chromatin structure and function - unifr.ch · 2012-03-12 · unfolded nucleosomes...
TRANSCRIPT
Fribourg 120224 -120316
Chromatin Structure and Function in
Transcription, Replication, Repair
1FT Fribourg12F
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/
120309 1615 -1900
FT Fribourg12F 2
(Ball 2003, Nature)
Packaging Genomes
Space Questions
Volume of the DNA?
Volume between chromatin ("interchromatin" space")?
Volume of the chromatin?
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Chromatin Dynamics
How and how tighty are histones bound to DNA ?
How and how tighty NHCPs bound to DNA ?
1 2 3 4 5 6 1
Thoma and Koller(1981) J Mol Biol 149, 709
Protein PAGE (coomassy blue)
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H1 is removed at about 0.5 M NaCl H2A, H2B, H3, H4 remain bound to DNA
H1
H3 H2B H2A
H4
(1) Soluble rat liver chromatin (Histones; Non-Histone Chromosomal Proteins, NHCP). Histones are the major protein components of chromatin.
Stoichiometry: about H1:H3:H2B:H2A:H4 = 1:2:2:2:2
(2) Supernatant after high speed centrifugation of chromatin in 300 mM NaCl (NHCP). NHCP are loosely bound and dissociate from chromatin < 300 mM NaCl
(3) Supernatant after high speed centrifugation of chromatin in 500 mM NaCl: H1 and NHCP are released.
(4) Chromatin fraction in a sucrose gradient in 500 mM NaCl: contains all core histones
Chromosomal Proteins
Chromatin of rat liver nuclei was fractionated on sucrose grdients containing different ionic strength
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Van Holde, K. E. (1989) Chromatin, Springer Verlag, Berlin
Stepwise Histone Binding to DNA
DNA binding strength: H1 < H2A, H2B < H3, H4
Ionic Interactions
Dissociation
Histone Basic Amino Acids
Acidic Amino Acids
MW
Lys Arg
H1 29% 1% 5% 23000
H2A 11% 9% 15% 13960
H2B 16% 6% 13% 13774
H3 10% 13% 13% 15342
H4 11% 14% 10% 11282
Assembly
H2A H2B are loosely bound towards the ends H3H4 are firmly bound in the center
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How to investigate localization and histone-DNA binding in
living cells?
Chromosomal Proteins
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Dynamic binding of histone H1 to chromatin in living cells Misteli et al (2000) Nature, 408, 877.
FRAP: Fluorescence Recovery After Photobleaching Cell expressing GFP-tagged protein
Photobleaching (seconds)
Distroys fluorescence, but not the proteins
Recovery of fluorescence (time)
Redistribution of proteins
HMG14-GFP: High Mobility Group 1 („Transcription“ Factor), fast recovery
H1-GFP: recovery in minutes strong binding (long residence time)
GFP alone: fast recovery
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Method • Cell lines expressing histones and HMGs (a High Mobility Group Protein; NHCP) tagged with green fluorescent protein (GFP) • Photobleaching (FRAP)
Results
HMG14-GFP: exchange in seconds
H1c-GFP : exchange in minutes
H2B-GFP: 3% of H2B exchanged within minutes, 40% did so slowly (t1/2 approximately 130 min).
H3-GFP: exchange many hours H4-GFP: exchange many hours
Dynamic binding of HMGs and histones to chromatin in living cells Misteli et al (2000) Nature, Kimura and Cook (2001) J Cell Biol, Kimura(2005) DNA Rep.
Dynamic binding: HMGs < H1 < H2B < H3, H4
dito: nucleotide excision repair factors, transcription factors, glucocorticoid receptor
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General Properties of Histones
Histones are highly conserved in evolution
Histones are basic proteins and positively charged
Bind to negatively charged DNA by ionic interactions
DNA binding strength: H1 < H2A, H2B < H3, H4
Histones are abunant (DNA/histones = 1/1 (w/w))
Histone stoichiometry about H1:H2A:H2B:H3:H4 = 1:2:2:2:2
Histone form (H2A H2B) and (H3 H4) dimers, (H3 H4)2 tetramers, octamers...
