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Nucleosome Positioning and Organization

02-‐715 Advanced Topics in Computa8onal 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 regula8on

• The binding of other proteins affect the posi8ons of nucleosomes

• Dynamic nature of nucleosome posi8oning influenced by the dynamic gene regula8on

• Sta8c nature of nucleosome posi8oning 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 replica8on, etc.

Dynamic Nucleosomes: Chromatin Remodeling Complex

• Nucleosome sliding – ATP-‐dependent chroma8n remodeling complexes bind to nucleosome

core proteins and DNA that wraps around it, and use the energy of ATP hydrolysis to move DNA rela8ve to the core.


• Chroma8n remodeling complex replacing histone proteins with other variants


• Chroma8n remodeling complex (with histone chaperones) replacing/removing histone proteins with other variants


• As genes are turned on and off, chroma8n remodeling complex are brought to specific regions of DNA to locally influence chroma8n structure

• Certain chroma8n structure can be inherited during cell division

Dynamic Nucleosomes: Chromatin Remodeling with Code Reader-Writer

Complex • Spreading chroma8n

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


• Spreading wave of chroma8n condensa8on to form a long range heterochroma8n

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 configura8on – 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’


• Nucleosome organiza8on: a probability distribu8on over nucleosome configura8ons

– P: nucleosome organiza8on

– C: a set of nucleosome configura8ons

– P(c): the probability of a nucleosome configura8on c


• Nucleosome occupancy: the sum of the probabili8es of the configura8ons in which the base pair is covered by a nucleosome

– Occ(x): the occupancy at basepair x – C: nucleosome configura8on

– P(c): nucleosome organiza8on

Illustration of Different Terminology


• Nucleosome posi8oning: the degree to which the posi8ons of individual nucleosomes vary across the different configura8ons of a nucleosome organiza8on. – a perfectly posi8oned nucleosome is one that adopts the same

posi8on across all measured configura8ons

– 30% posi8oning? – 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 organiza8on

• 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 chroma8n 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: rela8vely protected by nucleosomes

• Measure the diges8ng paeern with microarray or sequencing of the nucleosome-‐protected DNA segments

– Occ(x): occupancy at base pair x – ri: read counts at basepair i


• Challenges – Bias introduced by MNase’s preference of TA/AT dinucleo8de 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 popula8on of cells

• We get to measure only the average of the dynamically changing nucleosome posi8ons


• 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


• 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 posi8oned

– 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


• Nucleosome-‐free regions common in TF binding sites.

Microarray data for nucleosome posi8ons

DNA Sequence conserva8on score

Inferred nucleosome posi8ons with boxes for a TF binding mo8f


• Func8onal transcrip8on factor binding mo8fs are more accessible than unbound mo8fs

Nucleosome Maps

• DNA sequence is significantly predic8ve of nucleosome organiza8on in vitro and in vivo – discussion on Wednesday!

Summary

• Dynamic nature of nucleosome posi8oning

• Sta8c nature of nucleosome posi8oning

• Challenges in measuring nucleosome occupancy

Reference

• Genome-‐Scale Iden8fica8on of Nucleosome Posi8ons in S. cerevisiae. Science 2005, 309:30.

• Contribu8on of histone sequence preferences to nucleosome organiza8on: proposed defini8ons and methology. Genome Biology 2010.

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