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120224-FT Fribourg 06 Slides Chromatin Structure and Function Fribourg 120224 -120316 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 Chromosome Territories Recombination Heterochromatin DNA-Repair Euchromatin 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- Histones: Histone-Modifications Chromosomal H1 „Histone Code“ Proteins 2x(H2A, H2B, H3, H4) FT Fribourg12F Lodish et al 5th ed 3 FT Fribourg12F 4 Chromatin Structures Locus Specific Heterogeneity in Structure and Function Genome-wide patterns of histone modifications in yeast. Millar, C.B., and Grunstein, M. (2006). Nat Rev Mol Cell Biol 7, 657-666. FT Fribourg12F 5 FT Fribourg12F 6 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 FT Fribourg12F 7 FT Fribourg12F 8 Chromatin Definition and Composition Chromatin Structure and Function Definition When and where is chromatin made? • 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) 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 Lodish et al 5th ed FT Fribourg12F 11 FT Fribourg12F 12 Genome - Organization 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 ? Lodish et al 5th ed FT Fribourg12F 14 FT Fribourg12F 13 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-) genetics 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 FT Fribourg12F 17 FT Fribourg12F 18 DNA Nature 421, 421-422. FT Fribourg12F 19 FT Fribourg12F 20 Remember DNA ? Different Forms of Double Stranded DNA „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 FT Fribourg12F 21 FT Fribourg12F 22 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. FT Fribourg12F 23 FT Fribourg12F 24 Packaging Genomes Packaging Genomes 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 FT Fribourg12F 25 FT Fribourg12F 26 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) Karyotype: 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 FT Fribourg12F 27 FT Fribourg12F 28 How Many DNA Molecules per Eukaryotic Chromosome? 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 FT Fribourg12F 29 FT Fribourg12F 30 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. FT Fribourg12F 33 FT Fribourg12F 34 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
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