CAREER: Single-Molecule Analysis of

Genomic DNA and Chromatin in Eukaryotic Transcription Project Summary, Page 1 of 1

The long-term mission of our laboratory is to enable and make important discoveries in molecular cell
biology by innovating new biophysical methods and culturing interdisciplinary research and education
partnerships. 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 this
proposal, which will multiply the impact of our research goals. These include participation in Open
Science, 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 this
PI as leader of an exciting biophysics research laboratory collaborating with leading chromatin and
transcription biologists, successful university educator, and recruiter of underrepresented minorities to
research careers.

Intellectual Merit Optical

nucleosome Trap
In order to understand the dynamics of chromatin remodeling RNA Pol II
during gene transcription, new biophysical tools are needed
ssDNA
for single-molecule analysis of native chromatin. Therefore,
we are developing a unique single-molecule method (see Fig. 1) Coverglass
for mapping proteins on native chromatin that will provide this
capability. Our specific hypothesis is that optical tweezers Figure 1 Proposed unzipping of
single chromatin fibers with optical
unzipping of native chromatin will allow mapping of tweezers. Optical tweezers use laser
histones and polymerases with near base pair resolution on light focused through a microscope
the same individual fiber. The specific aims have been objective to apply and measure small
designed to achieve this goal of native chromatin mapping by forces on microspheres attached to
biomolecules. Monitoring the length
unzipping. Each aim can produce high-impact results and
of ssDNA and the unzipping forces will
develop new useful biophysical techniques, independent of the reveal the position of nucleosomes
success of the other aims: and polymerases with close to base
pair resolution.
1. Develop Shotgun DNA mapping (SDM) as a new
single-molecule method for identifying unknown DNA fragments. 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.

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 will
include Open Notebook Science, Open Access publishing, free availability of raw data and all
stages 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 physics
demos that mirror our “real life” demos will be developed for “Second Life.” I will continue
transforming Junior Lab course into an open science course and develop new biophysics
experimental modules.
3. Community and school outreach in Albuquerque. PI participation in outreach and
development of new partnerships with middle school teachers as well as encouragement of
strong outreach participation by graduate and undergraduate researchers in the lab.
CAREER: Single-Molecule Analysis of
Genomic DNA and Chromatin in Eukaryotic Transcription Project Description, Page 1 of 15

I. Specific Aims
(2)
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. In
order to modulate this barrier, nucleosomes can be remodeled (moved or removed) or modified by a
variety of post-translational modification (PTMs). During transcription, both remodeling and PTMs
(3)
regulate RNA Polymerase II (Pol II) . 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
(4)
transcription very important to cancer biology . However,
understanding these processes during gene transcription is Optical
currently limited by the inability to sensitively characterize nucleosome Trap
with high spatial resolution the positions of polymerases RNA Pol II
and nucleosomes on individual chromatin fibers in cells.
ssDNA
Therefore, we are developing a unique single-molecule method
(see Fig. 1) for mapping proteins on native chromatin that will Coverglass
provide this capability. Our specific hypothesis is that optical
tweezers unzipping of native chromatin will allow mapping Figure 1 Proposed unzipping of
single chromatin fibers with optical
of histones and polymerases with near base pair resolution tweezers. Optical tweezers use laser
on the same individual fiber. We have several reasons to light focused through a microscope
believe we can prove our hypothesis and shed light on these objective to apply and measure small
questions. First, The PI of this proposal is the co-inventor of the forces on microspheres attached to
biomolecules. Monitoring the length
technique for mapping protein binding by unzipping single DNA of ssDNA and the unzipping forces will
molecules and an expert in constructing tools for manipulating reveal the position of nucleosomes
(5-8)
single DNA molecules . It has been shown that bound and polymerases with close to base
proteins can be detected because the force required to unzip pair resolution.
protein-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 vitro
(9, 10)
assembled mononucleosomes with close to base pair resolution . Third, we are collaborating with
labs with extensive expertise in characterizing chromatin remodeling and Pol II elongation with ensemble
(11-14)
methods such as Chromatin Immunoprecipitation (ChIP) .

The specific aims for this CAREER proposal have been designed to achieve our goal of native
chromatin mapping by unzipping. Each aim can produce high-impact results and develop new
useful biophysical techniques, independent of the success of the other aims:

1. Develop Shotgun DNA mapping (SDM) as a new single-molecule method for identifying unknown
DNA fragments. 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
(15)
published genome. We have preliminary indication that it will work well in the yeast genome and it is
an 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 identify
clones. 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 sets
of possible fragments (larger genome than yeast or more frequent cutter than XhoI), the sensitivity
and specificity need to be increased. We will do so by increasing the sophistication and quality of the
simulations and matching algorithms.
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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 fully
eliminate 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. In
this aim, we will determine the in vitro Pol II unzipping signature for use in identifying native complexes in
Aim 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 the
Adelman 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 simulations

of the unzipping of naked DNA and a model protein-DNA system will be carried out. Results will
guide structural interpretation of Pol II data from aim 2.1 and will be used to initiate a separate project
with 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 is
high-resolution, single-molecule, site-specific mapping of nucleosome and polymerase positions on native
chromatin. Various independent approaches will be taken towards native chromatin unzipping in this aim
and significant progress towards the overall goal will be achieved, independent of success of the other
aims.
(16)
3.1 Unzipping of yeast plasmid chromatin. Yeast plasmid chromatin will be isolated with and
(17, 18)
without induction of the PHO5 gene encoded in the plasmid and ligated to unzipping constructs.
Single-molecule unzipping will be used to determine the locations of the nucleosomes and compared
with well-known locations from ensemble measurements.

3.2 Unzipping of native yeast chromatin from a single genomic site. Genomic chromatin
containing 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. Chromatin
structure will be mapped in wild type, an spt16 mutant, a mutant deficient in histone H2B
(12)
ubiquitylation, and the double mutant . Correlated nucleosome and polymerase occupancy on
individual chromatin fibers will be measured.

3.3 Shotgun chromatin mapping. Yeast native chromatin will be digested with XhoI and random
fragments will be ligated onto unzipping constructs. Shotgun DNA mapping will be used to identify
individual fragments. Nucleosome and polymerase positions will be identified at high resolution on
each individual fiber. High throughput experiments will reveal genome-wide correlations between
polymerases and histones and also identify divergent and cryptically initiated polymerases in wild
(12)
type, spt16 mutants, H2B-ubiquitylation mutants, and double mutants .

