Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Probing accessibilitySlide 26Theory InterludeSlide 28Slide 29Slide 30Slide 31Slide 32Slide 33The EndSlide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Nucleosomes: what, why and where?Rob BrewsterOutlineWhat is a nucleosome?- how is DNA packaged/organized in Eukaryotes?Why do nucleosomes form?- DNA is stiff, how do ~100 bp loops form so readily?Where do nucleosomes form?- What controls the spacing and structure of nucleosomes on the chromosome?Accessibility of DNA in the nucleosomes- How is DNA inside a nucleosomes accessed?DNA Organization in EukaryotesDNA is packaged and condensed into chromosome-Human genome is big, nucleus is small~ 2 billion basepairs ≈ 2mnucleus radius ~ 6 µm- Many different levels of organization- Compacts chromosome- Regulates transcription by making portions of the chromosome more/less accessible (up to 80% is inaccessible to protein binding)- We will focus on nucleosome formation(Alberts, Essential Cell Biology 1998)(Lee W et al., Nat. Genetics 2007)Electron micrograph of chromatin at low ionic strength- Nucleosomes appear as “beads on a stringBasic repeating structure can be probed (protect and seq method)- Digestion enzyme cuts accessible regions of DNA- DNA protected by nucleosome is not cutWhat is a nucleosome?(Furuyama and Biggins ,PNAS)(Electron Micrograph from Olins and Olins)MonoDualMonoMonoEnzymatic digestsParticular enzymes can cut double stranded DNA between basepairs- Most have specific recognition sites- Micrococcal nuclease cleaves everything it can (no specific seq.)Dilution of nuclease 1:3(pictures from NEB)(Peter J. Russell, iGenetics)Repeating structure contains 5 different proteins - Main body is an octomer formed from: two copies each of H2A, H2B, H3, H4 - DNA wraps 147 bp (1.75 turns) around the core-14 non specific adhesive contacts with histone (major groove – histone)- H1 attaches to the linker region and changes the conformation; required for chromatin formation- H1 covers 30-50bpStructure of individual nucleosomeDNA rigidityDNA is stiff…- ~150bp persistence length, λ, for lysed bacterial genome- loops smaller than λ should be rare… but not that stiff- single turn around histone core ~100 bpBack of the powerpoint calculationEnergy paid to bend loop of 147 bp DNA in 16.5 bp circleAssume λ=150 bp then: This cost must be balanced out (and then some) by the 14 contacts so each contact must contribute ~3 KTHowever, nucleosome contacts are ~ 1.5 KT (Polach and Widom, 2005; Schiessel, 2003)Reversing this and solving for maximum stiffness for stable nucleosomes, λ < ~ 80 bp What gives?(Cloutier and Widom, Molecular Cell 2004)Looping probability for small fragments is larger than expected- ~100 bp fragments form loops more readily than predicted- certain sequences are more flexible than others- sequences which loop more readily also more readily form nucleosomes (as much as 10^3-fold difference)Does this sequence dependence control where nucleosomes are positioned?Bending of short DNA fragmentsThe Eukaryotic genome!- Formation on Yeast genome is more probable than for e. coli genome- Implies Euk. Genome has sequence preferential for nucleosomesEukaryotic genome shows specific patterns- AA/AT/TT dinucleotide frequency~10 bp (one DNA twist)Where are Nucleosomes?(Zhang et al., Nature struct. & mol. bio., 2009)Power Spectrum of AA/AT/TT repeatsNucleosome code10bp Periodicity is evident both in vivo and in vitro- GC is 5bp out of phase with AT dinucleotides- due to bending differences in GC and AT?(Kaplan et al, nature 2009)Major grooveMajor grooveWrap 5bpMore examples(Segal et al., Nature , 2006)Nucleosome vs random sequencePeriodicity is missing from arbitrary sequences(Segal et al., Nature , 2006)Method: From in vivo occupancy data, calculate theprobability of any dinucleotide pair at a given nuc.position.Probability that 147bp sequence S is wrapped:The statistical weight of a longer sequence with multiple nucleosomes is just the product of the probability for every basepair to be in that state,The probability must now be computed computationally due to the enormous number of possible configurations (C)(Segal et al., Nature , 2006)Probabilistic model of nucleosome occupancyProbabilistic landscape for occupancyAt a particular nucleosomal coverage can predict where stable nucleosomes will be located(Kaplan et al, nature 2009)Predictions from thermodynamic modelProb. of coverage by nucPredicted stable nucExp. Determined nucSuccess rate within 32bp of predicted positionStatistical Positioning – A (semi)competing view(Zhang et al., Nature struct. & mol. bio., 2009)Various Transcribed GenesNucleosomes pattern emerges from steric exclusion on a line- Promoter region is always “nucleosome free”- DNA sequence appears to play only a small roleLessmoreIn VitroBarrierHard SpheresRandom 1D hard sphere gas?OccurrenceDistance from TSSPhasing in vivo resembles RDF of (for instance) LJ Gas(Zhang et al., Nature struct. & mol. bio., 2009)Can a simple model of a 1D hard sphere gas predict nucleosome positioning pattern?Conclusions on positioningDNA sequence matters for nucleosomes positions…- preferentially bind to particularly repeating sequences- avoid long tracks of A/T… however, this effect seems to be minimized in vivo- Simple 1D gas model fits in vivo nucleosome positions well(data not shown)How does one resolve the apparent conflict in these views?Accessing nucleosomal DNAHow is protected DNA accessed?- DNA replication, repair and transcription all require access to occluded DNA- Specialized motors called “remodeling factors” disassemble and perturb nucleosomes to allow access-However, even without these motors, wrapped DNA has some accessibility- Digestion assays probe equilibrium accessibility- Fret probes dynamic accessibilityIn vitro accessibility mechanismWhat is the mechanism to access a site buried deep in the nucleosome?Translation along dna???Transient unwrappingBulged structuresIn vitro accessibility mechanismWhat is the mechanism to access a site buried deep in the nucleosome?Translation along dna??Transient unwrappingBulged structuresIn vitro accessibility does not show length dependence beyond ~100 bp(Anderson et al, 2002)Fret on nucleosomal complexOne fret dye is put on the
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