Handouts Chromatin Structure and Function in Transcription, - - PowerPoint PPT Presentation

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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 thoma@cell.biol.ethz.ch http://www.cell.biol.ethz.ch/research/thoma/

120224 1615 -1900

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• Handouts

Chromatin - Organization

Lodish et al 5th ed

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Nucleosome Filament Low Salt

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

• f

Chromatin Fibers (30nm) Physiological Chromosome Territories Heterochromatin Euchromatin Structures Foci & Factories Nuclear Compartments

Chromatin Controls DNA

dynamic dynamic

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Chromatin Structures

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Genome-wide patterns of histone modifications in yeast.

Millar, C.B., and Grunstein, M. (2006). Nat Rev Mol Cell Biol 7, 657-666.

Locus Specific Heterogeneity in Structure and Function

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Focus

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Chromatin Structures Aims Concepts - Facts – Fiction Terms, Keywords Key Experiments Approaches Methods "Feeling for Chromatin" Motivation to Read Chromatin Papers Transcription Replication Repair Recombination

www.tropechopf.ch

Chromatin Structure and Function

www.tropechopf.ch 8

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Chromatin Structure and Function

Chromatin ?

• Ready stainable material in nuclei, substance of chromosomes (W. Flemming, 1880)
• The packaged state of eukaryotic genomes (in nuclei and chromosomes)
• (Not correct: chromatin = nucleosomes)

Chromatin Definition and Composition

Definition Composition

• DNA
• Histone Proteins
• "linker histones" H1
• "core histones" H2A H2B H3 H4
• "histone variants" with specific functions
• Non-Histone-Chromosomal Proteins (NHCPs)
• with various structural and functional roles
• RNA
• nascent RNAs during transcription
• structural RNAs (involved in silencing)
• DNA : histones = 1 : 1 (w/w); NHCP, RNA variable
• H1 : H2A : H2B : H3: H4 = 1 : 2 : 2 : 2 : 2

Stoichiometry

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Heterogeneity in structure and composition

• Locus dependent
• Time dependent (µs – minutes – hours – years – generations)

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Chromatin Structure and Function

When and where is chromatin made?

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• chromatin

CdkC = Cyclin dependent kinase- Complex = heterodimer Cdk+cyclin APC = Anaphase Promoting Complex Lodish 13-02

Replication of Chromatin

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• Genome – Organization
• Lodish et al 5th ed

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• Genome - Organization
• Lodish et al 5th ed

How are genomes packaged in nuclei & metaphase chromosomes

• to allow precise and accurate
• expression of genes
• douplication and segregation of genomes
• repair of DNA damage
• ?
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• Chromatin

is Complex Fascinating Multidisciplinary

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• Lodish 5e-1-12

Chromatin Structure and Function

lampbrush chromosome polytene chromosomes metaphase chromosomes interphase chromosomes transcription foci "factories" replication foci repair foci repair foci silencing phenotype FISH FISH Immunofluorescence EM Immuno fluorescence Immuno fluorescence Immuno fluorescence Immuno fluorescence DAPI

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Chromatin Structure and Function

biophysics biochemistry (reverse-) genetics cell biology systems biology disease molecular biology Molecular Life Science in vivo genomics epi-genomics in vitro cancer

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• T

B P TFI IB RNA Polymerase II

Basic Chromatin Concepts Enzymes Multi- Protein Complexes Protein – Protein Interactions Protein – DNA Interactions RNA – DNA Interactions RNA – Protein Interactions Proteins Protein Modifications DNA Modifications Interactions Protection DNA Structures Accessibility Precisely Coordinated in Time and Space Assembly - Disassembly Structures Turnover Synthesis - Degradation

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• Chromatin
• Felsenfeld, G. and Groudine, M. (2003) Nature 421, 448-453.

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• DNA

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• Nature 421, 421-422.

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• Remember DNA ?

Denaturation ("melting") Renaturation, Hybridization Temp < Tm Formamide Urea

In vivo:

• Helicases
• Transcription
• Replication
• Recombination

(homologous)

• DNA Repair

In vitro:

• PCR
• Hybridizations
• Identification of DNA

sequences with probes

• FISH (fluoresence in situ

hybridization)

„Normal“- B-Form DNA

5' 3' 3' 5'

negatively charged

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• Different Forms of Double Stranded DNA
• 3'

5' 5' 3'

http://www.fli-leibniz.de/IMAGE_DNA_MODELS.html FT Fribourg12F 23

• DNA-Flexibility and DNA-Bending
• Loops of Double

Stranded DNA

• DNA structure

containing a junction between left-handed Z-DNA and right-handed B-DNA. Ha et al. Nature 437, 1183-6 (2005).

