- Email: [email protected]
Ensemble and Single Molecule Biophysical Studies of TDP43
Monday, February 13, 2017 1031-Pos Board B99 Intrinsically Disordered Protein Dynamics Uncovered through Dynamic Flexibility Index (DFI) Analysis ul H. Zerze2, Jeetain Mittal2, Sara M. Vaiana1, Tushar Modi1, G€ S. Banu Ozkan1. 1 Department of Physics, Arizona State University, Tempe, AZ, USA, 2 Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA. Calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP) are two intrinsically disordered proteins (IDP) of the calcitonin peptide family. CGRP is an important new target for antimigraine drugs, it is involved in vasodilation and transmission of pain signals. IAPP regulates blood glucose levels and forms amyloid fibers in type II diabetes. They share up to 40% sequence similarity and bind to very similar receptors, but are found in different types of tissue and carry out different functions. Recent experimental studies measuring end-to-end contact formation by tryptophan triplet quenching, show a significant difference in the conformational dynamics of CGRP and IAPP. We investigate this through Dynamic Flexibility Index (DFI). DFI quantifies the resilience of a given position to the perturbations that occur at different parts of the chain, using linear response theory. This captures multidimensional effects when the chain is displaced out of equilibrium. Thus, it identifies flexible and rigid positions that govern the function. Our DFI profiles obtained from equilibrated atomistic MD simulations reveal distinct patterns for CGRP and IAPP. We investigate the effect of amino acid substitutions on the conformational dynamics of the unbound disordered IDP, and pinpoint the positions that give a significant difference in dynamics. 1032-Pos Board B100 A Study of Disorder-to-Order Transition by Characterizing the Binding Partners using a Statistical Potential Iqbal Sumaiya, Hoque Md Tamjidul. Computer Science, University of New Orleans, New Orleans, LA, USA. It is crucial to understand the biophysical properties of intrinsically disordered proteins (IDPs) and protein regions (IDRs) as they participate in numerous biological functions by exhibiting a multitude of structural conformations and dynamics. While the molecular recognition functions of IDPs/IDRs include pathways to carry out signaling and regulation, the structural heterogeneity of IDPs are highly linked to the amyloid aggregation that is involved in critical human diseases such as Parkinson’s disease, Alzheimer’s disease, type II diabetes and others. The functional advantages of IDPs/IDRs are primarily facilitated by their disorder-to-order transitions, specifically in short regions, through low affinity binding with a variety of partners. We found that about 21% of the proteins in DisProt have structural models in PDB, either in full or in part. We developed and deployed a novel statistical potential, termed Position Specific Estimated Energy (PSEE), to study the mechanism of free energy change required for disorder-to-order transition, mediated by negative enthalpy change of the system and increased entropy of the surrounding water. PSEE computes the potential of each residue from protein sequence alone using the pairwise thermodynamic interactions and the solvent accessibility of the residues in local neighborhood. In this study of characterizing the partnerbinding sites within IDPs/IDRs and their transitions from disorder-to-order, we emphasize on determining the level of energy of the partners that induce the respective binding and, PSEE was effective in segregating the disordered and ordered residues by computing the energy gap in between. Further, we identified the binding region’s likelihood of being disordered in its unbound state using a Disorder Predictor (DisPredict) developed by us. DisPredict is a SVM model with optimized radial basis function kernel and optimized threshold that employs a set of distinguishing attributes for disorder and order characterization including the novel PSEE. DisPredict, PSEE and our novel mechanism of partner-based identification of binding regions together, can assist in the experiments for drug discovery exploiting binding sites within IDPs/IDRs. 1033-Pos Board B101 Single-Molecule FRET Probes a Transient Complex that Facilitates Binding of an Intrinsically-Disordered Protein Jae-Yeol Kim, Fanjie Meng, Hoi Sung Chung. NIDDK/NIH, Bethesda, MD, USA. Intrinsically-disordered proteins (IDPs) are unfolded at the native condition and fold only when attaching to their binding partners. Even though IDPs often undergo large conformational changes, which may slow the binding process, the binding kinetics are sometimes surprisingly fast, close to those of diffusioncontrolled reactions. IDPs usually contain a large number of charged residues and theory predicts that the association can be facilitated by the formation of a transient complex (TC) stabilized by the electrostatic attraction between an IDP and its binding target. Although the modulation of the association rate by ionic