Specialized properties and functions of histones, variants, modifications in chromosome structures, differentiation, development, transcription,
replication, repair, recombination
Histones
Chromatin Structures
Packaging Genomes
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Nucleosomes
Higher Order Structures
Thoma MGE05-L04 11
Discovery and Characterization of Nucleosomes by Electron Microscopy
Miller Spreading, Negative Stain, Olins (1974), Science
(McKnight and Miller)
Miller Spreading, Negative Stain Miller Spreading, Metal Shadowing
(Hamkalo and Rattner)
„30 nm Fiber“, 150 m0 NaCl „beads - on - string“, low ionic strength
Le5 10-19
Unfolding of chromatin in „low salt conditions“ and high pH
"Beads-on-string", repetitive units, diameter: about 10 nm
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The first biochemical indication of a subunit structure
Biochemical Discovery and Characterization of Nucleosomes
1. Rat liver nuclei
2. Incubation with 1 mM Ca2+, 10 mM Mg2+
3. DNA purification and gel electrophoresis
Hewish and Burgoyne (1973) BBRC52,504
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Digestion of nuclear chromatin with micrococcal nuclease (MNase), a Ca2+ dependent endonuclease, cuts ss DNA, ds DNA, RNA, some preference for AT verus GC rich DNA
Method: Finch, Noll and Kornberg (1975)PNAS 1. Nuclei, add 1mM Ca2+ 2. Add MNase, digest for a limited time, stop reaction by removal of Ca2+ 3. Purify DNA 4. Agarose gel, stained with Ethidium Bromide
200 bp
400 bp
600 bp
800 bp
3 Nucleosomes
4 Nucleosomes
2 Nucleosomes
1 Nucleosome
MNase cuts DNA between nucleosomes ("linker DNA")
Nucleosomes: a repetitive subunit of chromatin
„Nucleosome repeat length“ of a genome
Nucleosomal DNA resists digestion by MNase
Nucleosomes – "repressive" structures
MNase
Packaging Genomes – Nucleosomes
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DNA-analysis in agarose gels
Analysis by EM
Soluble rat liver chromatin was generated by MNase digestion and fractionated on a sucrose gradient 1 2 3 4
Bottom Top A260
Finch, Noll and Kornberg (1975)Proc Natl Acad Sci U S A, 72, 3320-3322.
Nucleosomes - A Repetitive Subunit of Chromatin
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DNA-Agarose Gel, Färbung Ethidium Bromid
1. Rat liver nuclei 2. MNase digestion 3. DNA Purification 4. Agarose Gel
Noll and Kornberg (1977). J Mol Biol
Nucleosome Particles - Subunits of Chromatin
DNA
Core Histones
Linker Histone
168 bp: Chromatosome
Chromatosome
168 bp 2 turns
Octamer
H1
147 bp: Nucleosome Core
Nucleosome Core Particle conserved from yeast to man
147 bp 1.7 turns
Octamer 2x(H2A, H2B, H3, H4)
-
200 bp: Nucleosome
Nucleosome
> 168 bp 2 turns + extensions
Octamer
H1
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Noll (1974) NAR
1. Rat liver nuclei 2. DNaseI digestion 3. DNA purification 4. Polyacrylamide gel
Only the outside of DNA is accessible to proteins
Nucleosome structure regulates accessibility of
DNA for proteins
in transcription replication
repair
10 bp DNA repeat DNA is on the outside of
nucleosomes
Testing DNA-accessibility by Nucleases
DNaseI: endonuclease, binds DNA in the minor groove, generates ss cuts in ds DNA
EG12 Thoma 17
Nucleosome cores can be reconstituted
Agarose-Gel, „Band Shift“
DNA
Nucleosome
Histone Octamers Labelled DNA Histone Octamer (e.g. recombinant histones)
Dissociation Assembly by Stepwise dilution to 0.1M NaCl
(> 1M NaCl)
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11 nm 6 nm
H3 H4 H2A H2B
DNA 147 bp DNA (palindromic alpha-satellite DNA) 1.7 left handed supercoils Outside, bent, distorted
