RPD3 histone deacetylase

CpG methylation

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)

JHW


This slide series has been used for teaching to students of the montpellier university in 2004

Histone Methylation

C

C

C

C

C

C

Histones can be methylated at lysines or arginines. Example: H3 K9 methylation

N

Lysine

C

C

C

g

e

N+

a

d

b

C

C

C

O

S-adenosylmethyionine

HMT (Histone

Methyl-Transferase)

Histone

demethylase?

N

e-N-monomethyl-Lysine

N+

C

C

DNA backbone

binding may not be strongly affected, but specific proteins may recognize these modifications

C

e

C

C

C

O

S-adenosylmethyionine

HMT (Histone

Methyl-Transferase)

N

e-N-dimethyl-Lysine

N+

C

C

C

e

e

S-adenosylmethyionine

C

C

C

O

HMT (Histone

Methyl-Transferase)

N

N+

C

C

C

e-N-trimethyl-Lysine

e

e

C

C

C

O


This slide series has been used for teaching to students of the montpellier university in 2004

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


This slide series has been used for teaching to students of the montpellier university in 2004

Tri-; di-; mono-HMTases?

S. pombe to man

(Tri-met K27)

and tri- met K9

Enhancer of zeste

N. crassa

Multiple roles of H3 K9 methylation

Modified from:

Lachner and Jenuwein (2002). Current Opin. Cell Biol. 14, 286-98

In contrast to histone acetylation, histone methylation is STABLE. This makes of this mark a candidate for inheritance of chromatin states.


This slide series has been used for teaching to students of the montpellier university in 2004

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.


This slide series has been used for teaching to students of the montpellier university in 2004

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.


This slide series has been used for teaching to students of the montpellier university in 2004

Heterochromatin formation involves histone H3 lysine 9 methylation by Su(var)3-9 and recruitment of HP-1

Ac Ac

Open chromatin

H3

K9 K14

HDAC

H3

K9 K14

Methylation of K9 (Clr4; Suvar39)

Me

H3

K9 K14

Recruitment of Swi6 or HP-1

Swi6

Me

H3

K9 K14

Recruitment of more Clr4 (Suvar39) molecules by Swi6 (HP-1)

Condensed chromatin


This slide series has been used for teaching to students of the montpellier university in 2004

Epigenetic regulation of centromere function and RNAi

Outer repeats

Central domain

Outer repeats

S. Pombe Centromere

dsRNAs

Nucleosomes

Nucleosomes

K9 Me

K9 Me

Swi6

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:

K9 methylation, Swi 6 recruitment!

Nascent RNA

Ago (RISC), Clr4 Dcr complex

si RNA

See also: Grewal and Moazed (2003). Science 301, 798-802


This slide series has been used for teaching to students of the montpellier university in 2004

Opposing functions of H3 K9 versus K4 methylation

Lachner and Jenuwein. (2002) Current Opin. Cell Biol. 14, 286-98


This slide series has been used for teaching to students of the montpellier university in 2004

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


This slide series has been used for teaching to students of the montpellier university in 2004

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


This slide series has been used for teaching to students of the montpellier university in 2004

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


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