2009 Acs Irg Proposal, Submitted Research Plan

2009 American Cancer (ACS) IRG application from the lab from of Application forSociety an Individual Allocation Steven J. Koch at the University of New Mexico. [email protected] Cancer Society http://openwetware.org/wiki/Koch_Lab 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 with 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 first application, 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). 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 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 cell generation makes understanding chromatin remodeling during transcription very important to cancer biology(3). However, understanding these processes during gene transcription is currently limited by the inability to sensitively characterize with high spatial resolution the positions of polymerases and nucleosomes on individual chromatin fibers in cells. Therefore, we are developing a Optical single-molecule method (see Fig. 1) for mapping proteins on native Trap nucleosome chromatin that will provide this capability. Our specific hypothesis is RNA Pol II that optical tweezers unzipping of native chromatin will allow mapping of histones and polymerases with near base pair ssDNA resolution on the same individual fiber. We have several reasons to believe we can prove our hypothesis and shed light on these questions. Coverglass First, The PI of this proposal is the co-inventor of the technique for mapping protein binding by unzipping single DNA molecules and an Figure 1 Proposed unzipping of single chromatin fibers with optical expert in constructing tools for manipulating single DNA molecules(4-7). It tweezers. Optical tweezers use laser has been shown that bound proteins can be detected because the force light focused through a microscope required to unzip protein-bound DNA is substantially higher than for objective to apply and measure small naked DNA. Second, It has recently been shown that this DNA unzipping forces on microspheres attached to method can map the positions of in vitro assembled mononucleosomes biomolecules. Monitoring the length of ssDNA and the unzipping forces will with close to base pair resolution(8, 9). Third, we are collaborating with reveal the position of nucleosomes labs with extensive expertise in characterizing chromatin remodeling and and polymerases with close to base Pol II elongation with ensemble methods such as Chromatin pair resolution. (10-13) Immunoprecipitation (ChIP) . For this one-year ACS IRG proposal, we are proposing two specific aims to generate key preliminary data for longer-term R01 funding: Specific Aim 1: Prove that shotgun DNA mapping (SDM) works with yeast genomic DNA and improve the SDM algorithms. Shotgun DNA mapping is the ability to identify the genomic location of a random DNA fragment based on its naked DNA unzipping forces compared with simulated unzipping forces of a published genome (14). It is an enabler of our goal of native chromatin mapping. Specific Aim 2: Determine the unzipping signature for RNA Polymerase II by unzipping through stalled in vitro transcription complexes. The innovative single-molecule results pursued in these one-year aims will provide strong preliminary data in support of our NIH R01 application. Success in each aim on its own will produce high impact publications and ACS IRG 2009 Proposal, Steven J. Koch, [email protected]

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open doors for further applications in genomics and in vitro Pol II studies. Success in our longer-term goal of single-molecule chromatin mapping will provide important insights into the open questions in Pol II transcription as well as provide a new tool for studying chromatin biology in cancer cells. Two cancer biology areas we wish to pursue are epigenetic control of gene transcription(3) and chromatin remodeling during DNA double-strand break repair(10, 15). Background and significance A. 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 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 (PTMs), but their functions in transcription are not yet well understood(16). In order to understand these processes, we need to know with basepair Reassembled precision the position of all polymerases promoter Nucleosomes and nucleosomes on the gene. Pol Furthermore, we need to know whether the II nucleosomes are completely assembled cryptic octamers or are tetrameric or hexameric. Transcription promoter Finally, we need to know this information for polymerases and Figure 2 Nucleosome remodeling during transcription. Nucleosomes are evicted in front of the polymerase and reassembled behind the nucleosomes on the same individual polymerase. Proper reassembly prevents initiation from cryptic promoters. gene—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, FACT (17-20), and a specific PTM, monoubiquitylation of histone H2B (H2B-ub)(11). Both FACT and H2B-ub are important to cancer biology(21). The Osley lab has discovered that when both FACT and H2B-ub are not functioning, some kind of improper chromatin structure is assembled 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, particularly as related to human cancers. 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. This is called cryptic initiation and it is rapidly becoming an important area of study in transcription. (22, 23) 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 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 possible explanation is that antisense-oriented polymerases quickly encounter sense-oriented polymerases, and these ACS IRG 2009 Proposal, Steven J. Koch, [email protected]