Histones: Octamer (recombinant Xenopus histones) (H3.H4.H4.H3) Tetramer 2 (H2A.H2B)-Dimers Histones, folded, inside,N-terminal tails protrude outside
Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Crystal Structure of a Reconstituted Nucleosome Core
6 nm
DNA-Distortion in Nucleosome Cores
Richmond and Davey (2003) Nature 423, 145
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Structural and Functional Subunit of Chromatin Dynamic Particle
Controls DNA packaging, accessibility, protein
Some Properties of Nucleosome Cores Small disc shaped particles of 10 nm diameter, 5.7 nm high, pseudosymmetric
Abundant (about 107 nucleosomes / haploid human genome) Conserved structure from yeast to man
Histones inside – N-terminal tails protrude outside
Binding to DNA: 14 contact sites, binding strength: (H2A.H2B) < (H3.H4)
DNA 146 - 147 bp, 1.7 left-handed turns, one negative supercoil / nucleosomes, 80 bp/turn on
the outside, brings distant sites together inner surface is protected, outer surface is accessible, DNA is distorted - affects DNA recognition by proteins and damage formation
Nucleosome Heterogeneity Sequence
Histone Variants Histone Modifications
Nucleosome positioning & stability regulate DNA sequence accessibility
Nucleosome Cores
Chromatin Structures
Packaging Genomes
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Nucleosomes
Higher Order Structures
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Method 1. Nuclei 2. MNase digestion 3. DNA Purification 4. Agarose Gel and
Ethidium Bromide Staining
200
400
600
800
1000
1200
1400
1 2 3 4 5 6 7
200
400
600
800
1000
1200
Nucleosomes
bp
200 bp / nucleosome
Average Nucleosomal Repeat / Genome
ca. 300 bp
ca. 400 bp Local variability of linker DNA
Nucleosome Repeat Length and Linker DNA
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Repeat bp
Seeigel Spermien ca 240 Seeigel Embryonen 222 Ratten Leber 196 Ratten Niere 196 Huhn Oviduct 196 Hühner Erythrocyten 207 Bäckerhefe S. cerev. 160
Linker bp 95 77 51 51 51 62
15
Linker DNA Commonly used: Linker DNA = DNA connecting nucleosome – cores Nucleosome-Core DNA is constant (147bp), but the linker DNA varies in different organisms, tissues, and between nucleosome cores.
Nucleosome Repeat Length and Linker DNA
What determines the linker length?
Not known. Possible contributions by histone H1
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30 nm Chromatin
Fibers
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chromatin with H1
Salt dependent condensation of chromatin and H1-depleted chromatin Roles of histone H1 in chromatin structure:
binds to the entry and exit site of linker DNA in nucleosomes and stabilizes nucleosomes stabilizes and organizes chromatin fibers
Structural Transitions In Soluble Rat Liver Chromatin Thoma, Koller, Klug (1979) J. Cell Biol. 83, 403
0 mM
open zig-zag nucleosome-filament
> 60 mM NaCl
"30 nm" compact fiber
unfolded nucleosomes
"beads-on-a-string" partally unfolded nucleosomes
compact clumps
chromatin without H1
removal of H1 on
sucrose gradients containing 0.5M NaCl
Dialysis against buffers containing different ionic strength, fixation by 0.1% glutaraldehyde, adsorbed on carbon coated grid and stained by rotary shadowing with Pt for EM
~ 5 mM
closed zig-zag nucleosome fiber
100 nm
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6-8 nucleosmes / turn (11 nm)
Histone H1 contacts stabilize the fiber
Path of the linker DNA is unknown
Salt-dependent folding of chromatin
"0" "~ 5"
"~ 5 " "~ 40 "
"~ 40 " "~ 100 " "~ 100 "
"~ 100 " "~ 100 " "~ 100 "
Structural Transitions In Soluble Rat Liver Chromatin Thoma, Koller, Klug (1979) J. Cell Biol. 83, 403
Irregular, heterogenous structures (due to different linker length, composition etc?)