New biophysical tools are needed for single-molecule analysis of native chromatin. Success in this
project 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-molecule
mapping of epigenetic chromatin marks, DNA damage repair and replication, structural genome mapping
by unzipping, single-molecule telomere mapping, and studies of alternative splicing by single-molecule
cDNA unzipping. Given this wealth of possibilities and the strong collaborations formed, this CAREER
project will lay a solid foundation for a successful research career.
CAREER: Single-Molecule Analysis of
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II. Background and significance

A. Chromatin remodeling during transcription elongation

During transcription, nucleosomes are removed from in front of the polymerase to prevent stalling of
(19)
elongation . Nucleosomes are then reassembled behind the polymerase, which is important for
(20, 21)
preventing improper transcription initiation within the gene (termed cryptic initiation, see Fig. 2) . This
remodeling process is coordinated by histone chaperones that assist in chromatin reassembly. During
transcription, lysine residues on the histone tails are marked with various post-translational modifications
(22, 23)
(PTMs), but their functions in transcription are not yet well understood . In order to understand these
processes, we need to know with basepair
precision the position of all polymerases and
nucleosomes on the gene. Furthermore, we promoter
Reassembled
Nucleosomes
need to know whether the nucleosomes are
Pol
completely assembled octamers or are II
tetrameric or hexameric. Finally, we need to cryptic
Transcription
promoter
know this information for polymerases and
Figure 2 Nucleosome remodeling during transcription. Nucleosomes are
nucleosomes on the same individual gene— evicted in front of the polymerase and reassembled behind the
polymerase. Proper reassembly prevents initiation from cryptic promoters.
so that their direct interactions can be seen.

The main tool for studying remodeling during transcription is chromatin immunoprecipitation (ChIP). ChIP
is a powerful method that can provide some of the needed information such as relative levels of
polymerase and nucleosome 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 information about single chromatin fibers. Thus, we are working to provide a much
needed single-molecule analysis method 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,
(24-27) (12)
FACT , and a specific PTM, monoubiquitylation of histone H2B (H2B-ub) . Both FACT and H2B-ub
(28)
are important to cancer biology . The Osley lab has discovered that when both FACT and H2B-ub are
not functioning, chromatin is improperly reassembled on active genes. The molecular nature of this
misassembled chromatin is very difficult to decipher with ChIP or other ensemble assays. Answering
questions about this process is a specific motivation for our single-molecule technique development, but
we expect to be able to study chromatin structure during many important cellular processes and in any
eukaryotic organism.

B. Antisense Transcription

The misassembled chromatin mentioned in the previous section can lead to improper Pol II transcription
initiation from promoters within genes or elsewhere that would normally be blocked by nucleosomes.
(29, 30)
This is called cryptic initiation and it is rapidly becoming an important area of study in transcription.
A particularly fascinating, and possibly crucial point of cryptic initiation is that polymerases may
initiate in the antisense direction, and this has indeed very recently been shown to be widely
(20)
prevalent . 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
(29)
direction don’t get very far.‖ One possible explanation is that antisense-oriented polymerases quickly
encounter sense-oriented polymerases, and these head-to-head collisions effectively stall both
complexes. The ability to detect sense orientation of Pol II complexes is difficult with ensemble
(31)
techniques. Due to the asymmetry of the Pol II-DNA complex (in contrast to the nucleosome) , it is
CAREER: Single-Molecule Analysis of
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possible that single-molecule unzipping will be able to clearly indicate the transcriptional
orientation of the Pol II complexes on individual chromatin fibers. This may open the door 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-binding proteins could be mapped with high
(5)
resolution by unzipping single DNA molecules with optical tweezers . Fig. 1 shows conceptually how
these experiments are carried out with optical tweezers—though instead of a chromatin fiber, I unzipped
DNA plus site-specific binding proteins such as EcoRI. To
Data from Shundrovsky,
enable these experiments, I designed and implemented a et al., Nature Structural
& Molecular Biology 13,
versatile unzipping anchoring construct that allows for p. 549 (Not Koch Data)

unzipping of any DNA molecule with a known 5’ or 3’

(5)
overhang (see Fig. 4) . These two achievements are the
foundation for our goal of mapping native chromatin by
unzipping. A former labmate has used my versatile unzipping
construct to unzip through reconstituted mononucleosomes
and tetrasomes in the Wang lab. Some of this data is shown ©2006 Nature Publishing Group
in Fig. 3. The authors clearly demonstrated that
mononucleosome positions can be mapped with about 3
basepair precision. Furthermore, they were able to easily Figure 3 The protein mapping method
distinguish between octameric and tetrameric developed by Koch has been shown to
nucleosomes
(9, 10)
. These results give us confidence that we be capable of measuring the positions of
nucleosomes with 3 bp precision. It was
will be able to map nucleosome positions on native also shown possible to clearly distinguish
chromatin. Because no such data exist for Pol II unzipping, between octames and tetramers.
one of the specific aims of this proposal is to perform
Dig Nick Biotin Sticky end
single-molecule unzipping of stalled RNA Polymerase II 5’ 3’
for ligation
5’
complexes. Success will provide strong evidence for
Figure 4 The versatile unzipping construct from Koch et
the ability to map and identify polymerases and al. 2002 will be used in both aims. Any sticky end desired
nucleosomes on individual native chromatin fibers. is easily created via choice of oligonucleotide sequence.

D. Single-molecule transcription and chromatin studies

The power of single-molecule analysis for studying transcription and chromatin structure has been one of
(9, 10, 32-47)
the famous successes of single-molecule biophysics . Most of these studies have used purified
proteins to reconstitute chromatin or in vitro transcription. As shown in Figure 5, these experiments reside
in the ―single-molecule biochemistry‖ quadrant on the bottom left, as is true for single-molecule
(48-54)
manipulation experiments in general . Single-molecule biochemical experiments strongly
complement the vast number of ensemble experiments in the two upper quadrants and have revealed
exquisite details about the structures and biochemical processes involved in transcription and chromatin.
By comparison, very few single-molecule experiments have been performed in the bottom-right quadrant.
I have termed this quadrant ―single-molecule genetics,‖ because it presents the opportunity to leverage
the many tools in ensemble genetics in combination with the power of single-molecule analyses. Of the
(36, 44-47,
few existing ―single-molecule genetics‖ studies, many have been chromatin or transcription studies
55)
. Those studies have demonstrated the value of this untapped quadrant of experiments, but as of yet,
no methods have been developed for site-specific, single-molecule, native chromatin analysis as we are
CAREER: Single-Molecule Analysis of
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proposing. The wealth of potential discoveries in “single-molecule genetics” remains for the most
part untapped, and is the main focus of our CAREER proposal.

in vitro systems in vivo systems

(Reconstituted from components) (Intact, native molecules)

Ensemble Biochemistry Ensemble Genetics

N=trillions & in vitro systems
Nmolecules=trillions
Example: in vitro transcription,
Ensemble assays analyzing RNA transcripts on gel
N= trillions & in vivo systems
Example: Chromatin
Immunoprecipitation (ChIP)
analysis of polymerase and
nucleosome occupancy

Single-molecule Biochemistry “Single-molecule Genetics”

Nmolecules =1 N=1 & in vitro systems N=1 & in vivo systems

Example: single-molecule Example: Our proposal for single-

Single-molecule
measurement of force versus molecule mapping of native
assays velocity for RNA polymerase chromatin fibers

The majority of the excellent We feel our niche in the single-

single-molecule research is going molecule research world will be in
on in this quadrant leading the way in this quadrant

Figure 5 Two by two matrix illustrating four classes of experiments in transcription

biology. “Single-molecule genetics” is our terminology for the relatively untapped
quadrant that uses single-molecule analysis on native molecules or cells.