The solution structure and dynamics of the complex of a dimeric lac repressor DNA binding domain with nonspecific DNA. The same set of residues can switch roles from a purely electrostatic interaction with the DNA backbone in the nonspecific complex to a highly specific binding mode with the base pairs of the cognate

• perator sequence. The protein-DNA interface of the nonspecific complex is flexible on biologically

relevant time scales that may assist in the rapid and efficient finding of the target site. Kalodimos et al.(2004). Science 305, 386-389.

• Chromatin Structures

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Chromatin Structure and Function

Packaging Genomes How much DNA?

Packaging Genomes Alberts A8-6

• 1 bp =1.077*10-9pg
• 1pg
• 10pg
• 100pg
• 0.1 pg
• 10-2pg
• 10-3pg
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• (Ball 2003, Nature)

Packaging Genomes Space Questions Volume of the DNA? Volume between chromatin ("interchromatin" space")? Volume of the chromatin?

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• How many chromosomes per cell?
• Isolation of

Metaphase Chromosomes

Replication Segregation Interphase decondensed chromosomes Metaphase condensed chromosomes Colchicine (colcemid) binds tubulin and prevents their polymeri- zation

Lodish 5e-1-12

Karyotype: Number, sizes, and shapes of the entire set of metaphase chromosomes of a eukaryotic cell Method: Chromosome painting by FISH

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• Genetic Map - „Linkage“ Map“

Meiotic recombination between homologous chromosomes The closer two loci, the lower the frequency of recombination Map Unit (m.u.): The distance between two linked gene pairs where 1 percent of the products of meiosis are recombinant = a unit of distance in a linking map. (also known as centi-Morgan, cM).

Lodish 5e-9-45

How many chromosomes per cell? Genetic Approach

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• 1. Cells were imbedded in

agarose

• 2. Digested with Zymolyase,

protease, RNAse 3.Pulsed-Field-Agarose- Electrophoresis

• 4. Gel stained with Ethidium

Bromide

Yeast chromosomes contain one linear ds DNA The DNA of yeast chromosomes can be physically separated by electrophoresis 16 bands correspond to 16 linkage groups Baker's yeast Saccharomyces cerevisiae 16 linkage groups Small genome Small chromosomes Karyotyping is not possible How Many DNA Molecules per Eukaryotic Chromosome?

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• Nuclei

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• Metaphase

DAPI-Staining Metaphase FISH FISH Color Set Interphase Chromosome Territories Chicken Chromosomes and Nuclei Chromosome-Painting by Multicolor FISH (Fluorescence In Situ Hybridization) Cremer, T. and Cremer, C. (2001) Nat Rev Genet, 2, 292-301.

Chromosome Territories

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• Localization of genes (DNA sequences) on chromosomes of higher eukaryotes
• Metaphase Chromosome

Interphase Nucleus

• 1. Immoblize and fix on cover slip
• 2. Denature DNA (high pH)
• 3. Hybridize with fluorescently labeled probes or

detect probes with fluorescent antibodies against probes

• 4. Stain DNA unspecifically with DAPI
• 5. Detect probes by fluoresence microscopie

FISH (Fluorescence-In-Situ-Hybridisierung)

A7-18/19FISH Multicolor FISH

(not shown)

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• 1.

Denature DNA (temperature above 95oC) 2. Anneal (hybridize) primers (1,2) (below Tm, melting temperature) 3. Elongate with DNA-Polymerase (e.g. Taq-polymerase) 4. Repeat steps 1 to 3

Making probes by incorporation of labelled nucleotides during DNA-synthesis

• DNA with Your Favorite Sequence

5' 5' Amplified DNA- template for generation of probes 1. Denature DNA (temperature above 95oC) 2. Anneal (hybridize) primers (below Tm, melting temperature) 3. Elongate with DNA-Polymerase and 32P-α-NTPs or modified nucleotides (Digoxygenin) 5' 5' 5' 5' 1 2 Primer 1: Probe to detect the top strand Primer 2: Probe to detect the top strand Top Strand Bottom Strand Top Strand Bottom Strand

A 7- 1 8

Digoxygenin

(not shown)

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• Nuclear Compartments

Hemmerich et al. (2010). Chromosome Res.

