Visualizing CTCF Mediated DNA Looping at the Single Molecule Level

Monday, February 13, 2017

Symposium: TRP Channels 830-Symp Mechanisms of TRPV1 Ion Channel Gating Sharona E. Gordon. University of Washington, Seattle, WA, USA. TRPV1 ion channels are multimodal integrators of diverse stimuli, including small molecule agonists and inhibitors, phospholipids, lysolipids and fatty acids, and increased temperature. They are also subject to cellular regulation via second messengers such as Ca2þ and by phosphorylation. We have studied the molecular mechanism underlying TRPV1 regulation to better understand its function in the complex environment of a cell membrane. 831-Symp Computational Approaches to the Study of TRPV Channel Activation and Modulation Carmen Domene1,2. 1 Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom, 2Chemistry, King’s College London, London, United Kingdom. Transient receptor potential (TRP) ion channels constitute a notable family of cation channels involved in the ability of organisms to detect noxious mechanical, thermal and chemical stimuli that gives rise to the perception of pain. One of the most experimentally studied agonist of TRP channels is capsaicin, which is responsible for the burning sensation produced when chili pepper is in contact with organic tissues. Understanding how TRP channels are regulated by capsaicin and other natural products is essential to high impact pharmacological applications, particularly those related to pain treatment. By selected examples from the work we have carried out, I will provide an overview of the current knowledge we have about activation, permeation and selectivity of one of these human molecular thermometers. 832-Symp New Insights into the Function of TRPV Channels Vera Moiseenkova Bell. Pharmacology, Case Western Reserve University, Cleveland, OH, USA. Transient receptor potential vanilloid 2 (TRPV2) is a non-selective Ca2þ-permeable cation channel that belongs to the vanilloid subfamily of the TRP superfamily. Despite its discovery as a thermoTRPV channel over 15 years ago, TRPV2 remains an ‘‘orphan TRP’’ due to its still unclear physiological function. Here we took advantage of our ability to generate stable, functional recombinant TRPV2 to gain new biological insights into the cellular function of TRPV2. Using tetrameric TRPV2 as an antigen, we generated TRPV2 monoclonal antibodies and used these antibodies to test the controversial hypothesis that growth factors, including insulin-like growth factor 1 (IGF-1), cause translocation of TRPV2 to the plasma membrane. We found that TRPV2 localizes to intracellular membranes in both the absence and presence of growth factors in multiple cell types. Additionally, we used these newly generated antibodies to explore the role of TRPV2 in neuronal cell development. A detailed analysis revealed that nerve growth factor (NGF) increases TRPV2 expression through the MAPK signaling pathway. Furthermore, we discovered TRPV2 acts as a novel ERK substrate, whereby phosphorylation of TRPV2 by ERK enhances TRPV2mediated Ca2þ signals and neurite outgrowth downstream of NGF. TRPV2 localizes to endosomal pools within neurites of developing neurons, where it may serve to alter local Ca2þ signals. Based on these data, we propose a previously uncharacterized mechanism in which TRPV2 acts as an endosomal Ca2þ channel regulated by NGF via the MAPK pathway to alter local Ca2þ signaling within neurites and enhance neurite outgrowth.

Platform: Protein-Nucleic Acid Interactions I 833-Plat Single Molecule FRET Studies on the Mechanism of ATP-Dependent RNA Unwinding by Dead-Box Helicases: An RNA-Induced Movement of the RNA Binding Domain of YxiN Regulates Unwinding by the Helicase Core Brighton Samatanga, Alexandra Z. Andreou, Dagmar Klostermeier. Physical Chemistry, University of Muenster, Muenster, Germany. DEAD-box proteins catalyze the ATP-dependent unwinding of RNA duplexes. Their common helicase core, formed by two RecA-like domains, is typically flanked by additional domains that modulate helicase function by hitherto unidentified mechanisms. To understand the regulation of the DEAD-box helicase YxiN by its C-terminal RNA binding domain (RBD), we investigated the effect of RNA binding to the RBD on its position relative to the core. To this end, we used distances from single molecule FRET experiments on freely diffusing YxiN molecules by confocal microscopy as restraints to model structures of YxiN in the absence and presence of RNA. RNA binding to the RBD causes

