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TFAM Regulates Mitochondrial Transcription through Sequence-Specific DNA Looping
516a
Wednesday, March 2, 2016
models for the process of mechanochemical force generation have been put forward, but there is no consensus on which, if any, of these is correct. All the existing models assume that protein-generated forces drive the DNA forward. The scrunchworm hypothesis proposes that the DNA molecule is the active forcegenerating core of the motor, not simply a substrate on which the motor operates (Harvey (2015) J Struct Biol 189:1-8). The protein components of the motor dehydrate a section of the DNA, converting it from the B form to the A form and shortening it by about 23%. The proteins then rehydrate the DNA, which converts back to the B form. Other regions of the motor grip and release the DNA to capture the shortening-lengthening motions of the B/A/B cycle (‘‘scrunching’’), so that DNA is pulled into the motor and pushed forward into the capsid. This DNAcentric mechanism provides a quantitative physical explanation for the magnitude of the forces generated by viral packaging motors. It also provides a simple explanation for the fact that each of the steps in the burst cycle advances the DNA by 2.5 base pairs. The scrunchworm hypothesis is consistent with a large body of published data, and it makes four experimentally testable predictions. Here I discuss progress in experimental and modeling tests of the scrunchworm hypothesis. 2542-Plat TFAM Regulates Mitochondrial Transcription through Sequence-Specific DNA Looping Divakaran Murugesapillai1, Maria F. Lodeiro2, Louis J. Maher III,3, Craig E. Cameron2, Mark C. Williams1. 1 Department of Physics, Northeastern University, Boston, MA, USA, 2 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA, 3Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA. Mitochondrial transcription factor A (TFAM) is an abundant human mitochondrial High Mobility Group Box (HMGB) protein. TFAM is an architectural protein that shapes mitochondrial DNA (mtDNA) and regulates mitochondrial transcription. Sequence-specific binding of TFAM to DNA upstream of the light-strand promoter (LSP) leads to bending that correlates with transactivation of this promoter. Here we use a dual promoter construct containing both LSP, HSP1 and the natural inter-promoter region (IPR) to understand how TFAM binding governs transcription activation. We show that the IPR contributes to TFAM transactivation of HSP1, while removal of the carboxyl-terminal tail of TFAM (TFAM-DCT26) leads to a complete loss of transactivation of HSP1, with only minimal effects on LSP, suggesting a different biophysical mechanism for TFAM-mediated transcription activation from HSP1. To understand the mechanism by which TFAM activates HSP1 transcription, we used atomic force microscopy (AFM) in liquid to probe the effect of TFAM on the IPR construct. We observe that at the low concentrations of TFAM where transcription activation is high, TFAM induces the formation of DNA loops in a region defined by the IPR, with TFAM present at the strand crossing point. Under the same conditions, TFAM-DCT26 fails to induce DNA looping. While random DNA also forms loops in the presence of TFAM, TFAM is twice as likely to mediate the loops when the IPR sequence is present. Optical tweezers experiments also demonstrate that TFAM stabilizes DNA loops, which require significant force to break. Taken together, our results are consistent with sequence-specific DNA looping contributing to TFAM transactivation of HSP1, suggesting that unique mechanisms are employed for TFAM-dependent transcription at LSP and HSP1. 2543-Plat Visualizing the Assembly of DNA Condensation Clusters by SMC using Single-Molecule Microscopy Hyeongjun Kim, Joseph J. Loparo. BCMP, Harvard Medical School, Boston, MA, USA. SMC (structural maintenance of chromosomes) family members play essential roles in chromosome condensation, sister chromatid cohesion, and DNA repair. Despite intensive efforts, it is still unclear how SMCs are loaded onto DNA and structure chromosomes. We employed single-molecule fluorescence microscopy to visualize how individual Bacillus subtilis SMC (BsSMC) dimers interact with flow-stretched DNAs. We find that BsSMC can vary its initial interaction with DNA, switching between static binding and sliding. Its diffusive properties are insensitive to the presence of ATP, suggesting that the initial loading of BsSMC onto DNA is ATP-independent. At higher concentrations, BsSMCs form distinct clusters that condense DNA by both wrapping and bridging distal DNA segments. SMC-mediated DNA compaction occurs in a weakly ATP-dependent manner. However, ATP increases the apparent cooperativity of DNA condensation, demonstrating that multiple BsSMCs can interact cooperatively through their ATPase head domains. Consistent with these results, BsSMC mutants that alter the ATPase cycle compact DNA more slowly than wild-type BsSMC in the presence of ATP, and an ATPase headless