III. Preliminary Studies

A. Optical tweezers instrumentation and data analysis software

Design and construction of our optical tweezers was completed in July of 2009, with the last key element
being a high quality 4 Watt TEM00 diode-pumped solid state laser (Coherent) that was generously
provided to us by Dr. Evan Evans. We had previously been attempting construction using a high-power
visible (690 nm) laser diode system, due to the added safety and potential superiority of a 690 nm optical
trap. However, we had to put this on hold due to the inability to acquire a diode with sufficiently high
power and high beam quality. Other key elements of the system are shown in Figure 6. There are two
critical software components of our optical tweezers system. The first is the optical tweezers feedback
(5, 56)
control software , which we have succeeded in upgrading to the newer DAQmx version of LabVIEW.
This software provides the necessary feedback modules (force clamp; velocity clamp; loading rate clamp)
for DNA unzipping and protein-DNA unzipping. The other critical software component is a suite of
automated data analysis LabVIEW programs. These programs convert the raw OT data into ―force v.
unzipping index,‖ and are further automated to identify locations and disruption forces of DNA binding
(6)
proteins . The suite has been upgraded to LabVIEW 8.5. The primary coders of the original software
applications were the PI (Koch) and his colleague Dr. Richard Yeh during their Ph.D work.

As of the time of this writing (July 2009), we have stretched single double-stranded DNA molecules with
our system but have not yet unzipped DNA. We have preliminary calibration of the optical trap, showing
a trap stiffness factor of 0.2 pN / nm per Watt of input laser power. We can input at least 2 W of laser
power without damaging the objective, giving a stiffness of 0.4 pN / nm. We cannot directly verify this yet,
CAREER: Single-Molecule Analysis of
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due to bandwidth limitations of the detector. The

linear extent of the trap is about 100 nm, giving us a
maximum force of at least 40 pN – well over the
necessary 17 pN maximum unzipping force. We are
currently finishing calibration and constructing an 7
2
enclosure to minimize drift and noise.
1 3
B. Shotgun DNA Mapping

We have made substantial progress towards the

capability we are calling shotgun DNA mapping
(SDM). SDM is the ability to identify the genomic
location of a random DNA fragment based on its
naked DNA unzipping forces compared with
4
simulated unzipping forces of a published genome.
SDM will be an important enabler of our ability to map 6 5
native chromatin by unzipping without the need for
site-specific genetic engineering. We call this
Figure 6 KochLab optical tweezers setup, July 2009. (1)
proposed method shotgun chromatin mapping
Coherent 4W diode-pumped solid state laser (2) Power
(SCM) and the method is outlined in Fig. 7. Native modulater (BEOC) (3) Thorlabs Variable Beam
chromatin will be digested with restriction Expander (4) Steering optics (5) Olympus IX-71
endonucleases and random fragments will be Microscope body (6) Piezo stage (Mad City Labs)(7)
unzipped, providing high resolution positional Quadrant photodiode (PhreshPhotonics)
information for nucleosomes and
polymerases (see Aim 3). The high
resolution information will then be
assigned to an exact location in the
genome based on the underlying
SDM method. A very appealing
aspect of this process is that it does
not require genetic engineering or
site-specific chromatin extraction.

SDM works because we can

accurately predict the unzipping
forces for any known sequence of Figure 7. Overview of proposed method for shotgun DNA and chromatin
(57) mapping. We have recently achieved proof-of-principle results important
DNA . Fig. 8 shows one
for the “Global Genome Location,” part of the process (lower right).
experimental unzipping trace (Koch
(5)
data , 2002) compared with two simulations: one from the correct sequence, and one from an incorrect
sequence. The correct match is easily picked out by eye. We have developed an algorithm that
quantifies the match between and experimental and simulated unzipping curve. The match score we use

exp
is , where N is the number of points in matching window, F is the
sim
experimental unzipping force, F is the predicted force, FG=17.56 pN is the unzipping required for
poly(dG), and FA = 10.23 pN for poly(dA). A perfect match gives m = 0, and the maximum possible
mismatch is m = 1.
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A Correct Match, Score 0.2 B Mismatch, Score 0.8

Match Score
18 Simulation
Simulated data 18 OT Simulated
Data data

Force (pN)
Force (pN)

OT Optical
Data tweezers Optical tweezers
Simulation
Data (Koch 2002) Data (Koch 2002)
Match

12 12 File Number (Arb.)

0 1500 0 1500 Figure 19 Compilation of match scores
Unzipping fork index (bp) Unzipping fork index (bp)
for a single experimental data set. The
Figure 8 Experimental unzipping data compared with (A) matching and (B) non-matching file number is an arbitrary, arising from
simulation. The green window indicates the region from j=1200 to j=1700 where the the order in which the library
match scores were computed. A lower match score indicates the better match. simulations were loaded. A perfect
match would have a score of zero, and
We tested the algorithm using existing experimental unzipping data the correct match can be seen as having
(5)
for pBR322 plasmid . Our algorithm easily identified the pBR322 the lowest score, very distinguishable
fragment out of a background of simulations of all ~2700 possible from the incorrect matches.
yeast XhoI digested fragments. Fig. 9 shows this result, with the correct match clearly standing out from
the incorrect matches. Furthermore, we repeated this success for approximately 32 separate pBR322
data sets with zero failures. We are currently preparing a manuscript describing our proof-of-principle
(15)
results for SDM. These preliminary results give us confidence that we will be successful in
performing SDM with yeast genomic DNA as outlined in Specific Aim 1.

C. Molecular Constructs and E. coli, Yeast strains

Shotgun Clones

Specific Aim 1.1 is to demonstrate

success of Shotgun DNA mapping in
Clone #1 #4 #1
yeast genomic DNA. In order to do #2 #5 #3 #4 #5
so, we needed to first create clones
that can be sequenced to validate pBlue
the SDM results. We have
successfully prepared a dozen “mix”
shotgun clones of yeast genomic
DNA fragments propagated in a
SapI-digested NotI-digested
pBluescript plasmid in E. coli. The shotgun clones shotgun clones
clones were created by digesting a Dig-labeled “anchor”
yeast genomic DNA prep and a (Incomplete
segment for unzipping
digestion lanes 2 & 6)
pBluescript prep with either XhoI, or construct (see figure 4)
a double digest of XhoI-EcoRI,
Figure 10 – Agarose gel analysis of some of the successful shotgun clones.
followed by ligation. Blue/white Lane 1 is 10 kb ladder. Lanes 2-6 are SapI digested plasmid miniprep of
selection was used to pick colonies clones 1, 2, 4, 5 and pBluescript alone. (Lanes 2 and 6 did not digest
with incorporated yeast genomic completely.) Lanes 7-11 are NotI digestions of pBluescript, clones 1, 3, 4, 5.
DNA. Single colonies were used for Lane 13 & 14 are SapI and NotI digestion (respectively) of the “mixture” of
colonies described in the text. Lane 16 is the PCR product for the dig -
creation of glycerol stocks and
labeled anchor uses in the unzipping construct.
miniprep DNA. Figure 10 shows
digested miniprep DNA. The variable sizes indicates different fragments of yeast genomic DNA.
Additionally, lanes 13-14 show digestion of miniprep DNA from a mixture of colonies. This DNA was
created by combining 100’s of individual colonies into one growth media, and will be used as a further test
case of the SDM method.
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Digested miniprep DNA has been ligated onto the versatile unzipping construct (Figure 4) and we have
(5, 58)
confirmed the ability to form single-molecule tethers from this DNA . We are thus now ready to
proceed with Specific Aim 1 by unzipping the unknown fragments and testing the capabilities of
SDM in identifying them.