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a substantial movement of the RBD, from a position close to the C-terminal RecA-domain of the core towards the N-terminal RecA-domain. This movement is independent of direct contacts of the bound RNA with the core. Importantly, RNA binding to the RBD is communicated to the core, and stimulates ATP hydrolysis and RNA unwinding activities. RNA unwinding by DEADbox helicases has been linked to a conformational cycle, involving switching of the helicase core between open and closed conformations. In single molecule FRET experiments on immobilized YxiN using total internal reflection microscopy, we investigated the conformational dynamics of the YxiN core when RNA is bound to the RBD. We show that the conformational space of the helicase core, and hence the effect of the RNA on core activities, depends on the identity of the bound RNA, pointing to a differential recognition of RNA elements in their structural context. Our results establish that the RBD does not merely act as a passive anchor for YxiN on the RNA substrate but instead actively modulates the functions of the helicase core. Regulation of core activities by RNA-induced movement of ancillary domains may constitute a general regulatory mechanism of DEAD-box protein activity. 834-Plat Direct Single Molecule Measurement of ATP Hydrolysis Substates in Hel308 DNA Helicase using Nanopore Tweezers Jonathan M. Craig, Andrew H. Laszlo, Henry D. Brinkerhoff, Ian M. Derrington, Matt Noakes, Ian C. Nova, Kenji M. Doering, Benjamin I. Tickman, Noah F. De Leeuw, Jens H. Gundlach. University of Washington, Seattle, WA, USA. Single-molecule picometer resolution nanopore tweezers (SPRNT) is a single molecule tool for studying enzymes that move on nucleic acids (NA). SPRNT measures NA position relative to the enzyme at subAngstrom spatial resolution on millisecond timescales, while simultaneously providing the NA sequence near the enzyme. We use this method to resolve two substates of the ATP hydrolysis cycle of a Hel308 DNA Helicase. By examining the dwell times of each state, we derive a kinetic model of Hel308’s translocation along single-stranded DNA, and find that the model’s rate constants depend on the DNA sequence within the enzyme. This is, to our knowledge, the first evidence of sequence-dependent translocase behavior in a helicase system. 835-Plat Measuring the Orientation of Single Proteins Interacting with DNA using Fluorescence Polarization Microscopy Emil Marklund, Elias Amselem, Kalle Kipper, Magnus Johansson, Sebastian Deindl, Johan Elf. Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden. The polarization of the light emitted from a fluorophore is determined by its dipole moment and hence by the direction of the molecule. We are using this phenomena to measure the orientation of labeled transcription factor molecules. We are studying the lac repressor, labeled with a fluorophore that is predicted to be rigid and oriented along the DNA when the protein is associated to the DNA substrate. In our experiments we measure polarization of the light emitted from single transcription factor molecules when they are bound specifically and sliding on flow stretched DNA. The polarization data acquired is shifted towards the direction of DNA with relatively small changes in the signal over several seconds when using an integration time of ~100 ms. This shows that the transcription factor has a preferred and maintained orientation while interacting with the DNA substrate on this timescale. For transcription factors sliding on DNA, very little polarization preference is observed in the direction that would be averaged out by rotation of the fluorophore around the DNA. Taken together these observations strongly support the model of rotational sliding for the lac repressor. By using a simplified model of dipole excitation and emission we have also been able to quantify the fluorophore orientation and wobbliness for individual transcription factor molecules sliding on DNA. This analysis shows that around 40 % of the labeled molecules have an average dipole orientation close to parallel to DNA (<20 ) with a standard deviation in the orientation of 40 -60 . The remaining 60 % have a more undefined orientation with larger standard deviation (>60 ). 836-Plat Visualizing CTCF Mediated DNA Looping at the Single Molecule Level Maria Eugenia Fuentes Perez1, Kotryna Bloznelyte2, Matthias Merkenschlager1,2, David Rueda1,2. 1 Medicine, Imperial College, London, United Kingdom, 2Clinical Science Center, Medical Research Council, London, United Kingdom. Long distance interactions in chromatin play important roles during replication and gene regulation, where distant DNA regions are brought together to organize the genome in 3D. CTCF is an essential nuclear protein highly conserved from