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head-to-head collisions effectively stall both complexes. The ability to detect sense orientation of Pol II complexes is difficult with ensemble techniques. Due to the asymmetry of the Pol II-DNA complex (in contrast to the nucleosome)(25), it is 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 senseand 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

Data from Shundrovsky, et al., Nature Structural & Molecular Biology 13, p. 549 (Not Koch Data)

In my dissertation work, I showed that positions of DNA-binding proteins could be mapped with high resolution by unzipping single DNA molecules with optical tweezers(4). 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 enable these experiments, I designed and implemented a versatile unzipping anchoring construct that allows for ©2006 Nature Publishing Group unzipping of any DNA molecule with a known 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 used Figure 3 The protein mapping method my versatile unzipping construct to unzip through reconstituted developed by Koch has been shown to mononucleosomes and tetrasomes in the Wang lab. Some of this data be capable of measuring the positions of is shown in Fig. 3. The authors clearly demonstrated that nucleosomes with 3 bp precision. It was mononucleosome positions can be mapped with about 3 basepair also shown possible to clearly distinguish precision. Furthermore, they were able to easily distinguish between octames and tetramers. between octameric and tetrameric nucleosomes(8). These results give us confidence that we will be able to map nucleosome positions on native chromatin. One of the specific aims of this proposal is to perform single-molecule unzipping of stalled RNA Polymerase II complexes. Success in this aim will provide strong evidence that Dig Biotin Nick we will be able to map and identify polymerases and Sticky end 5’ 3’ for ligation 5’ nucleosomes on individual native chromatin fibers. Progress and Preliminary Data

Figure 4 The versatile unzipping construct from Koch et al. 2002 will be used in both aims. Any sticky end desired is easily created via choice of oligonucleotide sequence.

Discussed below are progress and results funded by the first year of ACS IRG funding as well as those funded by startup and directly relevant to this renewal and future R01 funding. A. Optical tweezers instrumentation and data analysis software As of January 2009, we now have all of the components required for our optical tweezers instrumentation. The remaining important steps are assembly and calibration of optical tweezers and conversion of existing data acquisition software for modified hardware. We estimate approximately two months for these tasks, which will not be funded by this renewal. Important components we have on hand and milestones we have achieved include: An Olympus IX-71 inverted microscope with a PlanApo 1.4 NA oil immersion objective. A Mad City Labs 1-D piezo stage with 30 micron travel and sub-nanometer precision. An OEM laser diode system with a 690 nm (visible-red) laser diode.

ACS IRG 2009 Proposal, Steven J. Koch, [email protected]

Figure 5. Photo of summer 2008 532 nm optical tweezers successful prototype. Optical path outlined in green. We trapped and manipulated microspheres with this setup, but the laser stability and lack of piezo stage were not suitable for unzipping experiments.

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Quadrant photodiode position sensing detector and amplifier. A versatile optical tweezers feedback control LabVIEW application(26). Hardware-specific portions of this software remain to be updated. Fully-functioning data analysis LabVIEW software, customized for automated DNA unzipping applications (26). Prototyped optical tweezers successfully achieved in summer 2008 (See Fig. 5) B. Shotgun DNA Mapping (For the purpose of Shotgun Chromatin Mapping) We have made substantial progress towards a 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 simulated unzipping forces of a published genome. SDM will be an important enabler of our ability to map native chromatin by unzipping without the need for site-specific genetic engineering. We call this proposed method Figure 6. Overview of proposed method for shotgun DNA and chromatin shotgun chromatin mapping (SCM) and the mapping. We have recently achieved proof-of-principle results important method is outlined in Fig. 6. Native chromatin for the “Global Genome Location,” part of the process ( lower right). will be digested with restriction endonucleases and random fragments will be unzipped, providing high resolution positional information on nucleosomes and polymerases (see next section). 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—thus, the application of the technique to human cells and even primary tumor cells can be envisioned once success has been demonstrated in yeast.

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Figure 17 Experimental unzipping data compared with (A) correct and (B) incorrect simulation. The green window indicates the region from j=1200 to 1700 where the match scores were computed. The greatly increased separation of the two curves in the incorrect match is reflected in the higher match score of 0.8 versus 0.2 for the correct match.

