chromatin structure and function in transcription ... · chromatin structure of the his3 gene by...

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Chromatin Structure and Function in

Transcription, Replication, Repair

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Chromatin Structure and Function

Fritz Thoma Institute of Molecular Health Science

(previous Institute of Cell Biology) ETH-Zürich

Hönggerberg HPM-E42 +41-44-6333323

[email protected] http://www.cell.biol.ethz.ch/research/thoma/

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Chromatin Dynamics

Epi-Genome Stability & Maintenance

Transcription Control Regions & Hypersensitive Sites

Promoters Elongation

Nucleosome Positioning

Remodeling Histone modifications & "Histone code"

ATP-dependent remodeling Histone exchange

Nucleosome dynamics

Assembly Replication

Recombination Repair

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low phosphate (active)

2. Pho4 recruits SAGA complex (containing Histone Acetyl Transferase Gcn5) 3. Gcn5p acetylates promoter region

TATA

SAGA

Gcn5

4. Gcn5p (bromodomain, binds acetylated histones) and recruits/stabilizes binding of SWI/SNF to newly hyperacetylated histones

TATA

SWI/SNFSAGA

Gcn5

5. SWI/SNF uses ATP hydrolysis to remodel promoter nucleosomes. 6. Histones are evicted (lost). 7. RNAPII and GTF bind promoter and initiate transcription TATA

Syntichaki et al. (2000) Nature, 404, 414) Barbaric (2001) Embo J, 20, 4944-4951.

1. Transcription activator Pho4 binds UASp1 in linker between nucleosomes

TATA

Pho4

high phosphate (inactve)

TATA

UASp1 UASp2

3

PHO5: "Classic Example" of Chromatin Remodeling in a Promoter (II) Chromatin Controls DNA

Nucleosome Dynamics (time dependent changes in structure and/or

composition)

What determines DNA accessibility? How can proteins access binding sites in nucleosomes?

TATA

linker-DNA nucleosome surface

Luger et al (1997) Nature 389:251

Nucleosome Structure Steric hindrance by histones & histone tails

Steric hindrance by DNA Histone modifications might promote or prevent

interactions Nucleosome Positions (position of histone octamer on the DNA

sequence)

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5 bp


Illustration of how nucleosome occupancy and positioning differ.

The upper panel shows a cross-section of a nucleosome, in which occupancy is distinguished from positioning. The lower panel shows how the two are measured. Occupancy is the area under the curve and reflects the local density of nucleosomes in a population, as illustrated by the column of spheres. Positioningor fuzziness is reflected in the standard deviation of the curve and is illustrated by how well the spheres are aligned in a column. The position of a nucleosome relative to some standard is indicated by how closely two peaks are separated. Comparing peaks of curves having high standard deviations is not likely to be meaningful because both peak locations have very high uncertainty..

Locus Specific Heterogeneity: nucleosome occupancy and positioning

Pugh, B.F. (2010). A preoccupied position on nucleosomes. Nature structural & molecular biology 17, 923


High-Resolution Genome-wide Mapping of Nucleosomes Zhang and Pugh (2011). Cell 144, 175-186.

DNA sequencing Arrays


High-Resolution Genome-wide Mapping of Nucleosomes Zhang and Pugh (2011). Cell 144, 175-186.

Genome Wide Studies of Nucleosomes by MNase Digestion and Sequencing

HIS3

PET

DED

UNF

ARS1

TRP1

5'3'

5'5'

5'

MNase DNA CHR

Suter et al. (1997) Embo J

Chromatin Structure of the HIS3 Gene by MNase Digestion and Indirect Endlabelling

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Lee et al. (2007) Nature genetics 39

1) Lee et al. (2007). 2) Yuan (2005)

1) 2) 1) 2)

HIS3 Gene 5 positioned nucleosomes NFRs (nucleosome free regions) at 5' and 3'end

Nucleosome Positions

Decreasing Positioning

In its purest form, statistical positioning relies on a single positional barrier (left side), against which nucleosomes are ordered. A probabilistic density trace of where nucleosomes would reside in a population is shown.

Kornberg, R.D., and Stryer, L. (1988).


Mechanisms of Nucleosome Positioning

DNA sequence: •  bendability•  flexibility

Proteins:•  direct contact•  indirect contact

Boundaries:•  exclusion by

sequence•  exclusion by proteins

Boundaries

BoundariesBoundaries Limited space between two boundaries restricts randomization and favours postitioning. (e.g. URA3 and HIS3 gene with 6 and 5 nucleosomes, respectively, between 5' and 3' nucleosome free regions.)


