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 posions are non-random and conserved across the similar cell types

• Nucleosome posioning affects regulaon

• The binding of other proteins affect the posions of nucleosomes

• Dynamic nature of nucleosome posioning influenced by the dynamic gene regulaon

• Stac nature of nucleosome posioning influenced by DNA sequence Dynamic Nucleosomes

• Kinec 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 mes 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 transcripon, DNA replicaon, etc. Dynamic Nucleosomes: 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: Complex

• Chroman remodeling complex replacing 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 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 – 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 posions: the nucleosome start/center/end posions of the 147bp sequence wrapped around a nucleosome

• Nucleosome configuraon – a set of non-overlapping nucleosome posions on a single DNA molecule of defined length. – if a is in state 1, then both the preceding and following 146 base pairs (bp) must be ‘0’ Definitions of Terminology

• Nucleosome organizaon: a probability distribuon 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 probabilies 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 posioning: the degree to which the posions of individual nucleosomes vary across the different configuraons of a nucleosome organizaon. – a perfectly posioned nucleosome is one that adopts the same posion across all measured configuraons – 30% posioning? – Absolute vs. condional posioning Definitions of Terminology

• Absolute nucleosome posioning at basepair x: the probability of a nucleosome starng at basepair x

– Absolute nucleosome posioning does not uniquely determine nucleosome organizaon • Condional nucleosome posioning at basepair x: the absolute posioning 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

• Digeson of chroman by micrococcal nuclease (MNase), an endonuclease that preferenally 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 digesng 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 posion at a single nucleode resoluon • Naked DNA as a control, but linker DNA is TA/AT rich, reducing the ulity 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 posions are different – With low coverage in sequencing, it is difficult to obtain a reliable map of nucleosome posions. 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 -Scale Nucleosome Map

• Map of posions of 2278 nucleosomes over 482 kilobases of DNA, including almost all of III and 223 addional regulatory regions – Most of the nucleosome were well posioned – A nucleosome free region of ~200bp in the Pol II promoters – Nucleosome free regions had evoluonarily conserved sequences – Most TF binding mofs were nucleosome free regions Yeast Genome-Scale Nucleosome Map

• Nucleosome-free regions common in TF binding sites.

Microarray data for nucleosome posions

Inferred nucleosome posions 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 posions with boxes for a TF binding mof

DNA Sequence conservaon score Yeast Genome-Scale Nucleosome Map

• Funconal transcripon factor binding mofs are more accessible than unbound mofs Nucleosome Maps

• DNA sequence is significantly predicve 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 Idenficaon of Nucleosome Posions in S. cerevisiae. Science 2005, 309:30.

• Contribuon of histone sequence preferences to nucleosome organizaon: proposed definions and methology. Genome Biology 2010.