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• Nuclear Compartments

Hemmerich et al. (2010). Chromosome Res.

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• Chromatin Dynamics

Dynamic as well as stable protein interactions contribute to genome function and maintenance

• Hemmerich et al. (2010). Chromosome Res.

(Im)mobilities in the nucleus. Illustration of subnuclear compartments and

• verview of their

component‘s residence times (tres) determined by FRAP

Time Scale sec <> min <> hours

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• Chromatin Dynamics

Dynamic organization of the cell nucleus: macromolecular crowding.

• Rippe, K. (2007). Current opinion in genetics

& development 17, 373-380. The association of a particle into a heptameric complex in the nucleus environment is depicted schematically. The process is reversible and a fast exchange between free subunits in the nucleoplasm and in the complex state

• exists. The high concentration of nucleic acid ("chromosome territories") and protein components

reduces the accessible volume (‘macromolecular crowding’) so that the effective subunit concentration is increased, which favors association. If a complex is formed the volume excluded to the abundant

small particles is also increased. This represents a favorable entropic ‘depletion attraction’ force. Finally, the displacement of water from hydrophobic surfaces of the protein is also driving the association into a dynamic complex. Subcompartment assembly in the nucleus. Macromolecular Crowding due to volume exclusion by chromatin and other macromolecules

• Proteins move by 3D

diffusion

• Reduced accessible

volume (excluded volume) enhances concentration and favours interactions between proteins and/

• r DNA

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• Chromatin Dynamics

Mobility and immobility of chromatin in transcription and genome stability.

• Soutoglou, E., and Misteli, T. (2007). Current
• pinion in genetics & development 17,

435-442.

Time Scale sec <> min <> hours <> days

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Chromatin Structure and Function

Packaging Genomes

Histones

Do eukaryotic cells need histones? How many histones?

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• Histones

Sea urchins package sperm DNA in nucleosomes using histone H2B and H1 variants. These histones are proteolysed during pronuclear decondensation and replaced with maternal cleavage-stage histones

Talbert and Henikoff (2010) Nat Rev Mol Cell Biol.

One major function of both sperm-specific histones and protamines might be to erase paternal epigenetic states. Nucleosomes that survive chromatin remodelling in the sperm and pronucleus have the potential to maintain epigenetic information at particular loci, with CENPA being the most notable example. Animal sperm DNA is tightly packaged with basic proteins such as variant histones and protamines (small, highly basic proteins). Human sperm: Nucleosome-bound DNA content is estimated at only 4–15%, and sperm contains all four canonical histones plus H2A.X, H2A.Z, H3.3 and cenH3

Histone Genetics

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Gene Cluster H1 H4 H2B H3 H2A 300 -600x 6kb P.miliaris (Seeigel) Spacer (not transcribed) Genes Chicken 1 cluster 39 histone genes Mouse 3 clusters 45 histone genes Humans 2 clusters 52 histone genes Ahn and Gruen (1999) 4.8 kb Günesdogan (2010) H1 H2B H2A H4 H3 23 x Drosophila melanogaster

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Histone Genes high copy number of histone genes prevents reverse genetics approach Yeast S. cerevisiae has only few genes coding for histones. Therefore, histones can be studied by manipulation of histone genes

HH01 a histone H1 like protein Chromosome XVI HH01 Role in recombination, no known effect on chromatin structure H2AZ (HTZ1) - a histone H2A variant Chromosome XV HTZ1 Synthesized independent of S-phase. Exchanged for histone H2A in nucleosomes by the SWR1 chromatin remodelling complex. Localized in promoter nucleosomes of regulated promotors Prevents spreading of silent heterochromatin Chromosome II Chromosome XIV H3 (HHT1, HHT2) and H4 (HHF1, HHF2) HHT1 and HHT2 code for identical proteins HHF1 and HHF2 code for identical proteins Chromosome IV Chromosome II H2A (HTA1, HTA2) and H2B (HTB1, HTB2) HTA1 and HTA2 code for nearly identical proteins HTB1 and HTB2 code for nearly identical proteins Core Histones standard histones of nucleosomes synthesized in S-phase

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Yeast Histone Genes and Synthesis

Gene Disruption / Repacement in Yeast S. cerevisiae

Haploid cell

Lodish 5th 9-37

Gene Disruption Construct If the disrupted gene is essential, the cells will die

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• Dito: one H2A, H3, H4 is required: Yeast needs at least one copy of each histone

However deletion of subtypes affects specific functions, e.g. mating type switching, sporulation…..