Nucleosome Positioning Mechanisms & DNA Accessibility

unwrapping

bulge diffusion

twist defect

twist diffusion

dissociation

reassembly

Structural and dynamic properties are affected by

Histone variants

Histone modifications

Remodeling complexes

NHCPs interacting with chromatin

DNA-damage

Cha

nge

of P

ositi

on "

Nuc

eoso

me

Mob

ility

"

DNA

H2A H2B

H2A H2B

H3H4 H3H4

"Binding Site Protected"

"Binding Site Exposed"

H1


Chromatin Dynamics




Nucleosome Positioning



Nucleosome dynamics

Assembly Replication


Histone Post Translational Modifications (PTMs) Felsenfeld, G., and Groudine, M. (2003). Nature 421, 448, Kouzarides, T. (2007). Cell 128, 693.

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Many amino acids of histones in the 'tails' and on the nuceosome surface are chemically modified.

Crosstalk between histone modifications: a modification facilitates / inhibits modification at an other site on the same or different histones /nucleosomes

Histone modifications may alter the "intrinsic properties of nucleosomes and higher order structures

Histone modifications constitute a set of markers (PTMs) of the local state of the genetic material, which has been called the 'histone code' (Strahl and Allis , 2000).

Histone modification is a dynamic and reversible process.

Histone modifications are binding sites for proteins (recruit NHCPs) Their presence on histones can dictate the higher-order chromatin structure in which DNA is packaged and can orchestrate the ordered recruitment of enzyme complexes to manipulate DNA

Modification and demodification are done by enzymes included in (large) complexes

120316 FT Fribourg 13Kouzarides, T. (2007)Cell, 128, 693

Modification

De-Modification

M

(Large) Enzyme-Complexes found for (almost) all reactions

Reversible, Transient, Dynamic

Posttranslational chemical modifications of histones (PTM)

L Y S

N H 3 +

L Y S

N H - C O - C H 3 Acetyl-CoA

HAT (Histone Acetyl Transferase)

HDAC (Histone Deacetylase) Inhibitors: butyrate

Acetylation of lysine residues neutralizes positive charge

Loss of charges may destabilize nucleosomes or higher order chromatin structures

Histone Modifications: Acetylation


Ac Ac Ac

Ac Ac Ac

AC

120316 FT Fribourg 15Kouzarides, T. (2007)Cell, 128, 693

Post Translational Modifications: Recruitment & Binding of Proteins

Specialized chromatin stuctures containing heterochromatin specific sets of histone modifications and heterochromatin specific proteins (e.g. HP1)


Histone Modifications (PTM): Crosstalk Felsenfeld, G., and Groudine, M. (2003). Nature 421, 448, Kouzarides, T. (2007). Cell 128, 693.

.

Crosstalk between histone modifications: a modification facilitates / inhibits modification at an other site on the same or different histones /nucleosomes

Modes of multivalent chromatin engagement. To distinguish among several potential mechanisms of multivalent association, we propose the following nomenclature.

a | Intranucleosomal association can be subdivided into two distinct classes124: cis-histone, when more than one discrete binding contact is made to a single histone, in particular the same tail; and trans-histone, whereby contacts are made to different histone protomers or attendant DNA within the same nucleosome.

b | By contrast, internucleosomal binding modes crosslink two nucleosomes that are either adjacent or discontinuous in DNA sequence. Most of these crucial interactions are envisioned as modification dependent; however, DNA interactions and modification-independent contacts may have a vital energetic role.

BPTF, bromodomain PHD finger transcription factor; HP1, heterochromatin protein-1; TAF1, TATA-binding protein-associated factor-1.

Ruthenburg et al. (2007). Nat Rev Mol Cell Biol 8, 983-994.

Post Translational Modifications: Recruitment & Binding of Proteins

Crosstalk in Reading PTMs

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Crosstalk in Chromatin "Writing & Reading"

Transcription is controlled by various distant and proximal control elements ("enhancers") that recruit factors for activation/repression

DNA

Nucleosomes

Chromatin Fibers

Loops / Domains

MetaphaseChromosome

Euchromatin

Hetero-chromatin

Few bp

one supercoil80 bp

6-8 nucleosomesabout 1 to 2 kb

one loop5 - 50 kb (?)

