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Application for an Individual Allocation from American Cancer Society Institutional Research Grant #IRG-92-024 
 
This research plan is for a renewal of our first-year ACS IRG funding. An important note is that during our preliminary work in collaboration w
ith our mentor’s lab, we have decided our most productive route is to pursue
chromatin mapping applications during gene transcription. Thus, our research plan is distinct from our firstapplication, and our original title is misleading.
Abstract and Specific Aims
DNA in eukaryotic cells exists as chromatin, of which the fundamental unit is the nucleosome
(
1
)
. Nucleosomesconsist 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. In order to modulatethis barrier, nucleosomes can be remodeled (moved or removed) or modified by a variety of post-translationalmodification (PTMs). During transcription, both remodeling and PTMs regulate RNA Polymerase II (Pol II)
(
2 
)
.The key role in transcription combined with the fact that many histone PTMs are heritable to the next cellgeneration makes understanding chromatin remodeling during transcription very important to cancer biology
(
3 
)
.
However, understanding these processes during gene transcription is currently limited by the inabilityto sensitively characterize with high spatial resolution the positions of polymerases and nucleosomeson individual chromatin fibers in cells
.
 
Therefore, we are developing asingle-molecule method (see Fig. 1) for mapping proteins on nativechromatin that will provide this capability.
Our specific hypothesis isthat optical tweezers unzipping of native chromatin will allowmapping of histones and polymerases with near base pairresolution on the same individual fiber
. We have several reasons tobelieve we can prove our hypothesis and shed light on these questions.
First
, The PI of this proposal is the co-inventor of the technique for mapping protein binding by unzipping single DNA molecules and anexpert in constructing tools for manipulating single DNA molecules
(
4-7 
)
. Ithas been shown that bound proteins can be detected because the forcerequired to unzip protein-bound DNA is substantially higher than for naked DNA.
Second
, It has recently been shown that this DNA unzippingmethod can map the positions of in vitro assembled mononucleosomeswith close to base pair resolution
(
8, 9 
)
.
Third
, we are collaborating withlabs with extensive expertise in characterizing chromatin remodeling andPol II elongation with ensemble methods such as ChromatinImmunoprecipitation (ChIP)
(
10-13 
)
.
For this one-year ACS IRG proposal
, we are proposing two specific aims to generate key preliminary datafor longer-term R01 funding:
Specific Aim 1:
Prove that shotgun DNA mapping (SDM) works with yeast genomic DNA and improve theSDM algorithms.Shotgun DNA mapping is the ability to identify the genomic location of a random DNA fragment based onits naked DNA unzipping forces compared with simulated unzipping forces of a published genome
(
14 
)
. It isan enabler of our goal of native chromatin mapping.
Specific Aim 2:
Determine the unzipping signature for RNA Polymerase II by unzipping through stalled invitro transcription complexes.
 
The innovative single-molecule results pursued in these one-year aims will provide strong preliminary data insupport of our NIH R01 application. Success in each aim on its own will produce high impact publications and
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.
ACS IRG 2009 Proposal, Steven J. Koch, sjkoch@unm.edu January 23, 20091
 
2009 American Cancer Society (ACS) IRG application from the lab ofSteven J. Koch at the University of New Mexico.sjkoch@unm.eduhttp://openwetware.org/wiki/Koch_Lab
 
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 transcriptionas well as provide a new tool for studying chromatin biology in cancer cells. Two cancer biology areas we wishto pursue are epigenetic control of gene transcription
(
3 
)
and chromatin remodeling during DNA double-strandbreak repair 
(
10, 15 
)
.
Background and significanceA. Chromatin remodeling during transcription elongation
During transcription, nucleosomes are removed from in front of the polymerase to prevent stalling of elongation. Nucleosomes are then reassembled behind the polymerase, which is important for preventingimproper transcription initiation within the gene (termed cryptic initiation, see Fig. 2). This remodeling processis coordinated by histone chaperones that assist in chromatin reassembly. During transcription, lysineresidues on the histone tails are marked with various post-translational modifications (PTMs), but their functions in transcription are not yet wellunderstood
(
16 
)
. In order to understand theseprocesses, we need to know with basepair precision the position of all polymerasesand nucleosomes on the gene.Furthermore, we need to know whether thenucleosomes are completely assembledoctamers or are tetrameric or hexameric.
Finally, we need to know thisinformation for polymerases andnucleosomes on the same individualgene
—
so that their direct interactions canbe seen.The main tool for studying remodeling during transcription is chromatin immunoprecipitation (ChIP). ChIP is apowerful method that can provide some of the needed information such as relative levels of polymerase andnucleosome occupancy, as well as information about PTMs and nucleosome structure (tetramer v. octamer).However, the spatial resolution of ChIP is limited to about 100 base pairs and it is unable to provide informationabout single chromatin fibers. Thus,
we are working to provide a much needed single-molecule analysismethod based on unzipping of chromatin fibers with optical tweezers
.
Mary Ann Osley’s lab has recently been studying the interaction of a particular histone chaperone, FACT
(
17-20 
)
,and a specific PTM, monoubiquitylation of histone H2B (H2B-ub)
(
11
)
. Both FACT and H2B-ub are important tocancer biology
(
21
)
. The Osley lab has discovered that when both FACT and H2B-ub are not functioning, somekind of improper chromatin structure is assembled on active genes. The molecular nature of thismisassembled chromatin is very difficult to decipher with ChIP or other ensemble assays. Answeringquestions about this process is a specific motivation for our single-molecule technique development, but weexpect to be able to study chromatin structure during many important cellular processes and in any eukaryoticorganism, particularly as related to human cancers.
B. Antisense Transcription
The misassembled chromatin mentioned in the previous section can lead to improper Pol II transcriptioninitiation from promoters within genes or elsewhere that would normally be blocked by nucleosomes. This iscalled cryptic initiation and it is rapidly becoming an important area of study in transcription.
(
22, 23 
)
A particularlyfascinating, and possibly crucial point of cryptic initiation is that polymerases may initiate in the antisensedirection, and this has indeed very recently been shown to be widely prevalent
(
24 
)
. Buratowski says in his
perspective, “The mystery is why RNA
polymerase II traveling in one direction can produce RNAs thousands of nucleotides long, whereas polymerases moving in the
opposite direction don’t get very far.”
(
22 
)
One possibleexplanation is that antisense-oriented polymerases quickly encounter sense-oriented polymerases, and these
Figure 2
Nucleosome remodeling during transcription. Nucleosomes areevicted in front of the polymerase and reassembled behind thepolymerase. Proper reassembly prevents initiation from cryptic promoters.
PolIITranscriptionReassembled
Nucleosomespromotercrypticpromoter
ACS IRG 2009 Proposal, Steven J. Koch, sjkoch@unm.edu January 23, 20092
 