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Reconstituted Tetranucleosome
DNA: 4 copies of a 147 bp long DNA sequence ("601") with strong nucleosome positioning properties, separated by 20 bp linker DNA
Histones: Recombinant core histones of Xenopus laevis
X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Schalch, T., Duda, S., Sargent, D.F. and Richmond, T.J. (2005) Nature, 436, 138-141.
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30 nm Fiber Models
Excluded by mass/unit length
and electric dichroism (McGhee et al.
(1980). Orientation of the nucleosome within
the higher order structure of chromatin.
Cell 22, 87-96.)
Thoma, Koller, Klug (1979) J. Cell Biol. 83, 403
"Solenoid" Finch and Klug (1976)
PNAS 73, 1897
"Helical-Ribbon" Woodcock et al.(1984).
J Cell Biol 99, 42-52.
a) depends on linker length
a)
"Crossed-Linker" Williams et al. (1986). Biophys J
49, 233
diameter depends on linker length
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In metaphase chromosomes & intephase
Structure(s) unknown
Diameter about 30-50 nm, irregular, dependent on linker length (?)
Nucleosomes arranged radially
Linker DNA path unknown
Packaging (mass / length) about 6-8 nucleosomes / 10 nm
H1 probably inside
Pronounced Heterogeneity structural, functional, compositin histone variants, histone modifications, NHCPs
Some properties of 30 nm fibers
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Loops
Chromosome Scaffold
Nuclear Matrix
Thoma101019 30Alberts8-16
Lampbrush chromosomes in amphibian oocytes
Interphase chromosomes in amphibian oocytes Paired homologous chromosomes, after replication, prior to meiotic divisions (4n)
Very active in transcription loops
open chromatin, transcribed condensed chromatin
(chromomeres)
Spreading on water
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Chromatin Loops - Chromosome Scaffolds – Nuclear Matrix
(Paulson & Laemmli, 1977)
Metaphase Chromosome
Removal of histones with heparin
Adsorption on EM grids Contrast by metal shadowing
Are chromatin loops attached to a nuclear matrix ? No components idenified so far MARs = Matrix Associated Regions in DNA
Are chromatin loops attached to a scaffold in metaphase chromosomes? Scaffoldprotein: Topoisomerase II & others ? SAR = Scaffold Associated Region (DNA)
Thoma MGE05-L06 32Holm et al (1985) Cell 41 553
DNA topoisomerase II is required at the time of mitosis in yeast
top2ts
26o
top2ts
35o
Yeast (top2ts), temperature sensitive mutation in the gene coding for topoisomerase II • Cells were blocked in G1 by alpha-factor • Released in fresh medium at 26° or 35° • Stained with DAPI
Role in segregation von ds-DNA after replication
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Metaphase
Chromosomes
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Indian Muntjac Cells Blocked in Metaphase with Colcemid
Chromosome 2
Saitho and Laemmli (1994)Cell 76, 609
Structure of Metaphase Chromosomes
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Metaphase Chromosome Structure(Saitho and Laemmli (1994)Cell 76, 609)
Bands arise from a differential folding path of the highly AT-rich scaffold. Evidence by differential staining approaches
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Packaging
P (smallest human chromosome) = 14 mm DNA / 2 µm = ca. 7000
Packaging Ratio (P) = Length of DNA (nm) / Length of chromatin
P (nucleosome) = (200bp.0.34nm/bp) / 10nm = ca. 6
P (30 nm fiber) = (6 to 8 nucleosomes) / 10nm = ca. 36 to 48
P (DNA) = 1
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Roles of Packaging
(In-) accessibility of DNA/proteins
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 (?)