Yeast plasmid chromatin

As described above in the Background and Significance, prior work has demonstrated that the position of
in vitro reconstituted mononucleosomes can be precisely measure by unzipping with optical tweezers. We
feel this is strong evidence that nucleosome positions on native chromatin can be measured by
unzipping. As part of Specific Aim 3, we will seek to prove this hypothesis. In order to do so, we need a
system with very well characterized in vivo nucleosome positions. The PHO5 gene promoter has 4 very
(17, 18)
well positioned nucleosomes that are thoroughly evicted upon gene induction . Furthermore, Korber
et al. have shown that when the gene is placed in a small yeast plasmid, the chromatin structure is
indistinguishable from that on the genomic PHO5 promoter. Therefore, we have set out to use the PHO5
plasmid system as an initial test of our native nucleosome mapping capabilities.

A B Wild-type
I-SceI site inserted here for our (no signal)
pDRP100 construct Plasmid Lanes 6-9
size markers*
Lanes 2-5 pDRP100 chromatin
digests
Lanes 10-13

nicked

linearized

supercoiled

* Mistakenly used plasmid before removal of pCU19 insert

Figure 11 – (A) Diagram of pTAP5C from Korber et al. (2004). We inserted an I-SceI recognition in the DNAse hyper-sensitive
region next to the EcoRI site, naming the plasmid pDRP100. The filled gray circles indicate the 4 nucleosomes evicted upon
induction of the PHO5 gene. (B) Southern blot analysis of DNA run on an agarose gel after restriction endonuclease digestion
of yeast nuclei. Probe hybridizes to Trp region of pDRP100. Lanes 2-5 are digested plasmid miniprep DNA (unfortunately we
mistakenly used the plasmid before removing the pUC19 region used for propagation in E. coli). Lanes 6 -9 and 10-13 are for
digested yeast chromatin. Absence of signal in lanes 6-9 (Wild-type W303A) and presence in 10-13 (pDRP100) demonstrates
successful propagation of pDRP100 plasmid . Lane 10: I-SceI digest; Lane 11: I-SceI/BamHI; Lane 12:I-SceI/ClaI; Lane 13: ClaI.
All lanes 10-13 show innefective digestion, with perhaps only ClaI showing increased linear DNA (see text).

We have engineered a unique I-SceI site near the EcoRI hypersensitive site in the pTAP5C PHO5
(17)
plasmid (kindly provided by Korber et al., see figure 11A). This plasmid was then transformed into
yeast, a strain which we are calling pDRP100. Propagation of the plasmid was confirmed by Southern
blot, using a probe against the Trp marker in the plasmid. We have begun to assess the ability to digest
the native chromatin and have preliminary indications of limited success. Yeast spheroplasts were
generated by digesting the cell walls with lyticase. Nuclei were purified via centrifugation and
resuspended in one of four restriction digest solutions: I-SceI; I-SceI+BamHI; I-SceI + ClaI; or ClaI.
Figure 11B shows a Southern blot analysis of DNA purified after nuclei digestion. Clearly demonstrated
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is the presence of the plasmid in the pDRP100 nuclei (no probe hybridizes in the wild type lanes). None
of the enzymes digested the plasmid chromatin effectively. However, limited cutting may be seen by ClaI
from the increase in linearized band in last visibile lane. This could be due to sub-optimal endonuclease
digestion conditions or possibly due to altered PHO5 chromatin structure (and thus shifted hypersensitive
sites) due to our insertion of the I-SceI site. This will be explored further as part of Specific Aim 3.

IV. Research Methods

The specific aims for this CAREER proposal have been designed to achieve our goal of native
chromatin mapping by single-molecule unzipping. The first specific aim is to develop “shotgun
DNA mapping,” (SDM) which is our name for 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
(15)
published genome . SDM will allow us to perform chromatin mapping of yeast and other organisms
without the need for site-specific genetic engineering. The second aim is to obtain the characteristic
force pattern for unzipping through RNA Pol II complexes. There already exists excellent data on the
(9, 10)
unzipping force pattern of reconstituted mononucleosomes , but not for Pol II. Obtaining this pattern
for in vitro Pol II complexes will be essential for identifying in vivo Pol II complexes by unzipping. The
third aim is to map native yeast chromatin by single-molecule unzipping. We will leverage
successes in the first two aims, to increase the value of success in the third aim.

Specific Aim 1: Develop shotgun DNA mapping (SDM).

rd
Lead graduate student: Larry Herskowitz (3 year Ph.D.) followed by future Ph.D. student.

Sub Aim 1.1: Use a shotgun cloning strategy to prove that SDM works for yeast genomic DNA.
So far, we have demonstrated with existing pBR322 data that the pBR322 fragment can easily be
(15)
identified from a background of the approximately 2700 yeast XhoI sites . We believe this is convincing
evidence that shotgun DNA mapping will be successful when applied to yeast genomic DNA, but the
possibility remains that there is something special about pBR322, or that there will be many difficult yeast
genomic sequences. Thus, in this sub aim, we will prove that SDM works for XhoI-digested yeast
genomic DNA. 1. Restriction digestion of yeast genomic DNA
2. Clone random fragments into plasmid pBR322
As shown in Figure 12, we will validate the shotgun 3. Prepare unzipping constructs from miniprep DNA
DNA mapping method in yeast via a shotgun cloning 4. Predict identity of clone based on shotgun DNA
method. Steps 1, 2, and 3 have been completed as mapping by unzipping
5. Sequence clones, compare results, and determine
discussed in Preliminary Studies. In step 4, we will
success rate
follow our standard published tethering and unzipping
(5, 6, 59)
protocol . Briefly, microspheres will be tethered Figure 12 Process for validating shotgun DNA mapping
to glass surfaces via dig-antidig and biotin-streptavidin method for yeast genomic DNA
linkages. Dozens of tethers will be selected by eye and unzipped automatically by feedback-controlled
optical tweezers. We will use the existing upgraded DNA unzipping data acquisition and analysis
(5, 6, 56)
software that was used for our pBR322 proof-of-principle . We will use the shotgun DNA mapping
(15)
software application we have developed with possible improvements from sub-aim 1.2. Finally, in step
5, we will unblind our experiments by revealing the identity of the chosen clones via standard DNA
sequencing. Overall, this method will be low-throughput, and we anticipate analysis of about 10 individual
clones.