head-to-head collisions effectively stall both complexes. The ability to detect sense orientation of Pol IIcomplexes is difficult with ensemble techniques. Due to the asymmetry of the Pol II-DNA complex (in contrastto the nucleosome)
(
25 
)
,
it is possible that single-molecule unzipping will be able to clearly indicate thetranscriptional orientation of the Pol II complexes on individual chromatin fibers
. This may open thedoor for answering critical open questions in Pol II transcription, such as: are head-to-head collisions of sense-and antisense-oriented polymerases common in vivo? Is this an important component of gene regulation or misregulation?
C. Mapping protein binding by single-molecule DNA unzipping
In my dissertation work, I showed that positions of DNA-bindingproteins could be mapped with high resolution by unzipping single DNAmolecules with optical tweezers
(
4 
)
. Fig. 1 shows conceptually howthese experiments are carried out with optical tweezers
—
thoughinstead of a chromatin fiber, I unzipped DNA plus site-specific bindingproteins such as EcoRI. To enable these experiments, I designed andimplemented a versatile unzipping anchoring construct that allows for unzipping of any DNA molecule with a kno
wn 5’ or 3’ overhang (see
Fig. 4). These two achievements are the foundation for our goal of mapping native chromatin by unzipping. A former labmate has usedmy versatile unzipping construct to unzip through reconstitutedmononucleosomes and tetrasomes in the Wang lab. Some of this datais shown in Fig. 3. The authors clearly demonstrated that
mononucleosome positions can be mapped with about 3 basepairprecision
. Furthermore, they were
able to easily distinguishbetween octameric and tetrameric nucleosomes
(
8 
)
. These resultsgive us confidence that we will be able to map nucleosome positions on native chromatin. One of the specificaims of this proposal is to perform single-molecule unzipping of stalled RNA Polymerase II complexes.
Success in this aim will provide strong evidence thatwe will be able to map and identify polymerases andnucleosomes on individual native chromatin fibers
.
Progress and Preliminary Data
Discussed below are progress and results funded by thefirst year of ACS IRG funding as well as those funded by startup and directly
relevant to this renewal andfuture R01 funding
.
A. Optical tweezers instrumentation and data analysissoftware
As of January 2009, we now have all of the components requiredfor our optical tweezers instrumentation. The remaining importantsteps are assembly and calibration of optical tweezers andconversion of existing data acquisition software for modifiedhardware. We estimate approximately two months for these tasks,which will not be funded by this renewal. Important componentswe have on hand and milestones we have achieved include:An Olympus IX-71 inverted microscope with a PlanApo 1.4NA oil immersion objective.A Mad City Labs 1-D piezo stage with 30 micron travel andsub-nanometer precision.An OEM laser diode system with a 690 nm (visible-red)laser diode.
5’
BiotinNickDig
3’5’
Sticky endfor ligation
Figure 4
The versatile unzipping construct from Koch etal. 2002 will be used in both aims. Any sticky end desiredis easily created via choice of oligonucleotidesequence.
Data from Shundrovsky,et al., Nature Structural& Molecular Biology
13
,p. 549 (
Not Koch Data
)
©2006 Nature Publishing Group
Figure 3
The protein mapping methoddeveloped by Koch has been shown tobe capable of measuring the positions of nucleosomes with 3 bpprecision. It wasalso shown possible to clearly distinguishbetween octamesand tetramers.
Figure 5
. Photo of summer 2008 532 nm opticaltweezers successful prototype. Optical pathoutlined in green. We trapped and manipulatedmicrospheres with this setup, but the laserstability and lack of piezostage were not suitablefor unzipping experiments.
ACS IRG 2009 Proposal, Steven J. Koch, sjkoch@unm.edu January 23, 20093
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