Structure and Function

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Chromatin Structure and Function Handouts in Transcription, Replication, Repair 120224 1615 -1900

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/ FT Fribourg12F 2 FT Fribourg12F 1

Chromatin - Organization Structural and Functional Heterogeneity of Chromatin

Structures Mechanisms dynamic Nuclear Compartments Transcription Foci & Factories Replication Territories Recombination Heterochromatin DNA-Repair Chromatin Controls Specialized Chromatin Loops DNA Gene Control Regions of Chromatin Promoters Fibers (30nm) Origins of Replication Physiological Centromeres Telomeres dynamic Remodelling Complexes Low Salt H2B H2A

H2B

Nucleosome H3 Filament RNAP H2A H4 H2B

Histone-Variants Non-- : Histone-Modifications Chromosomal H1 „Histone Code“ Proteins 2x(H2A, H2B, H3, H4)

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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.

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Chromatin Structure and Function Chromatin Structure and Function

Focus Chromatin ? Chromatin Structures

Transcription

Replication Repair Recombination

Aims

Concepts - Facts – Fiction Terms, Keywords Key Experiments Approaches Methods

"Feeling for Chromatin" Motivation to Read Chromatin Papers www.tropechopf.ch www.tropechopf.ch

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Definition When and where is chromatin made? • Ready stainable material in nuclei, substance of (W. Flemming, 1880) • The packaged state of eukaryotic genomes (in nuclei and chromosomes) • (Not correct: chromatin = nucleosomes) 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) Stoichiometry • DNA : histones = 1 : 1 (w/w); NHCP, RNA variable • H1 : H2A : H2B : H3: H4 = 1 : 2 : 2 : 2 : 2 Heterogeneity in structure and composition • Locus dependent • Time dependent (µs – minutes – hours – years – generations) FT Fribourg12F 9 FT Fribourg12F 10

Replication of Chromatin Genome – Organization

CdkC = Cyclin dependent kinase- Complex = heterodimer Cdk+cyclin

APC = Anaphase Promoting Complex

chromatin Lodish 13-02

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How are genomes packaged in nuclei & metaphase Chromatin chromosomes is to allow precise and accurate Complex expression of genes Fascinating douplication and segregation of genomes Multidisciplinary repair of DNA damage ?

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Chromatin Structure and Function Chromatin Structure and Function

cell biology Molecular Life Science lampbrush chromosome Immunofluorescence disease FISH polytene chromosomes in vivo

metaphase in vitro cancer chromosomes silencing EM interphase  phenotype chromosomes genomics FISH biochemistry (reverse-) biophysics

molecular biology epi-genomics Immuno repair foci fluorescence replication foci DAPI systems biology Immuno fluorescence Immuno fluorescence Immuno repair foci transcription foci fluorescence Lodish"factories" 5e-1-12  Lodish 5e-1-12 FT Fribourg12F 15 FT Fribourg12F 16 Basic Chromatin Concepts Chromatin

Precisely Coordinated in Time and Space Protection Enzymes Assembly - Disassembly Accessibility Interactions Multi- Protein Proteins Turnover Felsenfeld, G. and Groudine, M. (2003) Nature 421, 448-453. T Complexes B Synthesis - Degradation TFI P IB Protein Modifications

RNA Polymerase II Protein – DNA DNA Structures RNA – Protein Interactions Interactions Structures Protein – Protein Interactions DNA Modifications RNA – DNA Interactions

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DNA Nature 421, 421-422.

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„Normal“- B-Form DNA In vitro: In vivo: 3' 5' • PCR • Helicases 3' 5' • Hybridizations • Transcription • Identification of DNA • Replication sequences with probes • Recombination 5' • FISH (fluoresence in situ (homologous) 3' hybridization) • DNA Repair

3' 5' negatively charged

Denaturation ("melting") Renaturation, Hybridization

Formamide Temp Urea < Tm

http://www.fli-leibniz.de/IMAGE_DNA_MODELS.html

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DNA-Flexibility and DNA-Bending Chromatin Structure and Function

DNA structure Loops of Double containing a Stranded DNA junction between left-handed Z-DNA and right-handed B-DNA. Chromatin Structures Ha et al. Nature 437, 1183-6 (2005). Packaging Genomes

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 operator sequence. The protein-DNA interface of the nonspecific complex is flexible on biologically How much DNA? relevant time scales that may assist in the rapid and efficient finding of the target site. Kalodimos et al.(2004). Science 305, 386-389.