The success rate we achieve (we are expecting errors only in the case of obviously ambiguous
sequences) will predict the success rate of shotgun DNA mapping straight from genomic DNA or
chromatin—in which case the cloning steps will be eliminated.
CAREER: Single-Molecule Analysis of
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Sub Aim 1.2: Improve the algorithm for shotgun DNA mapping. The purpose of this sub-aim is to
increase the sensitivity and specificity of the shotgun DNA mapping algorithm we have developed.
Improving sensitivity and specificity will allow us to work with larger reference libraries, e.g. from
human genome fragments. We have three clear paths for achieving this: (a) improving the DNA
modeling, (b) developing a more sophisticated and better matching algorithm, and (c) significantly
improving optical tweezers data quality.
(57)
Modeling DNA unzipping. We are currently using a very simple model for the DNA energetics . This
simple model is analogous to the short-hand calculation for DNA melting temperatures, where only the AT
versus GC content of the primer is accounted for. This simplification usually works remarkably well for
DNA melting temperatures and it also works quite well for predicting DNA unzipping forces. However,
DNA base-stacking energy is important and by accounting for it we can improve the modeling of the
unzipping forces. Accounting for base-stacking energy is simple in principle as it only requires knowing
(60)
the energy values for the 10 nearest-neighbor interactions . The challenge we will address is optimizing
the energy values from SantaLucia for our case of DNA melting by unzipping, which differs slightly from
thermal melting.

Our current model is a quasi-equilibrium thermodynamic model that does not utilize any of the kinetics
information that is readily available in the data. We will work to implement a sequence-dependent kinetic
(61)
model of DNA unzipping. One such model is the Peyrard-Bishhop-Dauxois (PBD) model , a one-
(62)
dimensional model of DNA that has been shown to successfully model DNA bubble formation kinetics
(63)
and DNA unzipping . We have met with the lead PI of the latter reference, Dr. Rasmussen of Los
Alamos National Lab, and have obtained their Monte Carlo PBD unzipping code. Our first step will be to
see if the PBD code can produce averaged force predictions that are comparable to the thermodynamical
model. Next, we will look at kinetics predictions from the PBD model and develop data analysis methods
that can extract kinetics information from the data. We will do this in collaboration with Dr. Evan Evans, a
leader in the development of single-molecule bond kinetics analyses.

Optimizing the matching algorithm. Our current matching algorithm uses a straight-forward calculation
of the squared deviation of data from simulation. We believe it is likely we can improve the matching
algorithm via a number of independent avenues. The first avenue is to account for slight length errors in
experimental data due to microsphere size variation, drift, and other causes. We will allow for stretch and
(9)
shift in the data as in previous nucleosome mapping work . The next avenue is to include other
independent match criteria, which when combined with our existing match scoring may greatly increase
our sensitivity and specificity. There are several thermodynamical criteria we will look into, such as
average fluctuations in force or unzipping index, and cross-correlation techniques. Furthermore, as
discussed above, we will work to model and analyze the unzipping kinetics, which could dramatically
improve the matching algorithm.

Optimizing data quality. Drift and other noise in single-molecule unzipping data degrade the sensitivity
and specificity of the SDM process. The rate of unzipping and the mode of feedback (velocity clamp;
loading rate clamp) significantly impact these factors. For example, very slow stretch rate will optimize
the amount of kinetics information and allow for significant averaging of the Brownian noise. However,
drift will be much more significant for slow stretching. We will systematically study these effects in order
to find an optimum unzipping feedback program. Furthermore, we will develop more sophisticated
feedback algorithms—for example, dynamically switching from a velocity clamp to a force clamp in order
to extract DNA opening / closing kinetics from a particular unzipping fork location.

Sub Aim 1.3: Automate SDM data acquisition and analysis For our initial proof of principle, and
indeed many envisioned implementations, low-throughput unzipping and analysis is sufficient to obtain
CAREER: Single-Molecule Analysis of
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results. However, SDM automation will be required for genome-wide analyses and will greatly increase
the practicality of the method. Many of the components of the data acquisition and processing stream
have already been successfully automated. The goal of this aim is to fully eliminate the need for user
input, once a micro-flow cell has been manually mounted on the optical tweezers instrument. Currently,
there are three distinct steps that require user intervention and that need to be automated. (1) The first is
the selection of a tether for unzipping, which is currently done by eye. We will adapt our existing
LabVIEW bead tracking software in combination with an x-y stage and stepper motors to automatically
detect tethers with the correct amount of Brownian motion and initiate feedback controlled unzipping.
After unzipping the tether and storing the data, the stage will move to find the next tether. The find / unzip
/ store data process will repeat until the entire sample area has been scanned. (2) The next user
intervention step is the selection of ―good‖ data for automatic conversion from raw data to ―force‖ versus
―unzipping index‖ data. Currently ―junk‖ tethers or tethers that prematurely break are identified and
filtered out manually. We will develop algorithms to objectively filter the data automatically. (3) Finally,
the algorithms for identifying nucleosomes and polymerases need to be developed along with means for
accumulating and classifying this data. This is the most substantial step of the three, and will require
substantial development time in coordination with the other specific aims.

Specific Aim 2: Determine the unzipping signature for RNA Polymerase II transcription complexes.
rd
Lead graduate student: Anthony Salvagno (3 year Ph.D.) followed by future Ph.D. student.
(9, 10)
The unzipping signature of an in vitro assembled mononucleosome is already known and will be used
for identifying nucleosomes during native chromatin unzipping. In this aim, we will determine the in vitro
Pol II unzipping signature for use in identifying native complexes in Aim 3. Furthermore, new structural
information about in vitro transcription complexes may be obtained.

