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SPECIFIC AIMS
DNA in eukaryotic cells exists as chromatin, of which the fundamental unit is the nucleosome.Nucleosomes are DNA wrapped around octameric histone proteins and they play a fundamentalrole in both positive and negative regulation of proteins that require access to the DNA code.Nucleosomes can be remodeled (moved or removed) as well as labeled with a variety of post-translational modification (PTMs). These remodeling processes and PTMs play a critical role inregulation of gene transcription by RNA Polymerase II (Pol II) either via direct interaction withPol II, or via recruitment or inhibition of other transcription processes. The key role intranscription combined with the fact that many histone PTMs are heritable to the next cellgeneration makes understanding chromatin remodeling during transcription very important tocancer biology.
But understanding of the nucleosome-polymerase interaction duringgene transcription is currently limited by the inability to sensitively characterize withhigh spatial resolution the positions of polymerases and nucleosomes on individualchromatin fibers in cells
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Therefore, we are developing a single-molecule method (see Fig. 1)for mapping proteins on native chromatin that will provide this capability.
Our specifichypothesis is that optical tweezers unzipping of native chromatin will allow mapping of histones and polymerases with near base pair resolution on the same individual fiber 
.The specific open question we want to address is how histone H2B ubiquitylation interacts withFACT and Pol II during transcription, and what role antisense transcription plays in regulation of normal gene transcription at the molecular level. We haveseveral reasons to believe we can prove our hypothesis andshed light on these questions.
First
, The PI of this proposalis the co-inventor of the technique for mapping proteinbinding by unzipping single DNA molecules and an expert inconstructing tools for manipulating single DNA molecules.It has been shown that bound proteins can be detectedbecause the force required to unzip DNA is substantiallyhigher than for naked DNA.
Second
, It has recently beenshown that this DNA unzipping method can map thepositions of in vitro assembled mononucleosomes with closeto base pair resolution.
Third
, we are collaborating withlabs with extensive expertise in characterizing chromatinremodeling and Pol II elongation with ensemble methodssuch as Chromatin Immunoprecipitation (ChIP).
For this one-year ACS IRG proposal
, we are proposingtwo specific aims to generate the preliminary data necessary to pursue R01 funding for thisproject:
Specific Aim 1:
Prove that shotgun DNA mapping works in yeast cells and improve algorithms.Shotgun DNA mapping is the ability to identify the genomic location of a random DNA fragmentbased on its naked DNA unzipping forces compared with simulated unzipping forces of apublished genome. It is an enabler of our goal of native chromatin mapping.
Sub Aim 1
: Perform blinded unzipping of several yeast genomic DNA sequences from XhoIsites and attempt to identify sequences using shotgun DNA mapping method.
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Optical TrapssDNA
Coverglass
nucleosomeRNA Pol II
Figure 1
Proposed unzipping of single chromatin fibers with opticaltweezers. Optical tweezers use laser light focused through a microscopeobjective to apply and measure smallforces on microspheres attached tobiomolecules. Monitoring the lengthof ssDNAand the unzipping forces willreveal the position of nucleosomesand polymerases with close to basepair resolution.
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sjkoch4914

Thank you to Anthony, Andy, and Larry for help in writing this, and for all the help from our collaborators, Mary Ann, Kelly, and Karen!

reply01 / 21 / 2009
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