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DNA compactioncompaction in a human nucleus

1bp (0.3nm)

10,000 nm

30nm

11 nm

Compaction of DNA by histones Compaction by chromosome scaffold / nuclear matrix

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HISTONESare

highly conserved,small, basic proteins

H1

H2B

H2A

H3

H4

helix

variable

conserved

Histone acetylationis a reversible modificationof lysines in the N-terminiof the core histones. Result:• reduced binding to DNA• destabilization of chromatin

Linker histone

Core histones

N

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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)

The histone-fold

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Histone octamer assembly

H3-H4tetramer H2A-H2B

dimer

Histoneoctamer

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The Nucleosome as the fundamental chromatin unit

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• 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.

Nucleosome features

H2B H4H3H2A

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Chromatosome

5 mM

1 mM

Linker Histone and histone terminicontrol linker DNA entry/exit of

chromatosome in chromatin fiber.

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

H3

H1

N

C

H3

Zhou, Gerchman, Ramakrishnan, Travers,Muyldermans Nature 395, 402 (1998)

An, Leuba, van Holde, ZlatanovaPNAS 95, 3396 (1998)

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Chromatinfibers

+ charged N termini(bind DNA on neigboringnucleosomes)

highly acetylatedcore histones

(especially H3 and H4)

30 nmchromatin fiber

11 nm(beads)

• HIGH level of histone H1 • Reduced level of histone H1

• NO gene transcription • Gene transcription possible

Alternative chromatin fiber models

Zigzag fiber

Histone modifications

Cell (2002) 111, 285-91

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Acetylation of conserved lysines

H4 N-terminus

H3 N-terminus

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-+ + + + + ++ + + +

Ac or MeAcAcAcAc

5 8 12 16 20

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-

MeAc or Me

Ac Ac Ac Ac

++ + + + + + ++

4 9 14 18 23 27

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

Lysine

-N-Acetyl-Lysine

HAT (HistoneAcetyl-Transferase)

HistoneDeacetylase

reversible reactions

O

N

C

C C

C

C

CN+

P

OO

OP

O

-DNAbackbonebinding

no DNAbindingO

N

C

C C

C

C

C

N

O

C

C

Acetyl-CoA

CoAP-

--

--

---

--

--

--

--

--

-

-

-

-

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Acetylation of Chromatin Domains: analysis by the technique of Chromatin Immunoprecipitation (ChIP)

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

Example: the chicken -globin gene domain

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”

domainboundary

domainboundary

H A

0 10 20 30 kb

DNase I hyper-sensitive site

= -globin genes:

(Ac-Lys) antibody nucleosome ppt

DNA loop domain

General DNase I sensitivity

Control:inactive gene

chickenovalbumin

0 1 2 DNase I(U/ml)

DN

A r

em

ain

ing

DNase I

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Acetylation at Promoters leads to transcriptional activation

Thyroid Hormone Receptorexample of DNA-binding

Transcription Factor

TAF 250II

TBP

HAT

TATA

pol.II

HATGCN5

ACGGTC

ADA2ADA3

TranscriptionalACTIVATION

co-activator

HistoneAcetylTransferases

TH

ho

rmo

ne

+

HAT

P/CAF

p300CPB

HAT

TACCCG

TH

Hyper-acetylateddecondensed

Chromatin fiber

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TranscriptionalREPRESSION

co-repressor

TAF 250II

TBP

HAT

TATA

TACCCG

N-CoRSin3

RPD3

HistoneDeacetylase

Without Thyroid Hormone

Deacetylation at Promoters leads to Transcriptional repression

Hypo-acetylatedcondensed

Chromatin fiber

X

Acétylationdes histones

Chromatine décondensée:Etat transcriptionnel

actif

Déacetylationdes histones

Chromatine condensée:Etat transcriptionnel

réprimé

Acétylation vs déacetylation des histones

C C

Histone Methylation

Lysine

-N-monomethyl-Lysine

HMT (HistoneMethyl-Transferase)

Histonedemethylase

O

N

C

C C

C

C

CN+

O

N

C

C C

C

C

C

C

HMT (HistoneMethyl-Transferase)

S-adenosylmethyionine

O

N

C

C C

C

C

C

C

C

HMT (HistoneMethyl-Transferase)

O

N

C C

C

C

C

C

-N-dimethyl-Lysine

C-N-trimethyl-Lysine

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

N+

N+

N+

S-adenosylmethyionine

S-adenosylmethyionine

Histones can be methylated at lysines or arginines. Example: H3 K4 methylation

Histonedemethylase

Histonedemethylase

Histone demethylases found for K4, K9, K27 and K36

Histone methylation : Histone methyltransferases

Enhancer of Zeste H3 K27Enhancer of Zeste

Dm Trx, Ash1Mammalian MLL1-3

Euchromatin methylation, PcG-dependent gene silencing

Euchromatin methylation, gene activationtrxG-dependent gene silencing

Histone demethylases

2 classes:

LSD1

JmjC domain proteins

Histone demethylasesLSD1 demethylases target mono or dimethyl K4 and K9 of histone H3, depending

on their interacting cofactor

Histone demethylasesJmjC domain demethylases target many different lysines and are conserved

Histone methylationA reversible phenomenon with many dedicated components

Utx

JMJD3

Coffee break…