Nucleosome Positioning and Organization 02-715 Advanced Topics in Computaonal Genomics Nucleosome Core Nucleosome Core and Linker • 147 bp DNA wrapping around nucleosome core • Varying lengths of linkers between adjacent cores Linker Nucleosome Positions • Nucleosome posi8ons are non-random and conserved across the similar cell types • Nucleosome posi8oning affects gene regulaon • The binding of other proteins affect the posi8ons of nucleosomes • Dynamic nature of nucleosome posi8oning influenced by the dynamic gene regulaon • Stac nature of nucleosome posioning influenced by DNA sequence Dynamic Nucleosomes • Kine8c measurements show the DNA in an isolated nucleosome is surprisingly dynamic, rapidly uncoiling and then rewrapping around its nucleosome core. • This way, most of nucleosome-bound DNA sequence is accessible to other DNA-binding proteins Dynamic Nucleosomes • DNA in an isolated nucleosome unwraps around four 8mes per second, remaining exposed for 10-50 milliseconds before the DNA re-wraps around the nucleosome – Allows for other DNA-binding proteins to access the DNA for transcrip8on, DNA replicaon, etc. Dynamic Nucleosomes: Chromatin Remodeling Complex • Nucleosome sliding – ATP-dependent chroman remodeling complexes bind to nucleosome core proteins and DNA that wraps around it, and use the energy of ATP hydrolysis to move DNA relave to the core. Dynamic Nucleosomes: Chromatin Remodeling Complex • Chroman remodeling complex replacing histone proteins with other variants Dynamic Nucleosomes: Chromatin Remodeling Complex • Chroman remodeling complex (with histone chaperones) replacing/removing histone proteins with other variants Dynamic Nucleosomes: Chromatin Remodeling Complex • As genes are turned on and off, chroman remodeling complex are brought to specific regions of DNA to locally influence chroman structure • Certain chroman structure can be inherited during cell division Dynamic Nucleosomes: Chromatin Remodeling with Code Reader-Writer Complex • Spreading chroman changes – Gene regulatory protein recruits a code-writer enzyme, which modifies the histone code – The code-writer recruits code-reader, which then again recruits code-writer. – Reader and writer should recognize the same code • Barrier DNA sequence for blocking the long-range spreading Dynamic Nucleosomes: Chromatin Remodeling Complex • Spreading wave of chroman condensaon to form a long range heterochroman Nucleosomes and Chromatin Structure • H3 variant histone, called CENP-A replaces H3 in centromeric DNA sequences Definitions of Terminology • Nucleosome posi8ons: the nucleosome start/center/end posi8ons of the 147bp sequence wrapped around a nucleosome • Nucleosome configuraon – a set of non-overlapping nucleosome posi8ons on a single DNA molecule of defined length. – if a base pair is in state 1, then both the preceding and following 146 base pairs (bp) must be ‘0’ Definitions of Terminology • Nucleosome organizaon: a probability distribu8on over nucleosome configuraons – P: nucleosome organizaon – C: a set of nucleosome configuraons – P(c): the probability of a nucleosome configuraon c Definitions of Terminology • Nucleosome occupancy: the sum of the probabili8es of the configuraons in which the base pair is covered by a nucleosome – Occ(x): the occupancy at basepair x – C: nucleosome configuraon – P(c): nucleosome organizaon Illustration of Different Terminology Definitions of Terminology • Nucleosome posi8oning: the degree to which the posi8ons of individual nucleosomes vary across the different configuraons of a nucleosome organizaon. – a perfectly posi8oned nucleosome is one that adopts the same posi8on across all measured configuraons – 30% posioning? – Absolute vs. condi8onal posi8oning Definitions of Terminology • Absolute nucleosome posi8oning at basepair x: the probability of a nucleosome star8ng at basepair x – Absolute nucleosome posi8oning does not uniquely determine nucleosome organizaon • Condi8onal nucleosome posi8oning at basepair x: the absolute posi8oning at basepair x divided by the probability that a nucleosome starts anywhere within a larger region centered on x – the probability that a nucleosome starts at x given that a nucleosome starts somewhere between x - 73 and x + 73 Illustration of Different Terminology Experimental Technology for Measuring Nucleosome Organization • Diges8on of chroman by micrococcal nuclease (MNase), an endonuclease that preferen8ally cuts linker DNA rather than DNA wrapped around a nucleosome – highly digested DNA: depleted of nucleosomes – under-digested DNA: relavely protected by nucleosomes • Measure the diges8ng paern with microarray or sequencing of the nucleosome-protected DNA segments – Occ(x): occupancy at base pair x – ri: read counts at basepair i Experimental Technology for Measuring Nucleosome Organization • Challenges – Bias introduced by MNase’s preference of TA/AT dinucleode as its cleavage site • Cannot obtain the nucleosome posi8on at a single nucleo8de resolu8on • Naked DNA as a control, but linker DNA is TA/AT rich, reducing the u8lity of naked DNA as a control – Experiment is performed not on a single cell, but on a populaon of cells • We get to measure only the average of the dynamically changing nucleosome posions Experimental Technology for Measuring Nucleosome Organization • Challenges – In vitro and in vivo nucleosome posi8ons are different – With low coverage in sequencing, it is difficult to obtain a reliable map of nucleosome posi8ons. Currently, • 2 nucleosome read starts per base pair in a yeast in vivo map • 0.1-2 nucleosome read starts in yeast in vitro map • 0.07 nucleosome read starts in human in vivo map Experimental Technology for Measuring Nucleosome Organization • Robustness of nucleosome map: Are the two independently generated nucleosome maps highly correlated? Yeast Genome-Scale Nucleosome Map • Map of posi8ons of 2278 nucleosomes over 482 kilobases of Saccharomyces cerevisiae DNA, including almost all of chromosome III and 223 addi8onal regulatory regions – Most of the nucleosome were well posioned – A nucleosome free region of ~200bp in the Pol II promoters – Nucleosome free regions had evolu8onarily conserved sequences – Most TF binding mo8fs were nucleosome free regions Yeast Genome-Scale Nucleosome Map • Nucleosome-free regions common in TF binding sites. Microarray data for nucleosome posions Inferred nucleosome posi8ons with boxes for a TF binding mof DNA Sequence conservaon score Yeast Genome-Scale Nucleosome Map • Nucleosome-free regions common in TF binding sites. Microarray data for nucleosome posions Inferred nucleosome posi8ons with boxes for a TF binding mof DNA Sequence conservaon score Yeast Genome-Scale Nucleosome Map • Func8onal transcrip8on factor binding mo8fs are more accessible than unbound mo8fs Nucleosome Maps • DNA sequence is significantly predic8ve of nucleosome organizaon in vitro and in vivo – discussion on Wednesday! Summary • Dynamic nature of nucleosome posioning • Stac nature of nucleosome posioning • Challenges in measuring nucleosome occupancy Reference • Genome-Scale Iden8ficaon of Nucleosome Posions in S. cerevisiae. Science 2005, 309:30. • Contribu8on of histone sequence preferences to nucleosome organizaon: proposed defini8ons and methology. Genome Biology 2010. .