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Thursday, February 25, 2010

AGBT 2010 - Arend Sidow - Stanford University School of Medicine

Extremely High-Resolution Nucleosome Organization Maps and Gene Expression Analysis in Purified Human Cells

Interested in how sequence and regulation interact in the cell. Focused on the role of nucleosome organization. Long standing collaboration : Anton Valouev, Steve Johnson, and LifeTech.

[Review of Compaction of DNA. bases, wrapped on nucleosomes, clusters of nucleosomes, extended form of DNA, condensed sections, chromosomes...]

Very fancy picture of DNA-Nucleosome Structures - ~156 bp wrap around each nucleosome.

Require regulatory elements to release DNA from nucleosomes.
* what are most people thinking about? Histone modifications. However, this is not what they're interested in.
* Instead, looking at how DNA is organized on nucleosomes.
* Nice figure of Averaged coverage of nucleosomes areound promotor -> gene region. Shows open regions,etc
* Fits nicely pol II binding.
* Data for specific cell types

* DNA Sequencing
* Local gene regulatory functions/protein
* Global/cellular parameters

Work in the field:
* Yeast, (Segql, widom and colleagues)
** Predictions on how they work
* C. elegans : Fire, Sidow and colleages
** fits slightly differently than Yeast
* Human... may yet be different than other.

Org chart:
* Maps of nuclosome position -> parameters of cellularnucleosome organization -> merge with (sequence dictated preferences of nucleosome) -> form models

Show illustration technique:
* Micrococcal nuclease digest used
* Use high salt concentraton. ( 1nucleosome/kb?)
* Used 4 cell types (CD4 CD8 T lymphocites, granulocytes, in vitro reconstitution, & control)

10bp Rotational Setting
* Pronounced propensity for the AA/AT/TA rich to be where the minor grove contacts nucleosome
* CG rich where Nucleosome major grouve contacts nucleosome?
* Overall GC preference for Nucleosome binding.

[I think I missed something in the above explanation.]

Phasing isn't always pretty - it has gaps and isn't perfectly even.
Plot phasing - you get an aggregate data set
* peaks are not even and smooth, starts to look like signal/noise gets messy.
* called the phasing diagram: Phaseogram.

Positioning can drive phasing in a stereotypic way.

Core + linker (193bp)
linker ~ 47bp (at least in this cell type)

Linker distance tends to vary slightly in cell type (eg, granulocytes vs. CD4/CD8).
in CD4/CD8, it's closer to 55bp.

Thus, if linker lengths change slightly, then the rotational setting, and the seqeunce specificity must be weaker.

Sequence alone is incapable of putting nucleotides next to each other.
However, you DO see a sequence signature when nucleotides assemble spontaneously. This seems to TUNE the nucleotide placements.

In vivo, this still seems to happen. Specific signals are highly enriched for positioning sites. However, these are relatively sparse. Phasing is also clean in close proximity to these locations.

Binding Proteins:
* Looking at NRSF binding sites, this is a strong enrichment for phasing on either side,
* NRSF actually sits to this location, not a nucleosome,
* This however, nucleates the phasing location.
* Same thing with CTCF
* the actual spacing between the nucleosomes is still cell type dependent, however, that is dependent on the biology of the cell, not on the factor itself.

In vivo vs in vitro dinucleotide coverage
* GC likes nucleosomes in vitro
* AT does not like nucleosomes in vitro
* In vitro, none of these seem to matter at all, overrideing it.... with exceptions!
* CpG islands are really liked by nucleosides, however, the biology overrides these, as they are usually nucleotide free.

Expressed vs non-expressed genes.
* As expression level rises: coverage falls (though, still at 90%)
* However, spacing drops more obviously. (200bp is default when not expressed, drops by 10% when expressed) [I hope I got that in the right order]

* Sequence preferences
* Nucleation of nucleaosome arrays
* inter nucleosome distances

All combine to decide nucleosome spacing.



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