Sub Aim 2.1: Unzip through stalled Pol II in vitro transcription complexes. The lab of Dr. Karen
Adelman has extensive experience with in vitro Pol II transcription and will provide us with elongating
(13, 14, 64, 65)
transcription complexes stalled via nucleotide depravation . These complexes are known to be
very stable and thus can be shipped to our lab without dissociation. Importantly, the transcription
complexes will be prepared so as to have defined 5’-overhangs either upstream or downstream of the
elongating polymerase. These two different overhangs will allow us to unzip through the Pol II structure
from both directions. In contrast to the symmetric nucleosome structure, we anticipate that the
asymmetric elongating Pol II structure may provide distinct patterns depending on unzipping
direction…and thus we hope to be able to distinguish sense from antisense transcription complexes in
future in vivo mapping experiments.
(5)
The unzipping anchor designed by the PI permits unzipping of virtually any nucleic acid with a known 5’
or 3’ overhang with only simple changes required to an adaptor duplex. Thus, transcription complexes
will be ligated onto unzipping constructs using virtually the same methods as in the shotgun DNA
(58)
mapping experiments (Aim 1.1). Because it is highly stable, we do not expect the ligation process to
disturb the Pol II complex. If we do encounter trouble, we will first make the unzipping construct and then
initiate transcription on the full construct. This may offer further advantages by allowing for use of
streptavidin beads during the transcription initiation and elongation. Following ligation, constructs will be
(5, 6, 59)
tethered with our standard protocol and dozens of individual complexes will be unzipped, each
generating a Pol II disruption force pattern. We will generate computer algorithms to align these patterns
and to generate a ―master pattern‖ that we can use for identifying Pol II complexes in aim 3. Two
reference ―signatures‖ will be produced: one for each sense or antisense orientation of transcription.
CAREER: Single-Molecule Analysis of
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Stalled complexes will first be prepared by nucleotide depravation using a G-less cassette. Following
(66)
this, transcription will be stalled via lac repressor binding to an intra-gene lac operator sequence . It is
likely that the in vivo Pol II unzipping signature will be modified due to presence of different elongation
factors and chaperones, but to what extent, we do not yet know. In vitro experiments with the lac
repressor will provide information on how much the unzipping pattern changes due to interactions with
other DNA-binding proteins.

Sub Aim 2.2: Atomistic modeling of unzipping DNA and protein-DNA complexes. A big risk of our
goal of native chromatin mapping by unzipping is that the extracted molecules may be too
heterogeneous, and the complicated unzipping signals may be too difficult to decipher. We are
minimizing this risk by the systematic approach we are taking in this proposal. Given that Pol II is an
enzymatic machine with a wide array of interacting factors, it’s likely to present more of an identification
problem than nucleosomes. The ability to do atomistic modeling of the unzipping of arbitrarily large
protein-DNA complexes would allow for in silico predictions of unzipping force patterns of Pol II,
nucleosomes, including the array of possible in vivo varieties of histone post-translational modifications
(67)
and transcription elongation factors . However, this is a highly ambitious goal and well beyond the
scope of this CAREER proposal. It is a project that we are attempting to get funded to pursue in
collaboration with the lab of Dr. Chris Lorenz, an expert in computational modeling. It also ties into our
other funded project, in collaboration with Dr. Susan Atlas, which seeks to develop an atomistic model of
the molecular motor kinesin.

The goal of this sub-aim is to take small steps that will both help us initiate the larger project and will also
help us to interpret our new Pol II in vitro unzipping data. The student on this project will use our high-
priority free access to supercomputers at the UNM Center for Advanced Research Computing and will be
assisted by Drs. Atlas and Lorenz. The first step will be to build a simulation of naked DNA, and
implement molecular dynamics (MD) techniques for applying strain to the molecules that will cause
unzipping. These results will be tied in with the kinetics modeling in specific aim 1.2. Following naked
DNA MD simulations, a small model protein-DNA system with an existing crystal structure, such as
EcoRI-DNA will be modeled and subjected to in silico MD unzipping. Results from these simulations may
lend tremendous insight into the actual unzipping force patterns seen for nucleosomes and polymerases,
even though we are not able to simulate those large structures. In support of the MD simulations, and
kinetics modeling a number of naked DNA and model protein-DNA complexes will be unzipped. We
expect to use these experiments to produce important biophysical results enabled by the PI’s prior
(5, 6)
research that have not yet been investigated.

Specific Aim 3: Mapping native yeast chromatin by single-molecule unzipping.

Co-lead graduate students: A. Salvagno & L. Herskowitz, followed by future graduate students.
In these sub-aims, all steps prior to chromatin tethering and unzipping will be carried out in the
lab of Dr. Osley under the guidance of postdoctoral associate Dr. Kelly Trujillo.

The ultimate goal of this proposal is high-resolution, single-molecule, site-specific mapping of

nucleosome and polymerase positions on native chromatin. Various independent approaches will be
taken towards native chromatin unzipping in this aim, so that significant progress towards the overall goal
can be achieved, independent of success in the other aims.

Sub Aim 3.1: Unzipping of yeast plasmid chromatin. We have engineered a yeast plasmid containing
(17)
the PHO5 gene and an I-SceI restriction site and have transfected it into S cerevisiae. We will confirm
(17)
that I-SceI insertion did not change the chromatin structure by following the methods of Korber et al.
(see Preliminary Studies). Yeast nuclei will be prepared with and without PHO5 induction and digested
CAREER: Single-Molecule Analysis of
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(68)
with I-SceI . Native chromatin will be extracted (without sonication or cross-linking) and ligated onto the
unzipping construct (Fig. 4). Because the I-SceI site is unique, the only possible ligation will be from the
plasmid. If the I-SceI site is accessible, we feel this method will succeed, despite the ―needle in a
haystack‖ problem of all the background chromatin. However if the I-SceI insertion destroys the standard
PHO5 chromatin structure, I-SceI may no longer be accessible. If this is the case, or if other problems
(16)
arise we will pursue the plasmid chromatin purification methods of Clark before endonuclease
(18, 69)
digestion. The recombinase methods of Kornberg for endogenous PHO5 chromatin isolation are
also appealing, though we have not yet initiated communication with the Kornberg lab.
(5, 6, 59)
We will tether and unzip PHO5 chromatin using our standard protocol and determine nucleosome
(9, 10)
locations with close to basepair precision following methods established for reconstituted chromatin .
Single-molecule nuclesome occupancy on the PHO5 promoter with and without induction of the gene will
be compared with the known behavior from ensemble studies in order to validate our method.
Furthermore, important questions about the nucleosome remodeling mechanism during transcription
initiation can be answered with single-molecule measurements of native PHO5 chromatin, as recently
(18)
stated by Boeger et al .

Sub Aim 3.2: Unzipping of native yeast chromatin from a single genomic site. To study native
chromatin remodeling during transcription elongation, we will engineer an I-SceI restriction site upstream
of an endogenous yeast gene under galactose control. We will use the YLR454 gene, which is 8
kilobases long and has well-characterized polymerase and nucleosome dynamics during transcription
(21, 70)
elongation . Genomic chromatin will be double-digested with I-SceI and NotI and ligated onto
unzipping constructs complementary to the I-SceI overhang. We will initially attempt the process without
sonication or cross-linking, and if necessary to solubilize the YLR454 will apply light sonication and / or
cross-linking. Nucleosome positions on single chromatin fibers will be determined with close to basepair
(9, 10)
precision following methods established for reconstituted chromatin . Polymerase locations on the
same fibers will be determined following methods developed in Aim 2 above.

Chromatin structure will first be mapped in wild type I-SceI-YLR454, and compared with ensemble
(21, 70, 71)
measurements . Following this, I-SceI will be engineered into existing mutant strains in the Osley
(12)
lab: an spt16 mutant, a mutant deficient in histone H2B ubiquitylation (K123R), and the double mutant .
Correlated nucleosome and polymerase occupancy will be measured in single fibers, which will answer
(12)
questions about the unusual chromatin structure in these mutants seen by ensemble assays . Based
on Specific Aim 2, novel features of elongating polymerase may be identified, such as anti-sense oriented
polymerases from cryptic initiation.