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Monday, February 13, 2017

flies to human that it is implicated in multiple roles, including division of chromatin, gene activation, gene repression, and genome looping among others. CTCF contains 11 zinc fingers that can bind up to 35 bp of DNA including the well-conserved CCCTC sequence. Moreover, the binding sites orientation may play an important role in the regulation of enhancer-promoter choice, and the direction of looping formation. Here, we have used single molecule fluorescence resonance energy transfer (smFRET) to monitor and characterize CTCF-mediated DNA lopping. To this aim, we have designed a DNA substrate with a fluorophore-labelled (Cy3 and Cy5) binding site located at each end, such that loop formation brings together the two ends of the molecule thereby we will be able to observe FRET signal. Our data show that loop formation is only observed in the presence of two sequence specific binding sites and CTCF. Loop formation probability increases with protein concentration, yielding a dissociation constant of 10 nM and a Hill coefficient 1.6 in agreement with looping induced by dimers. Loop formation probability depends strongly on the distance between the binding sites, similar to previously reported models for the bacterial Lac operator. Contrary to what is observed in cells, our data show that CTCF does not favor any particular binding site orientation in vitro. Our results indicate that the preferred looping orientation observed in vivo likely results from additional regulatory looping factors. 837-Plat Protein-Mediated Loops in Supercoiled DNA Create Large Topological Domains Yan Yan1, David D. Dunlap1, Fenfei Leng2, Laura Finzi1. 1 Department of Physics, Emory University, Atlanta, GA, USA, 2Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA. Topological domains are structural units of practically all DNA genomes. They are formed of segments with constrained ends, usually by proteins. If these boundary elements restrict the free rotation of the DNA, they may be torsionally isolated. The existence of the topological domains helps to compact the DNA, reduce the amount of DNA that is relaxed by nicking, specifically regulate cellular processes, and partition the genome into active and non-active regions. A naı¨ve expectation is that the size of such domains would be as large as the distance between the protein binding sites. This appears to be the case in relaxed DNA. To investigate this magnetic tweezers were used to arbitrarily supercoil DNA segments containing high affinity LacI binding sites separated by 400 bp. As shown previously for lambda repressor-mediated loops, slight tension abolished looping unless the DNA was supercoiled and additional supercoiling compensated for increasing tension up to a threshold. Furthermore, the LacI protein junction of the loop was an effective barrier to the diffusion of supercoils even in DNA subject to the maximum torsional strain that can be produced under 0.5 pN of tension. Finally, loops that formed in supercoiled DNA often created topological domains that extended past the loop segment boundaries. Thus in considering loop-stabilized, topological domains it is important to consider the possibility that they include and affect the availability of sites outside the loop in supercoiled DNA. 838-Plat Toward Direct Observation of the DNA Binding Dynamics of Monomeric Type IIP Restriction Endonucleases Candice M. Etson. Physics Department, Wesleyan University, Middletown, CT, USA. Restriction endonucleases (REases) are enzymes that cleave duplex DNA at a recognition site specified by a particular nucleotide sequence. These enzymes are found in a wide range of bacterial species, and serve as a defense against infection by phage. Type IIP REases are typically antiparallel homodimers that recognize a palindromic DNA sequence and cleave within or close to the recognition site. However, some Type IIP REases cleave pseudopalindromic sequences, and some of these may be active as monomers. It is hypothesized that these monomeric REases sequentially cleave the two complementary strands of duplex DNA during a single DNA binding event. Since the two strands of DNA are antiparallel, after cleaving one strand, a monomeric REase would be required to rotate about the axis perpendicular to the DNA to be properly oriented to cleave the second strand. This type of reorientation is known a ‘‘flipping’’. Although only a small number of proteins have been observed reorienting in this way, protein flipping may play an important role in binding to a specific DNA sequence. Direct observation of flipping during DNA cleavage will greatly improve our understanding of how monomeric REases mediate double strand breaks. We are currently using total internal reflection fluorescence (TIRF) microscopy to observe REase-mediated cleavage of quantum dot-labeled DNA at the single-molecule level. In our highly multiplexed assay, the disappearance of a quantum dot indicates cleavage of the DNA that tethers it to a functionalized glass coverslip. We plan to utilize single-molecule Fo¨rster resonance energy