SDM works because we can accurately predict the unzipping forces for any known sequence of DNA. Fig. 7 shows one experimental unzipping trace 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 developed an algorithm that would quantify the match between and experimental and simulated unzipping curve. We tested this algorithm using experimental unzipping data for pBR322 plasmid. Our algorithm easily identified the pBR322 fragment from within a background of simulations of all ~2700 possible yeast XhoI digested fragments. Fig. 8 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.

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C. Collaborations. We have initiated collaborations with the labs of Dr. Mary Ann Osley (UNM School of Medicine, Dept. of Molecular Genetics and Microbiology) and Dr. Karen Adelman (NIH NIEHS, Laboratory of Molecular Carcinogenesis). These are two well-known labs with extensive experience in chromatin biology. Molecular biology work in this proposal will be carried out in Dr. Osley’s lab, and Dr. Adelman’s lab will provide us with stalled RNA Pol II complexes. Please see attached letters of support. Research Methods

Match Score

We have filed a provisional patent disclosure via STC.UNM and we are currently preparing a manuscript describing our proof-of-principle results(14) for SDM. One specific aim of this proposal is to extend our proof of principle and fully validate the method for XhoI digested yeast genomic DNA.

Match

File Number (Arb.) Figure 18 Compilation of match scores for a single experimental data set. The file number is an arbitrary, arising from the order in which the library simulations were loaded. A perfect match would have a score of zero, and the correct match can be seen as having the lowest score, very distinguishable from the incorrect matches.

Our 5-year goal for our NIH R01 proposal is the development of singlemolecule chromatin mapping based on optical tweezers unzipping. Below is a one-year research plan designed to provide preliminary data in two key areas. The first aim is to demonstrate “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 published genome(14). 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 unzipping force pattern of reconstituted mononucleosomes (8, 9), 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. Specific Aim 1: Prove that shotgun DNA mapping (SDM) works with yeast genomic DNA and improve the SDM algorithms. Sub Aim 1: Use a shotgun cloning strategy to prove that SDM works for yeast genomic DNA. Steps 1-3 and 5 in Fig. 9 will be carried out by Anthony Salvagno with guidance from Kelly Trujillo, a postdoc in Osley lab. Step 4 will be carried out by Larry Herskowitz in Koch lab. So far, we have demonstrated with existing pBR322 data that the pBR322 fragment can easily be identified from a background of the approximately 2700 yeast XhoI sites(14). 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 pBR322

3. Prepare unzipping constructs from miniprep DNA

4. Predict identity of clone based on shotgun DNA mapping by unzipping

5. Sequence clones, compare results and determine success rate

Figure 9 Process for validating shotgun DNA mapping method for yeast genomic DNA

As shown in Figure 9, we validate the shotgun DNA mapping method in yeast via a shotgun cloning method. In steps 1 and 2, we will use XhoI for digestion of genomic DNA and pBR322 as our vector. This will give us the flexibility in step 3 of using either the proven pBR322 unzipping construct methods(4), or alternatively the XhoI sites. We will follow our published ligation method in step 3, which involves ligation of the linear miniprep DNA to an “unzipping anchor,” which is appropriately labeled with digoxigenin and biotin for single-molecule ACS IRG 2009 Proposal, Steven J. Koch, [email protected]

