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2009 Proposal--CAREER: Single-Molecule Analysis of Genomic DNA and Chromatin in Eukaryotic Transcription

2009 Proposal--CAREER: Single-Molecule Analysis of Genomic DNA and Chromatin in Eukaryotic Transcription

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Published by Steven J. Koch
This is the technical part of my 2009 CAREER Proposal (just the Project Summary and Project Description). The Project Summary is required to be 1 page and to separately discuss "Intellectual Merit" and "Broader Impacts." The Project Description is 15 pages or less. Our overall goal is to develop single-molecule mapping of native chromatin based on optical tweezers unzipping.
This is the technical part of my 2009 CAREER Proposal (just the Project Summary and Project Description). The Project Summary is required to be 1 page and to separately discuss "Intellectual Merit" and "Broader Impacts." The Project Description is 15 pages or less. Our overall goal is to develop single-molecule mapping of native chromatin based on optical tweezers unzipping.

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Categories:Types, Research, Science
Published by: Steven J. Koch on Jul 22, 2009
Copyright:Attribution Non-commercial

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05/11/2014

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The long-term mission of our laboratory is to enable and make important discoveries in molecular cellbiology by innovating new biophysical methods and culturing interdisciplinary research and educationpartnerships. The specific research goal of this 5-year proposal is to develop new methods for 
site-specific single-molecule analysis of native DNA and chromatin extracted from yeast cells
. Our long-term mission is also supported by our specific educational and broader impacts goals in thisproposal, which will multiply the impact of our research goals. These include participation in OpenScience, integration of research and university education, and community and local school outreach.Together, the research and education goals in this proposal will establish a successful career path for thisPI as leader of an exciting biophysics research laboratory collaborating with leading chromatin andtranscription biologists, successful university educator, and recruiter of underrepresented minorities toresearch careers.
Intellectual Merit
In order to understand the dynamics of chromatin remodelingduring gene transcription,
new biophysical tools are neededfor single-molecule analysis of native chromatin
.
 
Therefore,we are developing a unique single-molecule method (see Fig. 1)for mapping proteins on native chromatin that will provide thiscapability.
Our specific hypothesis is that optical tweezersunzipping of native chromatin will allow mapping ofhistones and polymerases with near base pair resolution onthe same individual fiber
. The specific aims have beendesigned to achieve this goal of native chromatin mapping byunzipping. Each aim can produce high-impact results anddevelop new useful biophysical techniques, independent of thesuccess of the other aims:
1. Develop Shotgun DNA mapping (SDM)
as a newsingle-molecule method for identifying unknown DNA fragments. Shotgun DNA mapping is theability to identify the genomic location of a random DNA fragment based on its naked DNA unzippingforces compared with simulated unzipping forces of a published genome.
2. Determine the unzipping signature for RNA Polymerase II (Pol II) transcription complexes
.
3.
 
Map native yeast chromatin by single-molecule unzipping
.
Broader Impacts
1.
Open Science
. The entire CAREER project will be conducted as Open Science. This willinclude Open Notebook Science, Open Access publishing, free availability of raw data and allstages of processed data, and sharing of all data acquisition and processing software.Furthermore, the proposal will be published at the time of its submission.2.
Integration of research with undergraduate and graduate education
. Virtual reality physicsdemos that mirror our 
real life
demos will be developed for 
Second Life.
I will continuetransforming Junior Lab course into an open science course and develop new biophysicsexperimental modules.3.
Community and school outreach in Albuquerque
. PI participation in outreach anddevelopment of new partnerships with middle school teachers as well as encouragement of strong outreach participation by graduate and undergraduate researchers in the lab.
 
OpticalTrapssDNA
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.
CAREER: Single-Molecule Analysis of Genomic DNA and Chromatin in Eukaryotic TranscriptionProject Summary, Page 1 of 1
 
I. Specific Aims
DNA in eukaryotic cells exists as chromatin, of which the fundamental unit is the nucleosome
(
)
.Nucleosomes consist of an octamer of histone proteins which control access of other proteins to DNA.Therefore, nucleosomes are barriers in processes such as transcription, replication, and DNA repair. Inorder to modulate this barrier, nucleosomes can be remodeled (moved or removed) or modified by avariety of post-translational modification (PTMs). During transcription, both remodeling and PTMsregulate RNA Polymerase II (Pol II)
(
)
. The key role in transcription combined with the fact that manyhistone PTMs are heritable to the next cell generation makes understanding chromatin remodeling duringtranscription very important to cancer biology
(
)
.
However,understanding these processes during gene transcription iscurrently limited by the inability to sensitively characterizewith high spatial resolution the positions of polymerasesand nucleosomes on individual chromatin fibers in cells
.
 