Condensation of DNA
Distance between twobinding sites
(Dis-) favours (long-) distant interactions between proteins and/or DNA elements
Chromatin Structure and Function
Genes & Transcription TBP
TFIIB
RNA Polymerase II
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Control - Regulation activation – inactivation - silencing
Transcription Cycle initiation – elongation - termination
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rRNA
mRNA Protein
5s-rRNA, t-RNAs, snR6, 7SLRNA ...
Nucleolus
Nucleus
Nucleus
50-70%
20-40%
Ca.10%
RNA-Polymerase I
„Pol-I-Genes“
RNA-Polymerase II „Pol-II-Genes“
RNA-Polymerase III
„Pol-III-Genes“
Product Location Activity Class
Eukaryotic Gene Classes (see textbook)
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5‘ Enhancers
Proximal and distal regulation sites (Elements, Enhancers)
Internal control regions
RNA-Polymerase I
„Pol-I-Gene“
RNA-Polymerase II „Pol-II-Gene“
RNA-Polymerase III
„Pol-III-Gene“
TFI A,B,C, UBF
TFII A,B,D (TBP), E,F,H
TFIII A,B,C
TFIIH, TBP
TBP
• RNA-Polymerases: Enzymes for DNA-dependent RNA-synthesis
• General Transcription Factors (GTF): Formation of the initiation complex with RNA-polymerases. TBP, TFIIH are GTFs involved in different classes RNA-Polymerases and GTFs are required for correct initiation and elongation
• A combination of specific transcription factors interact with specific DNA elements and regulate transcription by interactions with GTFs and RNAP (Combinatorial control)
Eukaryotic Gene Classes (see textbook)
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Genes Transcribed by RNA Polymerase II (see textbooks)
Yeast+1 -90 Coding Region
TATA INR Box Cap-Site Promoter
Proximal UAS, URS Upstream Activating /Repressing Sequence
500 - 100
CpGIslands
Coding Region
house keeping genes
regulatedgenes
Higher Eukaryotes exon intron premRNA
+1 (TSS, Transcription Start Site)30TATA INR Box Cap-Site Promoter
Core Promoter: Coordinate assembly of General Transcription Factors (GRFIIs) and RNAPII to initiate transcription. Only about 40% of genes have a TATA box.
downstreamupstream
Control Regions: Enhancers, UAS (Upstream Activating Regions), Silencers,..... • DNA-Element: binding site on DNA• Recruit sequence specific transcription factors for activation or repression
1 - 50kbDistant Control Region
+10 bis +50kbDownstream Control Region
Internal Control Region
200 - 400bpProximal Control Region
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Locus Control Regions (LCR)
Locus Control Regions • a distant control region • regulates expression of globin genes
Human β-Globin Gene Cluster
Alberts et al.
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Insulators and Boundaries
Bell(2001)Science291,447
HS4, 3ʻ Hs: Insulators, enhancer blocking elements
Insulators (HS4, 3'HS) mark the boundaries of chromatin domains by limiting the range of action of
enhancers and silencers
LCR, Locus Control Region, regulates
transcription of globin gene
Enhancer regulates transcription of
folate receptor gene
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Long distance control - Genes transcribed by RNAPII
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
Promoter(DNA-)Elements
Regulatory(DNA-)Elements
Lodish et al. 5th edThoma111018 45
General Concepts: Genes transcribed by RNAPII
Chromatin-Remodelling
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Chromatin States of Genes
regulatory proteins regulatory elements RNAPolymerase Nucleosomes
Inactive in Euchromatin
Nucleosomal regions have reduced accessibility to nucleases (MNase, DNaseI)
MNaseDNaseI
Active in Euchromatin
MNaseDNaseI
Enhanced nuclease sensitivity in and around transcribed regionHypersensitive sites (HHS) in control regions: proteins disrupt nucleosomes
Transcription and histone modifications ‚open ʻ chromatin
Nucleosomal regions have slightly more reduced accessibility to nucleases (MNase, DNaseI)
MNaseDNaseI
Silenced in Heterochromatin
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Hypersensitive Sites
indicate interactions
of regulatory proteins and remodelling factors with DNA
Gene
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Transcription
+ induced
- repressed + constitutive + induced
- repressed
Firtel et al. In Architecture of Eukaryotic Genes, ed. G. Kahl (1988), VCH
DNaseI Hyper Sensitive Sites around the Chicken Lysozyme Gene
Hypersensitive sites are an indication for interactions of regulatory and remodelling factors with DNA.