construct compacts DNA non-cooperatively with substantially lower compaction rate. Our results suggest that transiently static BsSMC can nucleate the formation of functionally active clusters that locally condense the chromosome while forming long-range DNA bridges. 2544-Plat Single Molecule Imaging of p53’s Dynamic Interaction with Chromatin Vincent Wong1, Zhe Liu2, Sam Peng1, Charles Kenworthy1, Wei-Li Liu1, Robert A. Coleman1. 1 Albert Einstein College of Medicine, Bronx, NY, USA, 2Howard Hughes Medical Institute Janelia Research Center, Ashburn, VA, USA. P53 is a transcriptional activator that binds to its response elements (REs) on target promoters and activates expression of a large number of genes involved in tumor suppression. Previous genome-wide studies showed that p53 binds to its REs in regions densely populated with nucleosomes. However, it is unknown how p53 accesses its target REs in the context of chromatin to regulate transcription. To better decipher the interaction of p53 with chromatin targets, we utilized a combination of in vivo live cell and in vitro single molecule imaging along with bioinformatics and Next Generation Proteomic screens. Our live cell imaging studies indicate a dynamic interaction between p53 and chromatin that varies in distinct sub-nuclear compartments. In vitro, p53 binds a mononucleosome on a native p53 target gene with nearly identical residence times as our live cell measurements. Strikingly, p53 has a higher affinity for its RE when incorporated into nucleosomes compared to an RE on naked DNA, suggesting a novel role for p53 as a pioneer factor. In addition, p53 transiently interacts and alters the structure of nucleosomal DNA, presumably to facilitate access of additional p53 bound chromatin modifiers. Bioinformatic analysis reveal that p53 REs cluster specifically within 2 regions of the nucleosome. Interestingly, p53 interacts with peptides that have strong homology to histones H2A, H2B, and H4 in our next generation proteomic screens. Furthermore based upon crystal structures, these histone peptides are adjacent to the clustered p53 REs, strongly suggesting localization of two physiologically relevant binding platforms for p53 on the nucleosome. Collectively, our studies indicate that p53/histone contacts combined with p53’s interaction with REs may enhance p53 directed transcription by creating a stable multivalent platform for p53’s recruitment to target promoters.
Platform: Force Spectroscopy and Scanning Probe Microscopy 2545-Plat Acoustic Force Spectroscopy: An Instrument to Perform Highly Parallel Single Molecule Measurements Gerrit Sitters1,2, Felix Oswald2,3, Douwe Kamsma1, Jerom Langeveld2, Willem Peutz2, Erwin Peterman1, Gijs Wuite1, Olivier Heyning2. 1 Physics of Life, Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 2 Lumicks, Amsterdam, Netherlands, 3Vrije Universiteit Amsterdam, Amsterdam, Netherlands. Force-spectroscopy has become an indispensable tool to unravel the structural and mechanochemical properties of biomolecules. Acoustic Force Spectroscopy (AFS) is a new acoustic manipulation method, which consists of a resonator integrated into a micro-fabricated fluidic chip. An acoustical pressure gradient is created homogeneously throughout the sample enabling to exert forces on DNA-tethered microspheres. By changing the amplitude of the driving voltage the pressure gradient can be altered, allowing sensitive control of the force applied to the DNA molecules. This approach allows exerting acoustic forces from sub-pN to hundreds of pN applied to thousands of biomolecules in parallel, with sub-millisecond response time and inherent stability. Here we present the next step in validating this new technology, a standalone commercial grade prototype, that makes this method available to the wider scientific community. 2546-Plat A Novel Method for Multiplexed Nanometric Bead Tracking Thomas Brouwer, John van Noort. LION, Leiden University, Leiden, Netherlands. Single-molecule force spectroscopy is a powerful tool to resolve dynamics and interactions between large bio-molecules. Magnetic Tweezers, for example, probe the mechanics of a biopolymer by tethering it to a micrometer sized bead and tracking the bead using video microscopy and image processing. This technique strongly relies on accurate bead tracking in three dimensions