Therefore, we are developing a unique single-molecule method(see Fig. 1) for mapping proteins on native chromatin that willprovide this capability.
Our specific hypothesis is that opticaltweezers unzipping of native chromatin will allow mappingof histones and polymerases with near base pair resolutionon the same individual fiber
. We have several reasons tobelieve we can prove our hypothesis and shed light on thesequestions.
First
, The PI of this proposal is the co-inventor of thetechnique for mapping protein binding by unzipping single DNAmolecules and an expert in constructing tools for manipulatingsingle DNA molecules
(
5-8 
)
. It has been shown that boundproteins can be detected because the force required to unzipprotein-bound DNA is substantially higher than for naked DNA.
Second
, It has recently been shown that this DNA unzipping method can map the positions of in vitroassembled mononucleosomes with close to base pair resolution
(
9, 10 
)
.
Third
, we are collaborating withlabs with extensive expertise in characterizing chromatin remodeling and Pol II elongation with ensemblemethods such as Chromatin Immunoprecipitation (ChIP)
(
11-14 
)
.
The specific aims for this CAREER proposal have been designed to achieve our goal of nativechromatin mapping by unzipping. Each aim can produce high-impact results and develop newuseful biophysical techniques, independent of the success of the other aims
:
1. Develop Shotgun DNA mapping (SDM)
as a new single-molecule method for identifying unknownDNA fragments. Shotgun DNA mapping is the ability to identify the genomic location of a random DNAfragment based on its naked DNA unzipping forces compared with simulated unzipping forces of apublished genome. We have preliminary indication that it will work well in the yeast genome
(
15 
)
and it isan enabler of our goal of native chromatin mapping. In this aim, we will:
 
1.1
Validate SDM for identifying yeast genomic DNA fragments
. We have created a set of unzipping constructs from a library of S. cerevesiae shotgun clones. We will use SDM to identifyclones. SDM success rate will be characterized by subsequent DNA sequencing of the clones.1.2
Improve DNA unzipping simulations and SDM matching algorithms
. To work with larger setsof possible fragments (larger genome than yeast or more frequent cutter than XhoI), the sensitivityand specificity need to be increased. We will do so by increasing the sophistication and quality of thesimulations and matching algorithms.
OpticalTrapssDNA
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.
CAREER: Single-Molecule Analysis of Genomic DNA and Chromatin in Eukaryotic TranscriptionProject Description, Page 1 of 15
 
1.3
Automate SDM data acquisition and analysis
. SDM automation will be required for genome-wide analyses and will greatly increase the practicality of the method. The goal of this aim is to fullyeliminate the need for user input, once a micro-flow cell has been manually mounted to the stage.
2.
 
Determine the unzipping signature for RNA Polymerase II (Pol II) transcription complexes
. Inthis aim, we will determine the in vitro Pol II unzipping signature for use in identifying native complexes inAim 3. Furthermore, new structural information about in vitro transcription complexes will be obtained.2.1
Unzip stalled Pol II in vitro transcription complexes
. Stalled complexes provided by theAdelman laboratory will be unzipped from both upstream and downstream directions. Individual
traces will be aligned and two reference ―signatures‖ will be produced: one for each sense or 
antisense orientation of transcription.2.2
Atomistically model unzipping of protein-DNA complexes
. Molecular dynamics simulationsof the unzipping of naked DNA and a model protein-DNA system will be carried out. Results willguide structural interpretation of Pol II data from aim 2.1 and will be used to initiate a separate projectwith the long-term goal of full atomistic simulations of nucleosome and Pol II unzipping.
3.
 
Map native yeast chromatin by single-molecule unzipping
. The ultimate goal of this proposal ishigh-resolution, single-molecule, site-specific mapping of nucleosome and polymerase positions on nativechromatin. Various independent approaches will be taken towards native chromatin unzipping in this aimand significant progress towards the overall goal will be achieved, independent of success of the other aims.3.1
Unzipping of yeast plasmid chromatin
. Yeast plasmid chromatin
(
16 
)
will be isolated with andwithout induction of the
PHO 
5 gene encoded in the plasmid
(
17, 18 
)
and ligated to unzipping constructs.Single-molecule unzipping will be used to determine the locations of the nucleosomes and comparedwith well-known locations from ensemble measurements.3.2
Unzipping of native yeast chromatin from a single genomic site
. Genomic chromatincontaining the YLR454 gene will be digested with I-SceI and ligated onto unzipping constructs.Nucleosome and polymerase positions will be measured by single-molecule unzipping. Chromatinstructure will be mapped in wild type, an spt16 mutant, a mutant deficient in histone H2Bubiquitylation, and the double mutant
(
12 
)
. Correlated nucleosome and polymerase occupancy onindividual chromatin fibers will be measured.3.3
Shotgun chromatin mapping
. Yeast native chromatin will be digested with XhoI and randomfragments will be ligated onto unzipping constructs. Shotgun DNA mapping will be used to identifyindividual fragments. Nucleosome and polymerase positions will be identified at high resolution oneach individual fiber. High throughput experiments will reveal genome-wide correlations betweenpolymerases and histones and also identify divergent and cryptically initiated polymerases in wildtype, spt16 mutants, H2B-ubiquitylation mutants, and double mutants
(
12 
)
.
New biophysical tools are needed for single-molecule analysis of native chromatin.
Success in thisproject will develop such tools and will answer fundamental open questions in eukaryotic transcription.Furthermore,
we will have developed a unique and powerful single-molecule genetics technique
 that will enable many future high-impact research avenues. These may include studying single-moleculemapping of epigenetic chromatin marks, DNA damage repair and replication, structural genome mappingby unzipping, single-molecule telomere mapping, and studies of alternative splicing by single-moleculecDNA unzipping. Given this wealth of possibilities and the strong collaborations formed, this CAREERproject will lay a solid foundation for a successful research career.
CAREER: Single-Molecule Analysis of Genomic DNA and Chromatin in Eukaryotic TranscriptionProject Description, Page 2 of 15

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