1. Nuclei2. Digest with DNaseI (limited)3. Extract DNA4.Cut with restriction enzyme5. Gel and Southernblot6. Hybridize with probe close to one restriction site ("indirect endlabeling")
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Promoters
Regulation
Initiation of Transcription
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Stepwise assembly of the preinitiation complex and initiation of transcription by RNA-Polymerase II (in vitro and in vivo)
TFIID: TBP (TATA Binding Protein) binds TATA box TAFs (TBP associated proteins) interacts with Inr in TATA less promoters TFIIA: 3 subunits TFIIB: positions RNA-PolII TFIIF: 2 subunits
TFIIE: 2 subunits RNAP-II: 12 subunits
TFIIH: 5 subunits including 2 Helicases (opening of promoter) 1 Kinase (phosphorylation of RNAP-II-CTD)
TFIIA
Preinitiation Complex Initiation & Promoter Clearance (initiation to elongation transition) Phosphorylation of RNAP-II-CTD (Serine 5)
Mediator Elongation Recruitment of elongation factors, RNA-processing factors Phosphorylation of RNAP-II-CTD (Serine2)
Lodish 5th ed
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a, Open promoters ("preset promoters") have a depleted proximal nucleosome adjacent to the transcription start site (TSS, black arrow), a feature common at constitutive genes. (NDR Nucleosome depeted region or Nucleosome Free Region). ACT, transcription activator
b, Covered promoters (also remodelling promoters) have a nucleosome adjacent to the TSS in their repressed state, a feature common at highly regulated genes. The figure depicts features more common in each contrasting promoter type, but most yeast genes blend the features shown to provide appropriate regulation. Green nucleosomes contain canonical H2A, whereas brown nucleosomes bear H2A.Z. Binding sites (BS) for transcriptional activators (ACT) are shown. These are mainly exposed for open promoters and mainly occluded by nucleosomes (in the repressed state) at covered promoters. Covered promoters typically have nucleosome positioning sequence elements of varying strength and locations that help define nucleosome positions (faded green) and promoter architecture. NDR, nucleosome-depleted region.
Chromatin Concepts at PromotersCairns, B.R. (2009). Nature 461, 193; Wallrath & Elgin (1994)
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The ARS1 (yeast origin of replication): Nuclease sensitive, non-nucleosomal ‚open‘
The URA3 Gene: • 6 positioned nucleosomes in coding region, • non-nucleosomal ‚open‘ promoter (5') • non-nucleosomal ‚open‘ 3‘end
URA3 : an example of an open (non-nucleosomal) promoter
Nuclease (Hyper-) Sensitive Regions (no nucleosomes)
Positioned Nucleosomes: • protected regions of 145 - 200 bp = footprint of histone octamer • In all cells, the histone octamers sit on the same DNA sequence of the minichromosome
Chromatin Structure of the Yeast Minichomosome YRpTRURAP Method:
1. Yeast nuclei and DNA 2. Partial digest with MNase or DNase I 3. Mapping of cutting sites by indirect endlabelling (Purify DNA, cut with restriction enzyme e.g. EcoRI; Agarose gel, Southernblot; hybridize with short probe close to EcoRI site)
ARS1 3‘
URA3
TRP1
5‘
Thoma (1986) J. Mol.Biol.
