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. Dynamic Nucleosomes: Chromatin
Remodeling Complex
• Chroma8n remodeling complex replacing histone proteins with other variants Dynamic Nucleosomes: Chromatin
Remodeling Complex
• Chroma8n remodeling complex (with histone chaperones) replacing/removing histone proteins with other variants Dynamic Nucleosomes: Chromatin
Remodeling Complex
• 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 Dynamic Nucleosomes: Chromatin
Remodeling Complex
• 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’ Definitions of Terminology
• 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 Definitions of Terminology
• 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
Definitions of 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 Experimental Technology for Measuring
Nucleosome Organization
• 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 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 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 Yeast Genome-Scale Nucleosome Map
• Nucleosome-­‐free regions common in TF binding sites. Microarray data for nucleosome posi8ons Inferred nucleosome posi8ons with boxes for a TF binding mo8f DNA Sequence conserva8on score Yeast Genome-Scale Nucleosome Map
• Nucleosome-­‐free regions common in TF binding sites. Microarray data for nucleosome posi8ons Inferred nucleosome posi8ons with boxes for a TF binding mo8f DNA Sequence conserva8on 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 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.