2009 Acs Irg Proposal, Specific Aims Draft

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 fundamental role 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 posttranslational modification (PTMs). These remodeling processes and PTMs play a critical role in regulation of gene transcription by RNA Polymerase II (Pol II) either via direct interaction with Pol II, or via recruitment or inhibition of other transcription processes. The key role in transcription combined with the fact that many histone PTMs are heritable to the next cell generation makes understanding chromatin remodeling during transcription very important to cancer biology. But understanding of the nucleosome-polymerase interaction during gene transcription is currently limited by the inability to sensitively characterize with high spatial resolution the positions of polymerases and nucleosomes on individual chromatin fibers in cells. Therefore, we are developing a single-molecule method (see Fig. 1) for mapping proteins on native chromatin that will provide this capability. Our specific hypothesis 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 with FACT and Pol II during transcription, and what role antisense transcription plays in regulation of normal gene transcription at the molecular level. We have Optical several reasons to believe we can prove our hypothesis and Trap nucleosome shed light on these questions. First, The PI of this proposal RNA Pol II is the co-inventor of the technique for mapping protein binding by unzipping single DNA molecules and an expert in ssDNA constructing tools for manipulating single DNA molecules. Coverglass It has been shown that bound proteins can be detected because the force required to unzip DNA is substantially Figure 1 Proposed unzipping of higher than for naked DNA. Second, It has recently been single chromatin fibers with optical shown that this DNA unzipping method can map the tweezers. Optical tweezers use laser light focused through a microscope positions of in vitro assembled mononucleosomes with close objective to apply and measure small to base pair resolution. Third, we are collaborating with forces on microspheres attached to labs with extensive expertise in characterizing chromatin biomolecules. Monitoring the length of ssDNA and the unzipping forces will remodeling and Pol II elongation with ensemble methods reveal the position of nucleosomes such as Chromatin Immunoprecipitation (ChIP). and polymerases with close to base pair resolution.

For this one-year ACS IRG proposal, we are proposing two specific aims to generate the preliminary data necessary to pursue R01 funding for this project: 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 fragment based on its naked DNA unzipping forces compared with simulated unzipping forces of a published 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 XhoI sites and attempt to identify sequences using shotgun DNA mapping method. Page 1 of 2

Sub Aim 2: algorithm.

Improve the modeling of DNA unzipping forces and optimize the matching

Specific Aim 2: Determine the sense and antisense unzipping signature for RNA Polymerase II by unzipping through stalled in vitro transcription complexes. The innovative single-molecule results pursued in these one-year aims will provide strong preliminary data in support of our NIH R01 application. Success in each aim on its own will produce high impact publications and open doors for further applications in genomics and in vitro Pol II studies. Success in our longer-term goal of single-molecule chromatin mapping will provide important insights into the open questions in Pol II transcription as well as provide a new tool for studying chromatin biology in cancer cells. Two cancer biology areas we wish to pursue are epigenetic control of gene transcription and chromatin remodeling during DNA double-strand break repair. (The next section will be Background and Significance.) (These references will actually go at the end of the full proposal.)

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