>>> Mb (?)

Distance between twobinding sites

Chromatin folding brings distant DNA sites, nucleosomes, histones ... into close spacial proximity.

Chromatin structures might control modification ("writing"), recruitment and interactions ("reading") of NHCP (accessibility & crosstalk).

Modifications and/or recruited NHCPs might affect stability of structures

Clapier, C.R. and Cairns, B.R. (2009) Annu Rev Biochem, 78, 273-304.

ATP-dependent Nucleosome Remodelling

DNA Binding Protein


Nucleosome Positioning Mechanisms & DNA Accessibility

unwrapping

bulge diffusion

twist defect

twist diffusion

dissociation

reassembly

Structural and dynamic properties are affected by

Histone variants

Histone modifications

Remodeling complexes

NHCPs interacting with chromatin

DNA-damage

Cha

nge

of P

ositi

on "

Nuc

eoso

me

Mob

ility

"

DNA

H2A H2B

H2A H2B

H3H4 H3H4

"Binding Site Protected"

"Binding Site Exposed"

H1


Chromatin Dynamics




Gene Moblility and Transcriptional Memory



Nucleosome dynamics

Assembly & Maintenance Replication & Inheritance



Replication Genetics •  Douplication and segregation of DNA •  Minimizing error rates

Epigenetics •  Disruption of existing chromatin structures •  Reassemby of chromatin structures with old and new components •  Regeneration of epigenetic modifications marks Initiation •  ORIs (origins of replication) •  Controls, timing (early, late), once / cell cycle Elongation •  Bidirectional •  Leading- / lagging strands DNA synthesis •  Chromatin replication: "new & old" proteins, histones, NHCPs, specialized

structures, modification patterns Termination


Nucleosomes reassociate on the newly replicated DNA 225 to 285 ( +/- 120) nucleotides behind the replication fork (< seconds).

The 'old' and 'new' histones form nucleosomes within seconds on both chromatids

Structure of replicating SV 40 minichromosomes. Sogo et al. (1986). J.Mol.Biol. 189, 189.

nucleosome

trimethyl-psoralen crosslinking

DNA purification

Electron microscopy under denaturation conditions

Method

Nucleosome Assembly At the Replication Fork In Vivo In Human Cells


Parental Chromatin

H3H4

PTM H3-K9Me3 H4-K16ac

Assembly old and new mixed on both strands

CAF-1 cohesin

Deacetylation of H3-K56Ac by Hst3/Hst4 Methylation and Acetyl of H3-K9Me3; H4-K16ac

MINUTES - HOURS - G2/M

Maturation PTMs, NHCPs, Nucleosome Positioning

cohesin

SECONDS

PCNA

HAT? MCM

Disruption

Parental Histones

H3H4-ASF1

H1

H2AH2B-FACT

H3H4-ASF1

H2AH2B-NAP

NHCP H1

Synthesis of new histones and non histone chromosomal proteins in the cytoplasm

H4-K5acK12ac H3-K56Ac

H4 H3 (H3.1, H3.2)

PTMs B-type HATs

Hat1 Hat2 Rtt9

Cytoplasm

Nucleus

H1 H2A, H2B


Repair of Double Strand Breaks (DSB) by Homologous Recombination (I)

Cairns (2004) Cell

DNA ends are bound by yKu and MRX complex (Mre11/Rad50/Xrs2)

The checkpoint kinases (Mec1 and Tel1) are recruited and phosphorylate H2A

A histone acetyltransferase (NuA4) acetylates histones of the region.

Chromatin remodelers are recruited (INO80 and SWR1). INO80 may facilitate removal of nucleosomes.

SWR1 may cause exchange of histone H2A with the variant H2AZ (Htz1)

yKu and MRX (Mre11/Rad50/Xrs2

Take-home message (I): The first steps of HR require chromatin remodeling, histone modifications, nucleosome disruption and histone exchange

over long distances Lisby and Rothstein (2005) Biochimie

DSB

Resection

Strand invasion DNA synthisis

Resolvation


Repair of Double Strand Breaks by Homologous Recombination (II)

Linger and Tyler (2007) Mutat Res.

Take-home message (II): The later steps of HR require chromatin assembly and

remodelling similar to replication and over long distances. The impact on the fate of epigenetic marks is unknown.