Chr
om.
DN
A
Chr
om.
DN
A
Chr
om.
MNase
2 5 6
5 1 2
7 6 8
1 0 2 4 1 2 8 0 1 5 3 6 1 7 9 2 2 0 4 8 2 3 0 4 2 5 6 0
DNA Size
Marker
Yeast
ARS1 3'
5'
TRP1
URA3
5'
3'
H
H
YRpTRURAP
RI
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PHO5: "Classic Example" of Chromatin Remodeling in a Promoter (I)
Nuclei isolated from cells that had been grown in YPDA (- Induction) or no phosphate medium (+ Induction) were digested with 3, 6, 10 and 3 U/ml micrococcal nuclease (MNase) (lanes 2-5), or with 1, 1, 2 and 4 U/mol DNase I for 20 min (lanes 7-10, respectively). DNA was isolated, cut with ApaI, separated in a 1.5% agarose gel, blotted and hybridized with probe D ("indirect endlabelling"). The ApaI site used is about 1300 bp upstream of the PHO5 gene. A control digest of free DNA with 0.05 U/ml micrococcal nuclease, subsequently cleaved with ApaI, is shown in lane 1. Lane 6 contains three double digests (BamHI, HaeII, ClaI) and lane 11 the pBR322 reference fragments. The arrow denotes a prominent fragment characteristic of the active state that is not observed with free DNA (compare lanes 1 and 2). HS, hyper sensitive site.
MNase DNaseI "Removal of positioned nucleosomes from the yeast PHO5 promoter upon PHO5 induction releases additional upstream activating DNA elements."Almer et al. (1986). EMBO J. 5: 2689.
TATA
high phosphate (inactive)
low phosphate (active)
HS2
UASp1 and UASp2: Upstream Activating Sequences, binding sites for Pho4 activator. UASp1 is in a hypersensitive site (HS2), not folded in nucleosomes and readily accessible for Pho4.
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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
54
PHO5: "Classic Example" of Chromatin Remodeling in a Promoter (II)
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Remodeling of the PHO5 promoter occurs by histone dissociation and reassemby and not by nucleosome displacement in cis (mobility)
soluble histone pool
3. During phosphate starvation cells were shifted to galactose to express H3-FLAG and generate a soluble pool of H3-FLAG.
high phosphate (repression)
4. Phosphate was added to repress PHO5
5. Chromatin was analysed by ChIPs Promoter closure occurs very rapidly (minutes). The incorporated histones originate from the histone pool, H3-FLAG. TATA
TATA
TATA
TATA
low phosphate (actvation)
histone eviction? or nucleosome shift?
Schermer, U. J., Korber, P., and Horz, W. (2005). Mol Cell 19, 279 Strain with two differently tagged H3:, GAL-H3-FLAG expressed in glactose and H3-Myc expressed from the histone promoter in S-phase.
1. Cells were grown in glucose and high phosphate expressing H3-Myc.
2. Cells were starved for phosphate to induce PHO5. Starved cells are arrested in cell cycle and do not replicate. Chromatin contains only H3-Myc.
• Experiments with mutants showed that the histone chaperones Asf1 and Hir1 as well as the SWI/SNF nucleosome remodeling complex appear to be important for rapid reassembly of nucleosomes at the PHO5 promoter.
Asf1 Hir1 SWI/SNF
PHO5: "Classic Example" of Chromatin Remodeling in a Promoter (III)
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In one model (left), a combination of acetylation and chromatin remodeling directly results in the loss of Htz1-containing nucleosome, thereby exposing the entire core promoter to the GTFs and Pol II. SAGA and mediator then facilitate PIC formation through direct interactions. In the other model (right), which represents the remodeled state, partial PICs could be assembled at the core promoter without loss of Htz1. It is the binding of Pol II and TFIIH that leads to the displacement of Htz1-containing nucleosomes and the full assembly of PIC
Models of Chromatin Regulation during Transcription Initiation Li, B., Carey, M. and Workman, J.L. (2007) Cell, 128, 707-719.