Wednesday, March 2, 2016
models for the process of mechanochemical force generation have been put forward, but there is no consensus on which, if any, of these is correct. All the existing models assume that protein-generated forces drive the DNA forward. The scrunchworm hypothesis proposes that the DNA molecule is the active forcegenerating core of the motor, not simply a substrate on which the motor operates (Harvey (2015) J Struct Biol 189:1-8). The protein components of the motor dehydrate a section of the DNA, converting it from the B form to the A form and shortening it by about 23%. The proteins then rehydrate the DNA, which converts back to the B form. Other regions of the motor grip and release the DNA to capture the shortening-lengthening motions of the B/A/B cycle (‘‘scrunching’’), so that DNA is pulled into the motor and pushed forward into the capsid. This DNAcentric mechanism provides a quantitative physical explanation for the magnitude of the forces generated by viral packaging motors. It also provides a simple explanation for the fact that each of the steps in the burst cycle advances the DNA by 2.5 base pairs. The scrunchworm hypothesis is consistent with a large body of published data, and it makes four experimentally testable predictions. Here I discuss progress in experimental and modeling tests of the scrunchworm hypothesis. 2542-Plat TFAM Regulates Mitochondrial Transcription through Sequence-Specific DNA Looping Divakaran Murugesapillai1, Maria F. Lodeiro2, Louis J. Maher III,3, Craig E. Cameron2, Mark C. Williams1. 1 Department of Physics, Northeastern University, Boston, MA, USA, 2 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA, 3Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA. Mitochondrial transcription factor A (TFAM) is an abundant human mitochondrial High Mobility Group Box (HMGB) protein. TFAM is an architectural protein that shapes mitochondrial DNA (mtDNA) and regulates mitochondrial transcription. Sequence-specific binding of TFAM to DNA upstream of the light-strand promoter (LSP) leads to bending that correlates with transactivation of this promoter. Here we use a dual promoter construct containing both LSP, HSP1 and the natural inter-promoter region (IPR) to understand how TFAM binding governs transcription activation. We show that the IPR contributes to TFAM transactivation of HSP1, while removal of the carboxyl-terminal tail of TFAM (TFAM-DCT26) leads to a complete loss of transactivation of HSP1, with only minimal effects on LSP, suggesting a different biophysical mechanism for TFAM-mediated transcription activation from HSP1. To understand the mechanism by which TFAM activates HSP1 transcription, we used atomic force microscopy (AFM) in liquid to probe the effect of TFAM on the IPR construct. We observe that at the low concentrations of TFAM where transcription activation is high, TFAM induces the formation of DNA loops in a region defined by the IPR, with TFAM present at the strand crossing point. Under the same conditions, TFAM-DCT26 fails to induce DNA looping. While random DNA also forms loops in the presence of TFAM, TFAM is twice as likely to mediate the loops when the IPR sequence is present. Optical tweezers experiments also demonstrate that TFAM stabilizes DNA loops, which require significant force to break. Taken together, our results are consistent with sequence-specific DNA looping contributing to TFAM transactivation of HSP1, suggesting that unique mechanisms are employed for TFAM-dependent transcription at LSP and HSP1. 2543-Plat Visualizing the Assembly of DNA Condensation Clusters by SMC using Single-Molecule Microscopy Hyeongjun Kim, Joseph J. Loparo. BCMP, Harvard Medical School, Boston, MA, USA. SMC (structural maintenance of chromosomes) family members play essential roles in chromosome condensation, sister chromatid cohesion, and DNA repair. Despite intensive efforts, it is still unclear how SMCs are loaded onto DNA and structure chromosomes. We employed single-molecule fluorescence microscopy to visualize how individual Bacillus subtilis SMC (BsSMC) dimers interact with flow-stretched DNAs. We find that BsSMC can vary its initial interaction with DNA, switching between static binding and sliding. Its diffusive properties are insensitive to the presence of ATP, suggesting that the initial loading of BsSMC onto DNA is ATP-independent. At higher concentrations, BsSMCs form distinct clusters that condense DNA by both wrapping and bridging distal DNA segments. SMC-mediated DNA compaction occurs in a weakly ATP-dependent manner. However, ATP increases the apparent cooperativity of DNA condensation, demonstrating that multiple BsSMCs can interact cooperatively through their ATPase head domains. Consistent with these results, BsSMC mutants that alter the ATPase cycle compact DNA more slowly than wild-type BsSMC in the presence of ATP, and an ATPase headless
