Nucleosome Positioning and Organization
02-715 Advanced Topics in Computa onal 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 posi ons are non-random and conserved across the similar cell types
• Nucleosome posi oning affects gene regula on
• The binding of other proteins affect the posi ons of nucleosomes
• Dynamic nature of nucleosome posi oning influenced by the dynamic gene regula on
• Sta c nature of nucleosome posi oning influenced by DNA sequence Dynamic Nucleosomes
• Kine c 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 transcrip on, DNA replica on, etc. Dynamic Nucleosomes: Chromatin Remodeling Complex
• Nucleosome sliding – ATP-dependent chroma n remodeling complexes bind to nucleosome core proteins and DNA that wraps around it, and use the energy of ATP hydrolysis to move DNA rela ve to the core. Dynamic Nucleosomes: Chromatin Remodeling Complex
• Chroma n remodeling complex replacing histone proteins with other variants Dynamic Nucleosomes: Chromatin Remodeling Complex
• Chroma n remodeling complex (with histone chaperones) replacing/removing histone proteins with other variants Dynamic Nucleosomes: Chromatin Remodeling Complex
• As genes are turned on and off, chroma n remodeling complex are brought to specific regions of DNA to locally influence chroma n structure
• Certain chroma n structure can be inherited during cell division Dynamic Nucleosomes: Chromatin Remodeling with Code Reader-Writer Complex • Spreading chroma n 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 chroma n condensa on to form a long range heterochroma n Nucleosomes and Chromatin Structure
• H3 variant histone, called CENP-A replaces H3 in centromeric DNA sequences Definitions of Terminology
• Nucleosome posi ons: the nucleosome start/center/end posi ons of the 147bp sequence wrapped around a nucleosome
• Nucleosome configura on – a set of non-overlapping nucleosome posi ons 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 organiza on: a probability distribu on over nucleosome configura ons
– P: nucleosome organiza on – C: a set of nucleosome configura ons – P(c): the probability of a nucleosome configura on c Definitions of Terminology
• Nucleosome occupancy: the sum of the probabili es of the configura ons in which the base pair is covered by a nucleosome
– Occ(x): the occupancy at basepair x – C: nucleosome configura on – P(c): nucleosome organiza on Illustration of Different Terminology Definitions of Terminology
• Nucleosome posi oning: the degree to which the posi ons of individual nucleosomes vary across the different configura ons of a nucleosome organiza on. – a perfectly posi oned nucleosome is one that adopts the same posi on across all measured configura ons – 30% posi oning? – Absolute vs. condi onal posi oning Definitions of Terminology
• Absolute nucleosome posi oning at basepair x: the probability of a nucleosome star ng at basepair x
– Absolute nucleosome posi oning does not uniquely determine nucleosome organiza on • Condi onal nucleosome posi oning at basepair x: the absolute posi oning 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
• Diges on of chroma n by micrococcal nuclease (MNase), an endonuclease that preferen ally cuts linker DNA rather than DNA wrapped around a nucleosome – highly digested DNA: depleted of nucleosomes – under-digested DNA: rela vely protected by nucleosomes
• Measure the diges ng pa ern 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 dinucleo de as its cleavage site • Cannot obtain the nucleosome posi on at a single nucleo de resolu on • Naked DNA as a control, but linker DNA is TA/AT rich, reducing the u lity of naked DNA as a control – Experiment is performed not on a single cell, but on a popula on of cells • We get to measure only the average of the dynamically changing nucleosome posi ons Experimental Technology for Measuring Nucleosome Organization
• Challenges – In vitro and in vivo nucleosome posi ons are different – With low coverage in sequencing, it is difficult to obtain a reliable map of nucleosome posi ons. 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 posi ons of 2278 nucleosomes over 482 kilobases of Saccharomyces cerevisiae DNA, including almost all of chromosome III and 223 addi onal regulatory regions – Most of the nucleosome were well posi oned – A nucleosome free region of ~200bp in the Pol II promoters – Nucleosome free regions had evolu onarily conserved sequences – Most TF binding mo fs were nucleosome free regions Yeast Genome-Scale Nucleosome Map
• Nucleosome-free regions common in TF binding sites.
Microarray data for nucleosome posi ons
Inferred nucleosome posi ons with boxes for a TF binding mo f
DNA Sequence conserva on score Yeast Genome-Scale Nucleosome Map
• Nucleosome-free regions common in TF binding sites.
Microarray data for nucleosome posi ons
Inferred nucleosome posi ons with boxes for a TF binding mo f
DNA Sequence conserva on score Yeast Genome-Scale Nucleosome Map
• Func onal transcrip on factor binding mo fs are more accessible than unbound mo fs Nucleosome Maps
• DNA sequence is significantly predic ve of nucleosome organiza on in vitro and in vivo – discussion on Wednesday! Summary
• Dynamic nature of nucleosome posi oning
• Sta c nature of nucleosome posi oning
• Challenges in measuring nucleosome occupancy Reference
• Genome-Scale Iden fica on of Nucleosome Posi ons in S. cerevisiae. Science 2005, 309:30.
• Contribu on of histone sequence preferences to nucleosome organiza on: proposed defini ons and methology. Genome Biology 2010.