transfer (smFRET) to trace individual monomeric REases in real time through the entire process of DNA cleavage from substrate binding to product release. Our observations should provide significant insight into the mechanism by which these enzymes cleave duplex DNA. 839-Plat Single Molecule Studies on G-Quadruplex, Protein, and Small Molecule Interactions Hamza Balci, Sujay Ray, Jagat Budhathoki, Parastoo Maleki. Physics, Kent State University, Kent, OH, USA. Recent single molecule studies have revealed a number of significant insights on the mechanism of protein and G-quadruplex (GQ) interactions. In particular, our work on GQ interactions with single stranded DNA binding proteins, such as RPA and POT1, and helicases, such as BLM and RECQL5, showed significant variations in terms of the underlying dynamics and efficacy of these molecules in unfolding GQ structures. On the other hand, GQ-stabilizing small molecules have attracted attention due to their potential use as telomerase inhibitors and anticancer drugs. Such small molecules are expected to promote GQ folding and stability while inhibiting the activity of proteins that unfold GQ. A comparative single molecule study of how such small molecules influence GQ folding and stability and inhibit the activity of GQ-destabilizing proteins will be presented. 840-Plat High-Resolution Single Molecule Rotation Tracking of RecBCD using DNA Origami Rotors Benjamin D. Altheimer1, Pallav Kosuri2, Mingjie Dai1, Peng Yin3,4, Xiaowei Zhuang2,5. 1 Biophysics, Harvard University, Boston, MA, USA, 2Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA, 3Wyss Institute, Harvard University, Boston, MA, USA, 4Systems Biology, Harvard University, Boston, MA, USA, 5Physics, Harvard University, Cambridge, MA, USA. We have invented a new high throughput, high resolution single molecule method that enables tracking the rotation of DNA molecules with millisecond time resolution. Origami Rotational Beacon Image Tracking (ORBIT) uses DNA origami rotors that can be ligated to a DNA duplex of interest. We track the rotation of the DNA duplex, as amplified by the DNA origami, using fluorescent labels on one arm of the origami. ORBIT does not rely on an externally applied force but instead achieves high spatiotemporal resolution mainly due to the low drag of the origami rotor and the stiffness of the short attached DNA duplex. Using ORBIT, we report the first direct measurements of DNA rotation by the helicase RecBCD. We observe pausing and backtracking and account for our observations with a simple translocation model. We also report high resolution measurements of RecBCD initiation, discovering unexpected reversibility in DNA unwinding and rewinding prior to translocation. Our results using substrates with single stranded overhangs indicate that the RecB motor domain is responsible for initiating processive translocation.

Platform: Membrane Receptors and Signal Transduction II 841-Plat The Allosteric Site is Required for Voltage Dependence of Muscarinic GPCRs Anika Hoppe1, Moritz B€unemann2, Andreas Rinne1. 1 Department of Cardiovascular Physiology, Ruhr-University Bochum, Bochum, Germany, 2Department of Pharmacology and Clinical Pharmacy, Philipps-University Marburg, Marburg, Germany. G-protein coupled receptors (GPCRs) encompass the largest group of membrane proteins in eukaryotes and regulate numerous intracellular pathways. Class A GPCRs are activated by binding of specific agonists to an orthosteric binding site, which is formed by the seven trans-membrane helices. In contrast, so called allosteric modulators bind to a site on the extracellular surface, the allosteric site. There is an interaction of both sites to modify receptor function and allosteric modulators either enhance or attenuate receptor signaling. Previous work from our laboratory showed that muscarinic receptors (M-Rs) are voltage sensitive. Voltage dependence was only evident for active receptors and may represent allosteric modulation of M-Rs. In this study we compared the voltage dependencies of M1-Rs and M3-Rs to allosteric modulation of both receptors. Changes in receptor signaling were quantified in single HEK 293 cells with a FRET biosensor for the Gq protein cycle. In the presence of acetylcholine (ACh), a depolarization of the membrane from 90 mV to þ40 mV potentiated M1-R signaling, whereas it attenuated M3-R signaling. Likewise, the positive allosteric modulator BQCA potentiated ACh/M1-R signaling, whereas the negative allosteric modulator Gallamine attenuated