Sub Aim 3.3: Shotgun chromatin mapping. Shotgun chromatin mapping (SCM) is the use of shotgun
DNA mapping (SDM; Aim 1) as a means for identifying the underlying DNA sequence of random
chromatin fibers. This is the ultimate goal of the proposal, and if successful it will allow mapping of
polymerases and nucleosomes in any mutant yeast strain, without the need for engineering an I-
SceI site. Full success depends on success in Aims 1 and 2. However, even partial success will be very
valuable and will be possible independent of the other aims. (For example, non-site-specific mapping, or
nucleosome-only mapping.)

The first step is to create unzippable chromatin fragments from yeast genomic chromatin. Yeast nuclei
(68)
will be prepared as described in preliminary studies and chromatin will be digested by XhoI . XhoI
(15)
recognizes approximately 1350 sites in the genome . However, many of these sites will be occluded by
chromatin structure—thus there is an inherent bias in this step (which can be measured by SDM of
genomic DNA following digestion). Strategies for reducing this bias will be pursued in the future, though
likely it will be a persistent issue with SCM. The versatile unzipping construct (Fig. 4) with XhoI sticky end
CAREER: Single-Molecule Analysis of
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will be ligated to the XhoI-digested soluble chromatin component. Because XhoI digestion produces self-
complementary overhangs, concatamers will form during ligation. This is not expected to be a problem,
but to eliminate concatamers we can first digest the chromatin with another enzyme and treat with calf-
intestinal alkaline phosphatase.
(5, 6, 59)
Chromatin unzipping tethers will be formed using our standard procotol . Shotgun DNA Mapping
(Aim 1) will be used to exactly identify each fragment, a process we are calling shotgun chromatin
mapping (SCM). Because we are using native chromatin, we will need to generate a sophisticated
sequence of feedback-controlled optical tweezers unzipping steps. Our control software is ideally suited
for this and reconfiguring feedback sequences is easy and does not require modifications to the LabVIEW
(56)
code . We will use a feedback sequence as follows: (1) find tether center, (2) constant velocity
stretching until j=500 basepairs are unzipped, (3) reverse direction and constant slow-velocity ―rezipping,‖
until j = 0 (4) reverse direction again and continue unzipping until tether breaks or piezo runs out of range.
Steps 2 and 4 will produce data showing protein-DNA interactions. Nucleosome positions will be
(9,
determined with close to basepair precision following methods established for reconstituted chromatin
10)
. Polymerase locations on the same fibers will be determined following methods developed in Aim 2
above. SDM will be applied to map these high-resolution positions to an exact genome location.
This will be possible because in step 4 of the feedback sequence the first 500 bases will be free of
protein, and thus will provide a naked DNA unzipping curve for use in SDM.

With our existing software and hardware, it is possible but tedious to unzip over a thousand individual
(5, 6)
tethers . This will probably be sufficient for validating SCM as a new tool for high-resolution single-
molecule chromatin mapping. However, we want to explore genome-wide correlations between
polymerases and histones and look for divergent and cryptically initiated polymerases. This will require
high-throughput automation of data acquisition and analysis, which is specific aim 1.3. Because SCM
does not require genetic engineering of each strain studied, we will be able to quickly generate and
compare results in wild-type and strains deficient in FACT and H2B-ubiquitylation, as described in
Background Section A.

V. Educational Integration and Broader Impacts

Our lab believes strongly in our mission of advancing molecular cell biology, and therefore our goals are
well aligned with those of the NSF for integrating research with education and maximizing the broader
impacts of our research. Broadly communicating our results and methods, teaching at all levels, and
training future leaders in biophysics research together will greatly multiply the impact of our research
discoveries. Below, we describe our plans towards this goal in two areas: open science and integration
of research with undergraduate education.

One way of broadening our impact is in recruitment and training of underrepresented minorities to
biophysics, and this is an underlying component of our goals below. We are at an advantage in this
area, because of the unique demographics of the University of New Mexico and the surrounding area.
(72)
UNM is designated a Hispanic-Serving Institution by the US Dept. Ed. and other government agencies.
(73)
We participate in the UNM PREP program that recruits minorities to science and our lab employed
three Hispanic researchers so far. Additionally, the local population consists of a high proportion of
minorities underrepresented in science. I have participated in community scientific outreach since early in
my graduate career and it remains an enjoyable and valued activity. During my first three years at UNM, I
have judged the Central New Mexico Science and Engineering Challenge (Middle School Microbiology),
the Cleveland Middle School Science Fair, and given a presentation on nanomanipulation in the biological
sciences to local Middle and High School science teachers at the NNIN-sponsored summer ―nanocamp.‖
CAREER: Single-Molecule Analysis of
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I plan to grow my own outreach, and continue to encourage, value, and reward community outreach
by the graduate, undergraduate, and postdoctoral members of our lab.

Open Science Since early 2007, our lab has been actively participating in OpenWetWare (OWW), an
Open Science site hosted by MIT (http://www.openwetware.org/wiki/Koch_Lab). The goal of open
science is complete open sharing of all data and knowledge generated in the laboratory. As our
lab has matured, we have taken many steps and a few leaps towards open science. These include ―open
(74) (75) (76, 77)
notebook science‖ via OpenWetWare , detailed protocol publishing , and with this CAREER
(78)
proposal, a fully-open research proposal . If funded, we are committed to carrying out the proposed
research as openly as possible. This will include Open Notebook Science, Open Access publishing,
(79)
free availability of raw data and all stages of processed data , and sharing of all data acquisition and
processing software. While we are committed to this mission, exactly what the best methods are for
doing this is a difficult question that leaders in the open science movement are still figuring out. In the
past year, I have been fortunate to meet some of these leaders who have already helped our lab
tremendously in its goal for openness. I have attached letters (Cameron Neylon, Jean-Claude Bradley,
and Andrew Lang) expressing continued willingness to help with these difficult issues. I have also formed
a collaboration with two undergraduate students at UNM, Noel Fernando and Leslie Broyles, who are
majoring in digital media arts. These students have offered assistance in our open science and education
endeavors that would benefit from advanced digital media expertise. A specific idea is to make a video
presentation that will explain our lab’s open science activities to the general public. Our ability to
succeed in our open science goals will be greatly aided by the CAREER award and the stability
and assurance it will provide our lab.

Integration of research with undergraduate education. Many of the classes I have taught
or will teach are carried out in a lecture hall that has a wide array of fantastic hands-on physics demos.
While teaching physics 102 (a non-math based course intended for non-science majors), I have noticed
an unsurprising fact: students are very eager for an opportunity to play with the demos! Unfortunately, it
is not practical for students to have this opportunity. I often assign quiz questions or homework problems
that directly relate to demos we use in class. For example, the ―wave table‖ is a device made up of rods
tied together with wire. Beautiful waves are created and concepts such as wave interference, standing
waves, frequency, are illustrated. I believe that virtual reality is a very promising method for bringing
these demos into the students’ hands. Thus, I have budgeted to employ an undergraduate computer
engineer to build virtual versions of our demos in the virtual reality environment ―Second Life‖ (SL). Two
(80)
of the experts in leveraging SL for scientific purposes have offered their advice to me in getting this
project going (see letter from Jean-Claude Bradley and Andrew Lang).