construct compacts DNA non-cooperatively with substantially lower compaction rate. Our results suggest that transiently static BsSMC can nucleate the formation of functionally active clusters that locally condense the chromosome while forming long-range DNA bridges. 2544-Plat Single Molecule Imaging of p53’s Dynamic Interaction with Chromatin Vincent Wong1, Zhe Liu2, Sam Peng1, Charles Kenworthy1, Wei-Li Liu1, Robert A. Coleman1. 1 Albert Einstein College of Medicine, Bronx, NY, USA, 2Howard Hughes Medical Institute Janelia Research Center, Ashburn, VA, USA. P53 is a transcriptional activator that binds to its response elements (REs) on target promoters and activates expression of a large number of genes involved in tumor suppression. Previous genome-wide studies showed that p53 binds to its REs in regions densely populated with nucleosomes. However, it is unknown how p53 accesses its target REs in the context of chromatin to regulate transcription. To better decipher the interaction of p53 with chromatin targets, we utilized a combination of in vivo live cell and in vitro single molecule imaging along with bioinformatics and Next Generation Proteomic screens. Our live cell imaging studies indicate a dynamic interaction between p53 and chromatin that varies in distinct sub-nuclear compartments. In vitro, p53 binds a mononucleosome on a native p53 target gene with nearly identical residence times as our live cell measurements. Strikingly, p53 has a higher affinity for its RE when incorporated into nucleosomes compared to an RE on naked DNA, suggesting a novel role for p53 as a pioneer factor. In addition, p53 transiently interacts and alters the structure of nucleosomal DNA, presumably to facilitate access of additional p53 bound chromatin modifiers. Bioinformatic analysis reveal that p53 REs cluster specifically within 2 regions of the nucleosome. Interestingly, p53 interacts with peptides that have strong homology to histones H2A, H2B, and H4 in our next generation proteomic screens. Furthermore based upon crystal structures, these histone peptides are adjacent to the clustered p53 REs, strongly suggesting localization of two physiologically relevant binding platforms for p53 on the nucleosome. Collectively, our studies indicate that p53/histone contacts combined with p53’s interaction with REs may enhance p53 directed transcription by creating a stable multivalent platform for p53’s recruitment to target promoters.
Platform: Force Spectroscopy and Scanning Probe Microscopy 2545-Plat Acoustic Force Spectroscopy: An Instrument to Perform Highly Parallel Single Molecule Measurements Gerrit Sitters1,2, Felix Oswald2,3, Douwe Kamsma1, Jerom Langeveld2, Willem Peutz2, Erwin Peterman1, Gijs Wuite1, Olivier Heyning2. 1 Physics of Life, Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 2 Lumicks, Amsterdam, Netherlands, 3Vrije Universiteit Amsterdam, Amsterdam, Netherlands. Force-spectroscopy has become an indispensable tool to unravel the structural and mechanochemical properties of biomolecules. Acoustic Force Spectroscopy (AFS) is a new acoustic manipulation method, which consists of a resonator integrated into a micro-fabricated fluidic chip. An acoustical pressure gradient is created homogeneously throughout the sample enabling to exert forces on DNA-tethered microspheres. By changing the amplitude of the driving voltage the pressure gradient can be altered, allowing sensitive control of the force applied to the DNA molecules. This approach allows exerting acoustic forces from sub-pN to hundreds of pN applied to thousands of biomolecules in parallel, with sub-millisecond response time and inherent stability. Here we present the next step in validating this new technology, a standalone commercial grade prototype, that makes this method available to the wider scientific community. 2546-Plat A Novel Method for Multiplexed Nanometric Bead Tracking Thomas Brouwer, John van Noort. LION, Leiden University, Leiden, Netherlands. Single-molecule force spectroscopy is a powerful tool to resolve dynamics and interactions between large bio-molecules. Magnetic Tweezers, for example, probe the mechanics of a biopolymer by tethering it to a micrometer sized bead and tracking the bead using video microscopy and image processing. This technique strongly relies on accurate bead tracking in three dimensions