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tethering and unzipping(4, 27). In step 4, we will follow our standard published tethering and unzipping protocol. Briefly, microspheres will be tethered to glass surfaces via dig-antidig and biotin-streptavidin linkages. Dozens of tethers will be selected by eye and unzipped automatically by feedback-controlled optical tweezers. We will use the existing DNA unzipping data acquisition and analysis software that was used for our pBR322 proof-ofprinciple(4, 5, 26). We will use the shotgun DNA mapping software application we have developed(14) with possible improvements from sub-aim 2. Finally, in step 5, we will unblind our experiments by revealing the identity of the chosen clones via DNA sequencing available at UNM. Overall, this method will be lowthroughput, 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—where the cloning steps will be eliminated. Alternative plan: If problems arise from the cloning strategy, we will form unzipping constructs from specific genomic regions amplified via PCR. We will keep the identity of the primers concealed from the singlemolecule experimenters until the identities have been predicted by SDM. Sub Aim 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 two clear paths for achieving this: (a) improving the DNA modeling and (b) developing a more sophisticated and better matching algorithm. Modeling DNA unzipping. We are currently using a very simple model for the DNA energetics (28). 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 basestacking 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 the energy values for the 10 nearest-neighbor interactions(29). 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. 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 match score (and thus increase the sensitivity and specificity) via a number of independent avenues. The first avenue we will pursue 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 shift in the data as in previous nucleosome mapping work (8). Beyond this, we will pursue numerous other match criteria, which when combined with our existing match scoring may greatly increase our sensitivity and specificity. Specific Aim 2: Determine the unzipping signature for RNA Polymerase II by unzipping through stalled in vitro transcription complexes. Ligation reactions will be carried out by Anthony Salvagno with help from Kelly Trujillo, a postdoc in Osley lab. Unzipping and analysis will be carried out by Mr. Salvagno in Koch lab. In order to map the positions of Pol II on native chromatin by unzipping, we need to determine what the unzipping force pattern will be for Pol II complexes—similar to as has already been done for nucleosomes (see background section C). In order to calibrate the signal we expect to see from Pol II while unzipping native chromatin, we will unzip complexes prepared from in vitro transcription using purified components. The Adelman lab has extensive experience with in vitro Pol II transcription and will provide us with elongating transcription complexes stalled via nucleotide depravation(12, 13, 30, 31). 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 ACS IRG 2009 Proposal, Steven J. Koch, [email protected]

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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 two different 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.

Figure 10. Stalled Pol II in vitro transcription complexes will be provided by the Karen Adelman lab of the NIEHS. The transcription templates will be prepared with two distinct non-palindromic overhangs. This will allow ligation to unzipping constructs in either the sense or antisense orientation.

The beauty of the unzipping anchor designed by Koch is that it 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 mapping experiments (Aim 1). Ligated constructs will be tethered 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 (based on crosscorrelation) and to generate a “master pattern” that we can use for identifying Pol II complexes in future native chromatin experiments. Broader Impacts Success in development of this single-molecule chromatin mapping method will enable a wide array of new investigations relevant to cancer. Two areas we are planning on pursuing are gene transcription and DNA damage repair. Our technique lends itself to future automation as a high-throughput assay, and when this is implemented, we will enable possible studies of the epigenomics of cancer in transcription, damage repair and replication. Furthermore, we envision two other cancer related avenues. The first is applying shotgun DNA mapping as a tool for structural genome mapping. The unzipping method is very well suited for identifying many kinds of mutations, such as deletions, insertions, and even balanced mutations such as inversions. The second avenue is for studying telomere structure, which is important in cancer biology. Methods have been developed for isolating telomeres with defined cohesive ends, which would enable study of their structure via the unzipping method.

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Acknowledgments I thank Anthony Salvagno, Larry Herskowitz, Andy Maloney, Diego Ramallo, Kelly Trujillo, Karen Adelman, and Mary Ann Osley for significant help in generation of this research plan. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

14.

15. 16. 17. 18. 19. 20. 21. 22. 23.