Another course I teach is Junior Lab, which is the first ―real‖ physics lab course that physics majors at
UNM enroll in. The main innovation with this course is that I host this course completely as Open
(81)
Science, on OpenWetWare . The students keep all of their notes electronically on OWW, along with
their lab write-ups and formal reports. Additionally, all of the written feedback from me (with the exception
of letter grades) is also carried out in public on OWW. The results have been very rewarding: of the 31
students, at least 6 of them continued to use OWW subsequent to Junior Lab in their own coursework
and lab work. Furthermore, I think there are significant benefits to training students in open science
at this early stage in their research careers and will ultimately make a large impact on the next
generation of open scientists. In addition to the open science component to Junior Lab, I intend to add
new biophysics modules to the course. Specific modules I would like to add are: tethered particle motion
(TPM) analysis of single DNA molecules optical tweezers / magnetic tweezers experiments, and PDMS
microfluidics. I have commitment from the chair of UNM Physics Dept. to support our goals of adding
biophysics experiment modules with college and department funds when needed (see Chair letter).
CAREER: Single-Molecule Analysis of
Genomic DNA and Chromatin in Eukaryotic Transcription References, Page 1 of 4

References Cited

1. "Acknowledgments: I thank Andy Maloney, Anthony Salvagno, Larry Herskowitz, Brigette Black,
Linh Le, Diego Ramallo, Laurie Hudson, Janet Oliver, Kelly Trujillo, Chris Lorenz, Evan Evans,
Karen Adelman, and Mary Ann Osley for significant help in generation of this research plan.
Thank you to the American Cancer Society for project seed funding through UNM’s IRG
program."
2. Kornberg, R. D., "Chromatin structure: a repeating unit of histones and DNA." Science 184, 868
(May 24, 1974).
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of principle for shotgun DNA mapping by unzipping. Available from Nature Precedings
<http://hdl.handle.net/10101/npre.2009.2808.1> (2009)."
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35. Brower-Toland, B., Wacker, D. A., Fulbright, R. M., Lis, J. T., Kraus, W. L., Wang, M. D., "Specific
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36. Cui, Y., Bustamante, C., "Pulling a single chromatin fiber reveals the forces that maintain its
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55. Yu, J., Xiao, J., Ren, X., Lao, K., Xie, S., "Probing Gene Expression in Live Cells, One Protein
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56. "We have made or data acquisition code available via sourceforge and described it on
OpenWetWare. We will do the same with the analysis software, and all of our shotgun DNA
mapping applications.
http://openwetware.org/wiki/Koch_Lab:Publications/Drafts/Versatile_Feedback/Software/How_to_
obtain_the_software."
57. Bockelmann, U., EssevazRoulet, B., Heslot, F., "Molecular stick-slip motion revealed by opening
DNA with piconewton forces." PHYSICAL REVIEW LETTERS 79, 4489 (1997).
58. "Public protocol available on our OpenWetWare site:
http://openwetware.org/index.php?title=Koch_Lab:Protocols/Unzipping_constructs&oldid=212016
."
59. "Public protocol available on our OpenWetWare site:
http://openwetware.org/index.php?title=Koch_Lab:Protocols/Microsphere-
DNA_tethering/Glass%2C_dig%2C_biotin%2C_microsphere%2C_4kb_DNA&oldid=283740."
60. SantaLucia, J., Jr., "A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-
neighbor thermodynamics." PNAS 95, 1460 (February 17, 1998, 1998).
61. Dauxois, T., Peyrard, M., Bishop, A. R., "Dynamics and Thermodynamics of a Nonlinear Model
for DNA Denaturation." Physical Review E 47, 684 (Jan, 1993).
62. Ares, S., Voulgarakis, N. K., Rasmussen, K. O., Bishop, A. R., "Bubble nucleation and
cooperativity in DNA melting." Phys Rev Lett 94, 035504 (Jan 28, 2005).
63. Voulgarakis, N. K., Redondo, A., Bishop, A. R., Rasmussen, K. O., "Probing the mechanical
unzipping of DNA." Phys Rev Lett 96, 248101 (Jun 23, 2006).
64. Adelman, K., Wei, W., Ardehali, M. B., Werner, J., Zhu, B., Reinberg, D., Lis, J. T. (2006), vol. 26,
pp. 250-260.
65. Adelman, K., Marr, M. T., Werner, J., Saunders, A., Ni, Z. Y., Andrulis, E. D., Lis, J. T., "Efficient
release from promoter-proximal stall sites requires transcript cleavage factor TFIIS." Molecular
Cell 17, 103 (2005).
66. Deuschle, U., Hipskind, R. A., Bujard, H., "RNA polymerase II transcription blocked by
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68. Almer, A., Horz, W., "Nuclease hypersensitive regions with adjacent positioned nucleosomes
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69. Griesenbeck, J., Boeger, H., Strattan, J. S., Kornberg, R. D., "Affinity purification of specific
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70. Mason, P. B., Struhl, K., "Distinction and Relationship between Elongation Rate and Processivity
of RNA Polymerase II In Vivo." Molecular Cell 17, 831 (2005).
71. Fleming, A. B., Osley, M. A. (2008).
72. "http://www.ed.gov/about/offices/list/ocr/edlite-minorityinst.html."
73. "Postbaccalaureat Research and Education Program (PREP) @ UNM.
http://biology.unm.edu/PREP/index.asp."
74. "Wikipedia: Open Notebook Science http://en.wikipedia.org/wiki/Open_Notebook_Science."
75. "KochLab members' open lab notebooks http://openwetware.org/wiki/Koch_Lab:Notebooks."
76. "KochLab Protocols on OpenWetWare http://openwetware.org/wiki/Koch_Lab:Protocols."
77. "A KochLab graduate student, Andy Maloney, has posted instructions on how to construct our
OEM laser diode system for optical tweezers.
http://openwetware.org/wiki/Koch_Lab:Research/How_to_build_your_own_laser_diode."
78. "The technical portion of this proposal will be uploaded to the public document sharing site,
Scribd. It will be available after submission via the site: http://www.scribd.com/sjkoch4914."
79. "Using start-up budget, we have acquired a 2 TB file server and thanks to Caleb Morse (a
talented undergradaute ECE major), we already have a functioning system for sharing raw data
files. Data files are copied to the server daily and become immediately publicly available.
http://kochlab.org/files/data."
80. Lang, A. S. I. D., Bradley, J.-C., "Chemistry in Second Life
http://usefulchem.wikispaces.com/SLchemPaper." (2009).
81. "UNM Junior Physics Lab on OpenWetWare: http://www.openwetware.org/wiki/Physics307L."