Are Histone Genes and Variants Essential?

Kolodrubetz etal (1982). PNAS 79, 7814; Rykowski et al. (1981).Cell 25, 477

Systematic manipulation of histone genes in yeast S.cerevisiae (by M. Grunstein, M. Osley et al.) H2A.2 H2B.2 H2B.1 H2A.1 H4.1 H3.1 H3.2 H4.2 Knock out

Δ

H2A.2 H2B.2 H2B.1 H2A.1 H4.1 H3.1 H3.2 H4.2 alive with one H2B.2 H2A.2 H2B.2 H2B.1 H2A.1 H4.1 H3.1 H3.2 H4.2 alive with one H2B.1 H2A.2 H2B.2 H2B.1 H2A.1 H4.1 H3.1 H3.2 H4.2 dead with no H2B One copy of H2B is required for survival.

Δ Δ Δ Δ

H2A.2 H2B.2 H2B.1 H2A.1 H4.1 H3.1 H3.2 H4.2 alive One H2A,H2B, H3, H4 is sufficient for survival under laboratory conditions

Δ Δ Δ Δ

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• How many histones does the cell need?

How important is the stoichiometry? Approach? Overexpression of histone genes Imbalanced expression of histone genes

Histones

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• H2A.

2 H2B. 2 H2B. 1 H2A. 1 H4. 1 H3. 1 H3. 2 H4. 2 YEp-Plasmids (high copy number vectors; 30-50 copies / cell) URA3

Overexpression of histone genes in yeast S.cerevisiae

Genes in YEp: Chromosome loss YEpVector 10-5 normal H2A-H2B 10-3 high H3-H4 10-3 high H2A-H2B-H3-H4 10-5 normal Normal stoichiometry of histone dimer sets is necessary for high fidelity of mitotic chromosome transmission.

• Overexpression of histones using high copy number vectors (gene dosage) in a diploid yeast

strain

• Loss of chromosomes was recorded as a change in colony colors

Meeks-Wagner and Hartwell(1986) Cell

Roles of histones What happens if you remove histones ("histone depletion") ?

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How to deplete histones in a living cell ?

DAPI Anti-Tubulin 6 hours after glucose Shift of yeast cells to glucose media

• Cell cycle arrest after one generations
• Cells can't segregate chromosomes possibly due

to lack of packaging of DNA

• Activation of some promoters due to the loss of

histones (nucleosomes)

• Activation of cryptic promoters
• Conclusion: Repressive role of histones in

transcription initiation

Conditional histone depletion in yeast S.cerevisiae

Han et al. (1987) Cell 48, 589; Kim et al. (1988). EMBO J 7, 2211-2219. H2A.2 H2B.2 H2B.1 H2A.1 H4.1 H3.1 H3.2 H4.2 Knock out of H4 genes in genome

Δ Δ

Yeast cells in galactose media: H4.1 is expressed from plasmid Normal growth in galactose YCp = Yeast centromere vector for extrachromosomal maintenance; about 1 copy/cell URA3 GAL-H4.2 H4.2 gene fused to GAL1 promoter: transcribed in galactose, repressed in glucose = "conditional expression"

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H4.1 H3.1 H3.2 H4.2

Δ Δ

URA3 H4.1

How to make a histone mutant in yeast S.cerevisiae

HIS3 h4-mutant

• 1. Generate mutant in vitro
• 2. Transform

HIS3 h4-mutant H4.1 H3.1 H3.2 H4.2

Δ Δ

• 3. Select on media containing 5-FOA

(fluoro-orotic-acid). Yeast cells expressing URA3 will be killed. Cells that loose the URA3 plasmid can survive, if the h4-mutant is not lethal. HIS3 h4-mutant

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• S. cerevisiae histone H2 (HTA1 and HTA2) belong to H2A.X variants

DNA-damage (e.g. by MMS) dependent phosphorylation Survival on agar plates S129* = C-terminal deletion, S129A = serine to alanine, S129T = serine to threonine, S129E = serine to glutamic acid,

H2A.X Variant : a role of S. cerevisiae H2A in DNA-repair

(Downs et al. (00)Nature 408, 1001)

S129E is resistant to MMS, mimics phosphorylation hta1-mutants with enhanced sensitivity to MMS Mec1: a protein involved in damage checkpoint response

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• Chromatin Dynamics

How and how tighty are histones bound to DNA ? How and how tighty NHCPs bound to DNA ?

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