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Space Questions Volume of the DNA? Volume of the chromatin? Alberts A8-6 Volume between chromatin ("interchromatin" space")? (Ball 2003, Nature)

1 bp =1.077*10-9pg

10-3pg 10-2pg 0.1 pg 1pg 10pg 100pg

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How many chromosomes per cell? How many chromosomes per cell? Genetic Approach

Isolation of Genetic Map - „Linkage“ Map“ Metaphase Chromosomes Meiotic recombination Interphase between decondensed chromosomes homologous chromosomes Replication The closer two loci, the lower the frequency of recombination Metaphase Colchicine condensed (colcemid) : Map Unit (m.u.): The distance chromosomes binds Number, sizes, and shapes of the entire set of between two linked gene pairs metaphase chromosomes of a eukaryotic cell tubulin and Segregation where 1 percent of the prevents products of meiosis are their polymeri- recombinant = a unit of zation distance in a linking map. (also known as centi-Morgan, Lodish 5e-9-45 cM). Lodish 5e-1-12

Method: Chromosome painting by FISH

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1. Cells were imbedded in Baker's yeast Saccharomyces cerevisiae agarose 2. Digested with Zymolyase, 16 linkage groups protease, RNAse Nuclei 3.Pulsed-Field-Agarose- Small genome Electrophoresis Small chromosomes 4. Gel stained with Ethidium Karyotyping is not possible Bromide The DNA of yeast chromosomes can be physically separated by electrophoresis

16 bands correspond to 16 linkage groups

Yeast chromosomes contain one linear ds DNA

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

Metaphase Metaphase Metaphase Chromosome Interphase Nucleus (not shown)

A7-18/19FISH FISH (Fluorescence-In-Situ-Hybridisierung)

1. Immoblize and fix on cover slip

DAPI-Staining FISH FISH Color Set 2. Denature DNA (high pH) Interphase Chromosome Territories 3. Hybridize with fluorescently labeled probes or detect probes with fluorescent antibodies against probes

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

Chicken Chromosomes and Nuclei Chromosome-Painting by Multicolor FISH (Fluorescence In Situ Hybridization) FT Fribourg12F Cremer, T. and Cremer, C. (2001) Nat Rev Genet, 2, 292-301. FT Fribourg12F 31 32 Making probes by incorporation of labelled nucleotides during DNA-synthesis Nuclear Compartments

1 (not shown) Top Strand 5' Bottom 5' DNA with Your Favorite Sequence Strand 2 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 Amplified DNA- Digoxygenin template for 5' generation of 5' probes

A 7- 1. Denature DNA (temperature above 95oC) 1 8 2. Anneal (hybridize) primers (below Tm, melting temperature) 3. Elongate with DNA-Polymerase and 32P-α-NTPs or modified nucleotides (Digoxygenin)

Top Strand 5' Primer 1: Probe to detect the top strand

Bottom Strand 5' Primer 2: Probe to detect the top strand Hemmerich et al. (2010). Chromosome Res.

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Nuclear Compartments Chromatin Dynamics

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

(Im)mobilities in  the nucleus. Illustration of subnuclear compartments and overview of their component‘s residence times (tres) determined by FRAP

Time Scale sec <> min <> hours

Hemmerich et al. (2010). Chromosome Res. FT Fribourg12F Hemmerich et al. (2010). Chromosome Res. 35 FT Fribourg12F 36 Chromatin Dynamics Chromatin Dynamics

Dynamic organization of the cell nucleus: macromolecular crowding. Mobility and immobility of chromatin in transcription and genome stability. 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/ Time Scale or DNA sec <> min <> hours <> days 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. Soutoglou, E., and Misteli, T. (2007). Current opinion in genetics & development 17, Rippe, K. (2007). Current opinion in genetics 435-442. & development 17, 373-380.

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Chromatin Structure and Function Histones

Packaging Genomes Histones

Do eukaryotic cells need histones?

Talbert and Henikoff (2010) Nat Rev Mol Cell Biol. Sea urchins package sperm DNA in nucleosomes using and H1 variants. These histones are How many histones? proteolysed during pronuclear decondensation and replaced with maternal cleavage-stage histones

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

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.

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Gene Cluster

Spacer (not transcribed) P.miliaris (Seeigel) 300 -600x Histone 6kb Genes H1 H4 H2B H3 H2A Drosophila 23 x 4.8 kb melanogaster Günesdogan (2010) H1 H2B H2A H4 H3

Genetics Chicken 1 cluster 39 histone genes Mouse 3 clusters 45 histone genes Ahn and Gruen (1999) Humans 2 clusters 52 histone genes

high copy number of histone genes prevents reverse genetics approach

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Yeast Histone Genes and Synthesis Gene Disruption / Repacement in Yeast S. cerevisiae

Yeast S. cerevisiae has only few genes coding for histones. Therefore, histones can be studied by manipulation of histone genes Gene Disruption H3 (HHT1, HHT2) and H4 (HHF1, HHF2) Construct Haploid HH01 a histone H1 like protein cell Chromosome II Chromosome XVI