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Transcribing through
chromatin
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Loss of nucleosomes during transcription of the yeast GAL10 gene Cavalli and Thoma (1993)EMBO 12,4603
Nucleosomes In genome 1. Nuclei 2. MNase digestion (limited) 3. DNA purification 3. Agarose gel: reveals nucleosomal repeat of the genome in glucose and galactose
Southern blot / Probe: GAL10 gene
glucose galactose GAL10 gene
nucleosomal repeat of genome
glucose galactose Yeast genome GAL10 gene:
Repressed in glucose Transcribed in galactose
Nucleosomes on GAL10? Southern blot, probe with defined sequence (GAL10):
Loss of a regular nucleosome repeat upon transcription; (partial) loss of nucleosomes due to transcription (?)
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Transkription Elongation - Supercoiling - Topoisomerases
RNAP DNA
Topoisomerase I migrates with RNAP II and relaxes supercoils
DNA RNAP
DNA and RNAP can't freely rotate: RNAP generates 1 positive and 1 negative supercoil /10bp
Alberts et al.
Nucleosomes contain one negative supercoil Positive supercoils might destabilize nucleosomes (?)
Negative supercoils might favour reassembly of nucleosomes (?)
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Elongating Pol II requires HATs to acetylate the nucleosome in front of elongation machinery.
The passage of Pol II causes histone displacement. Subsequently, these histones are redeposited onto the DNA behind Pol II via concerted actions of histone chaperones. Alternatively, the free forms of histones in the nucleus are also available for reassembly.
Newly deposited nucleosomes are immediately methylated by Set2.
Methylation of H3K36 is then recognized by Eaf3, which in turn recruits the Rpd3S deacetylase complex.
Rpd3S removes the acetyl marks and leaves the nucleosome in a stable state.
Methylation of H3K36 is eventually eliminated by a histone demethylase when the gene turns off.
Transient Disruption and Reassembly of Nucleosomes During Transcription Elongation Li, B., Carey, M. and Workman, J.L. (2007) Cell, 128
Histone chaperone Asf1 binds H3.H4 dimers
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The distribution of histones and their modifications are mapped on an arbitrary gene relative to its promoter (5' IGR; Inter Genic Region), ORF (Open Reading Frame), and 3' IGR.
The curves represent the patterns that are determined via genome-wide approaches (ChIPs and microarrays "ChIP on Chip").
The squares indicate that the data are based on only a few case studies. With the exception of the data on K9 and K27 methylation, most of the data are based on yeast genes.
Genome-Wide Distribution Pattern of Histone Modifications from a Transcription Perspective
Li, B., Carey, M. and Workman, J.L. (2007) Cell, 128
"General" for active genes • acetylation of H3 and H4
"General" for inactive (often "heterochromatin"): • H3 K9me and H3 K27me
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"open chromatin domains"
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Active genes are more sensitive to DNase I than inactive genes
Active genes are in an „open“ chromatin conformation
1. Nuclei2. Digest with DNaseI (limited)3. Extract DNA4. Cut with BamH1 restriction enzyme5. Gel and Southernblot6. Hybridize with probe for adult globin gene
active inactive
globin gene
control
"Open" chromatin structure of active genes
Structural transitions occur during activation and inactivation of genes.
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Chromatin structure around the Chicken Lysozyme Gene Locus
Firtel et al. In Architecture of Eukaryotic Genes, ed. G. Kahl (1988), VCH
Transcribed Region
Open Domain (DNase I sensitivity)
The domain of DNase I sensitivity and hyperacetylation is larger than the transcription unit.
Histone hyperacetylation might destabilize chromatin structures.
Acetylated Histones ChIPs (Chromatin Immuno Precipitation) with antibodies against acetylated histones (H3)
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End of lecture120309