Lisby and Rothstein (2005) Biochimie

DSB

Resection

Strand invasion DNA synthisis

Resolvation


Nucleotide excision repair in mammals and yeast

Hanawalt, P.C. and Spivak, G. (2008) Nat Rev Mol Cell Biol, 9, 958-970.

Rad26 Rad2

Rad1-Rad10

Rad4-Rad23 Rad28?

Rad2

Rad14 RFA

Rad2

Rad14

Dst1

TFIIH

Rad7-Rad16

Transcription Coupled Repair (TCR)

Damage Recognition by

RNAP II stalled at the damage

Excision of about 30 nucleotides

containing the damage

DNA repair synthesis by replication enzymes

Global Genome Repair (GGR)

Damage Recognition by

Recognition Factors

3' 5'

3' 5'

RepairDNA-Synthesis

ChromatinRegeneration

5.

CAC?CAF1?

4.ReplicationFactors

Excision3. XPG

XPFERCC1

Rad2

Rad1Rad10

DNA-DamageRecognition

1.

ChromatinRemodeling

2.

Open ComplexFormationDamageVerification

Human: Yeast:

XPChHR23B

Rad4Rad23

Rad7Rad16

DDB?

??

RemodelingComplexes ?

XPARPA

TFIIH(XPD,XPB)

Rad14Rfa

TFIIH(Rad3,Rad25)

Nucleotide Excision Repair

NER9.99-2

Thoma, F. (1999) Embo J, 18, 6585

Nucleotide Excision Repair in Chromatin

m m m m m m

m m m m m

m = PTM of histone


•  Structural and dynamic properties of chromatin modulate accessibility of DNA for damage recognition •  UV induced acetylation of histones by Histone Acetyl Transferase (Gcn5?) (random or targeted at the damage site is unknown) may destabilize chromatin •  Recruitment of ATP-remodeling complex (Swi/Snf) may disrupt /shift nucleosomes to facilitate repair

•  NER excision reaction in vitro requires minimal substrate size of 100 bp. •  Repair patches can be labelled by incorporationa of BrdU or 3H-T (= "UDS, Unscheduled DNA Synthesis") •  DNA synthesis might shift of nucleosomes or evict histones around the damage site. How far is unknown.

CAFI

Access

Repair

Restore

Access

Repair

Restore

HAT

Swi/Snf

Take-home message: The size of the disrupted and restored

chromatin region as well as the impact on restoration of epigenetic marks is unknown.

repair patches

•  Early repair patches are nuclease sensitive and get slowly nuclease resistant due to incorporation in nucleosomes (Smerdon 1978) = chromatin rearrangement after repair. • Rearrangement may occur by repositioning of displaced nucleosomes (sliding back) and/or by reassembly through recycling or deposition of new histones with the help of chromatin assembly factors (CAF) •  Reestablishment of epigenetic marks (M) is unknown

Nucleosome Filament

Low Salt

H2BH4H2A

H2A

H3

H2B

H2B

Histones: H1

2x(H2A, H2B, H3, H4)

RNAP

Transcription

Replication

Recombination DNA-Repair

Mechanisms

Promoters

Origins of Replication

Centromeres

Telomeres

Gene Control Regions

Specialized Chromatin

Non-Histone-Chromosomal

Proteins

Remodelling Complexes

Histone-Variants Histone-Modifications

„Histone Code“

Structural and Functional Heterogeneity of Chromatin

Loops of Chromatin Fibers (30nm)

Physiological

Chromosome Territories

Heterochromatin

Euchromatin

Structures

Foci & Factories

Nuclear Compartments

Chromatin Controls DNA

dynamic

dynamic


Epigenetics

Epigenetic processes play a critical role in creating stable patterns of gene expression during normal growth and differentiation.


Chromatin & Epigenetics Kirchmaier, A.L. and Rine, J. (2006)Mol Cell Biol, 26, 852

Epigenetic processes regulate gene expression through heritable

chromatin structures, creating distinctly different states of gene expression in genetically identical cells.

Creating epigenetic influences on gene expression has three requirements:

(i) the assembly of a specialized chromatin structure at a locus or loci,

(ii) the maintenance of that structure throughout the cell cycle, and

(iii) the ability of that structure to template its own replication, akin to the ability of complementary strands of DNA to template their replication.

chromatin structure and function in transcription ... · chromatin structure of the his3 gene by...

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