Chromosome XIV HH01 Role in recombination, no known HHT1 and HHT2 code for identical proteins effect on chromatin structure HHF1 and HHF2 code for identical proteins H2AZ (HTZ1) - a histone H2A variant H2A (HTA1, HTA2) and H2B (HTB1, HTB2) Chromosome XV If the Chromosome II disrupted HTZ1 gene is Synthesized independent of S-phase. essential, Chromosome IV the cells Exchanged for histone H2A in will die nucleosomes by the SWR1 chromatin HTA1 and HTA2 code for nearly identical proteins remodelling complex. HTB1 and HTB2 code for nearly identical proteins Localized in promoter nucleosomes of Core Histones regulated promotors standard histones of nucleosomes Lodish 5th 9-37 synthesized in S-phase Prevents spreading of silent heterochromatin FT Fribourg12F 43 FT Fribourg12F 44 Are Histone Genes and Variants Essential? Histones

Systematic manipulation of histone genes in yeast S.cerevisiae (by M. Grunstein, M. Osley et al.) How many histones does the cell need? Δ Knock out H2B.1 H2A.1 H2A.2 H2B.2 H4.1 H3.1 H4.2 H3.2 How important is the stoichiometry?

H2B.1Δ H2A.1 H2A.2 H2B.2 H4.1 H3.1 H4.2 H3.2 alive with one H2B.2 H2B.1 H2A.1 H2A.2 H2B.2Δ H4.1 H3.1 H4.2 H3.2 alive with one H2B.1 Approach? H2B.1Δ H2A.1 H2A.2 H2B.2Δ H4.1 H3.1 H4.2 H3.2 dead with no H2B One copy of H2B is required for survival. Overexpression of histone genes  Dito: one H2A, H3, H4 is required: Yeast needs at least one copy of each histone

H2B.1Δ H2A.1 H2A.2Δ H2B.2 H4.1Δ H3.1 H4.2 H3.2Δ alive Imbalanced expression of histone genes  One H2A,H2B, H3, H4 is sufficient for survival under laboratory conditions

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

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

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Overexpression of histone genes in yeast S.cerevisiae Roles of histones

H2B. H2A. H2A. H2B. H4. H3. H4. H3. What happens if you remove histones ("histone depletion") ? 1 1 2 2 1 1 2 2

YEp-Plasmids (high copy number vectors; How to deplete histones in a living cell ? 30-50 copies / cell) URA3

• 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

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

FT Fribourg12F 47 FT Fribourg12F 48 Conditional histone depletion in yeast S.cerevisiae How to make a histone mutant in yeast S.cerevisiae Han et al. (1987) Cell 48, 589; Kim et al. (1988). EMBO J 7, 2211-2219. 1. Generate mutant in vitro Knock out of H4 genes in genome h4-mutant Δ Δ H2B.1 H2A.1 H2A.2 H2B.2 H4.1 H3.1 H4.2 H3.2 Δ Δ h4-mutant HIS3 GAL-H4.2 H4.1 H3.1 H4.2 H3.2 H4.2 gene fused to GAL1 promoter: transcribed in galactose, H4.1 2. Transform repressed in glucose = "conditional expression" HIS3 URA3 YCp = Yeast centromere vector for URA3 extrachromosomal maintenance; about 1 copy/cell Yeast cells in galactose media: H4.1 is expressed from plasmid Normal growth in galactose

Shift of yeast cells to glucose media 6 hours after glucose 3. Select on media containing 5-FOA

• Cell cycle arrest after one generations (fluoro-orotic-acid). Yeast cells Δ Δ • Cells can't segregate chromosomes possibly due expressing URA3 will be killed. H4.1 H3.1 H4.2 H3.2 to lack of packaging of DNA h4-mutant Cells that loose the URA3 plasmid • Activation of some promoters due to the loss of can survive, if the h4-mutant is not histones (nucleosomes) DAPI Anti-Tubulin lethal. HIS3 • Activation of cryptic promoters • Conclusion: Repressive role of histones in transcription initiation

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H2A.X Variant : a role of S. cerevisiae H2A in DNA-repair (Downs et al. (00)Nature 408, 1001)

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

DNA-damage Chromatin Dynamics (e.g. by MMS) dependent phosphorylation

Survival on agar plates How and how tighty are histones bound to DNA ?

hta1-mutants with enhanced sensitivity to MMS How and how tighty NHCPs S129E is resistant to MMS, mimics phosphorylation bound to DNA ? Mec1: a protein involved in damage checkpoint response

S129* = C-terminal deletion, S129A = serine to alanine, S129T = serine to threonine, S129E = serine to glutamic acid,

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