Kornberg, R. D., "Chromatin structure: a repeating unit of histones and DNA." Science 184, 868 (May 24, 1974). Fuchs, S., Laribee, R., Strahl, B., "Protein modifications in transcription elongation." Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1789, 26 (2009). Jones, P. A., Baylin, S. B., "The epigenomics of cancer." Cell 128, 683 (2007). Koch, S. J., Shundrovsky, A., Jantzen, B. C., Wang, M. D., "Probing protein-DNA interactions by unzipping a single DNA double helix." Biophys J 83, 1098 (Aug, 2002). Koch, S. J., Wang, M. D., "Dynamic force spectroscopy of protein-DNA interactions by unzipping DNA." Phys Rev Lett 91, 028103 (Jul 11, 2003). Koch, S. J., Thayer, G. E., Corwin, A. D., de Boer, M. P., "Micromachined piconewton force sensor for biophysics investigations." APPLIED PHYSICS LETTERS 89, 173901 (2006). Xia, D., Gamble, T. C., Mendoza, E. A., Koch, S. J., He, X., Lopez, G. P., Brueck, S. R., "DNA transport in hierarchically-structured colloidal-nanoparticle porous-wall nanochannels." Nano Lett 8, 1610 (Jun, 2008). Shundrovsky, A., Smith, C. L., Lis, J. T., Peterson, C. L., Wang, M. D., "Probing SWI/SNF remodeling of the nucleosome by unzipping single DNA molecules." Nature Structural & Molecular Biology 13, 549 (2006). Hall, M. A., Shundrovsky, A., Bai, L., Fulbright, R. M., Lis, J. T., Wang, M. D., "High-resolution dynamic mapping of histone-DNA interactions in a nucleosome." Nat Struct Mol Biol (Jan 11, 2009). Tsukuda, T., Fleming, A. B., Nickoloff, J. A., Osley, M. A., "Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae." Nature 438, 379 (2005). Fleming, A. B., Kao, C. F., Hillyer, C., Pikaart, M., Osley, M. A., "H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation." Mol Cell 31, 57 (Jul 11, 2008). Muse, G., Gilchrist, D., Nechaev, S., Shah, R., Parker, J., Grissom, S., Zeitlinger, J., Adelman, K., "RNA polymerase is poised for activation across the genome." Nature Genetics 39, 1507 (2007). Zeitlinger, J., Stark, A., Kellis, M., Hong, J. W., Nechaev, S., Adelman, K., Levine, M., Young, R. A., "RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo." Nat Genet 39, 1512 (Dec, 2007). "Herskowitz, Lawrence, Salvagno, Anthony, Maloney, R. Andy, Le, Linh, and Koch, Steven. Proof of principle for shotgun DNA mapping by unzipping. Available from Nature Precedings (2009)." Osley, M., Tsukuda, T., Nickoloff, J., "ATP-dependent chromatin remodeling factors and DNA damage repair." Chromatin: Repair, Remodeling and Regulation 618, 65 (2007). Li, B., Carey, M., Workman, J. L., "The Role of Chromatin during Transcription." Cell 128, 707 (2007). Orphanides, G., LeRoy, G., Chang, C. H., Luse, D. S., Reinberg, D., "FACT, a factor that facilitates transcript elongation through nucleosomes." Cell 92, 105 (1998). Orphanides, G., Wu, W.-H., Lane, W., Hampsey, M., Reinberg, D., "The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins." Nature 400, 284 (1999). Belotserkovskaya, R., Oh, S., Bondarenko, V., Orphanides, G., Studitsky, V., Reinberg, D., "FACT Facilitates Transcription-Dependent Nucleosome Alteration." Science 301, 1090 (2003). Saunders, A., Werner, J., Andrulis, E., Nakayama, T., Hirose, S., Reinberg, D., Lis, J., "Tracking FACT and the RNA Polymerase II Elongation Complex Through Chromatin in Vivo." Science 301, 1094 (2003). Espinosa, J. n. M. (2008), vol. 22, pp. 2743-2749. Buratowski, S., "TRANSCRIPTION: Gene Expression--Where to Start?" Science 322, 1804 (2008). Fleming, A. B., Kao, C.-F., Hillyer, C., Pikaart, M., Osley, M. A., "H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation." Mol Cell in press (2008).

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24. 25. 26.

27. 28. 29. 30. 31.

Core, L., Waterfall, J., Lis, J., "Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters." Science, 1162228 (2008). Gnatt, A. L., Cramer, P., Fu, J., Bushnell, D. A., Kornberg, R. D., "Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution." Science 292, 1876 (Jun 8, 2001). "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." "Public protocol available on our OpenWetWare site: http://openwetware.org/index.php?title=Koch_Lab:Protocols/Unzipping_constructs&oldid=212016." Bockelmann, U., EssevazRoulet, B., Heslot, F., "Molecular stick-slip motion revealed by opening DNA with piconewton forces." PHYSICAL REVIEW LETTERS 79, 4489 (1997). SantaLucia, J., Jr., "A unified view of polymer, dumbbell, and oligonucleotide DNA nearestneighbor thermodynamics." PNAS 95, 1460 (February 17, 1998, 1998). Adelman, K., Wei, W., Ardehali, M. B., Werner, J., Zhu, B., Reinberg, D., Lis, J. T. (2006), vol. 26, pp. 250-260. 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).

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