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strength of the solution has been observed for a number of IDPs, TC has not been directly probed during the course of binding. To experimentally investigate the role of TC in fast binding, we employed single-molecule Fo¨rster resonance energy transfer (FRET) spectroscopy that monitors the conformational evolution of an IDP from the unbound state to the bound complex. We studied binding of the transactivation domain (TAD) of the tumor suppressor protein p53 (negatively charged, 10) and the nuclear co-activator binding domain (NCBD) of CBP (positively charged, þ6). The N- and C-termini of TAD were labeled with Alexa 647 and Alexa 488 site-specifically and molecules were immobilized on a glass surface using a biotin-neutravidin linkage to monitor binding to unlabeled NCBD in solution. Because of the fast binding kinetics, the bound state, TC, and unbound state could not be clearly resolved in binned trajectories. Instead of binning, we used the maximum likelihood method (Gopich and Szabo, JPCB(2009)) to analyze photon trajectories collected at high illumination intensity and to measure the binding kinetics (photon count rate of 50 - 100 ms1) and the lifetime of TC (photon count rate of 500 - 1000 ms1). We found that the association rate reaches ~ 1 x 109 M1s1, which is close to the diffusion-limited rate, and the lifetime of TC is relatively long (~100 microsecond) compared to the kinetics at low ionic strength (~ 0.04 M). More importantly, both quantities decrease significantly with the increasing ionic strength, indicating that the destabilization of TC results in the slow association. This result demonstrates that the fast association of an IDP can be achieved by stabilizing TC through native/non-native electrostatic interactions, which would allow longer time for an IDP to interact with a target protein to fold and bind. 1034-Pos Board B102 Therapeutic Potential of Peptides Derived from Intrinsically Disordered Protein Regions Stephen Beesley1, Efrosini Artikis2, Ewa Bienkiewicz1. 1 Florida State University, Tallahassee, FL, USA, 2Biophysics Program, University of Michigan, Ann Arbor, FL, USA. The conformational duality of the prion protein translates into strikingly distinct fates this protein can impart on the living systems. In one case, the outcome is the irrevocable neurodegeneration and fatal disease. In stark contrast, the cellular form of the prion protein (PrPc) partakes in lifesustaining cellular functions that include neuroprotection, signal transduction, and angiogenesis. Importantly, PrPc has been shown to be involved in a natural response to vascular injury, with the cell damage and death due to stroke being significantly increased in its absence. In vascular injury, including stroke, one of the key cell death-triggering events is the release of toxic levels of free hemin. A potential solution to this damage is a peptide therapeutic agent that would neutralize hemin, thus reducing deleterious effects of bleeding. Both the hemorrhagic and ischemic stroke (with ‘‘microbleeds’’ inflicting secondary damage) would benefit from this approach. Using biophysical methods, we have identified a peptide derived from the intrinsically disordered PrPc N-terminus and tested a hypothesis that this hemin-sequestering fragment could bind and neutralize hemin leading to reduction in brain damage due to bleeding. We employed fluorescence, surface plasmon resonance, and a well-established mouse model of intracerebral hemorrhagic stroke (ICH) to carry out these studies. Neurological/behavioral deficiencies and brain tissue damage caused by stroke were tested in the presence and absence of the peptide treatment. Our results indicate that the peptide derived from the intrinsically disordered prion domain binds hemin and reduces deleterious effects of stroke in-vivo, making it a strong candidate for further development as a novel therapeutic intervention in vascular injury events. 1035-Pos Board B103 Ensemble and Single Molecule Biophysical Studies of TDP43 Phoebe S. Tsoi, Kyoungjae Choi, Josephine C. Ferreon, Allan Chris M. Ferreon. Pharmacology, Baylor College of Medicine, Houston, TX, USA. Life expectancy has been significantly extended through innovation in modern medicine. Ironically, with longevity comes the greater threat of neurodegenerative diseases: chronic, debilitating illnesses with no cures and limited treatments. The World Health Organization has projected neurodegenerative diseases to overtake cancer in the number of annual disease-related deaths by 2040, second only to cardiovascular disease. TDP43 is a 43 kDa RNA splicing regulator, as well as one of the best biomarkers for several neurodegenerative diseases including Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). TDP43 aggregates have been found in 97% of ALS patients and up to 50% of AD patients. We have studied TDP43 using single molecule Fo¨rster resonance energy transfer (smFRET) combined with ensemble optical (UV Vis and far UV CD) and NMR spectroscopy to determine the structure of TDP43 domains and extrapolate the correlation between structure and pathology. The extent of order or disorder of the N terminal domain has been
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Monday, February 13, 2017
controversial, but we have conclusive data that this domain is fully folded and plays a critical role in TDP43 oligomerization. The C terminal domain is an intrinsically disordered region that aggregates readily. Our studies show that this domain is fully unstructured, and we have been able to induce folding using the osmolyte/chemical chaperone trimethylamine N oxide (TMAO). 1036-Pos Board B104 GRP78 is a Major Ribosylated Protein in CHO Cells Rong_Qiao He1, Yang Lu2, Bei Bei Wu1, Le Xiang YU1, Chan Shuai Han1, Yan Wei1. 1 State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Science, Beijing, China, 2School of Life Science, University of Science and Technology of China, Hefei, Anhui, China. Over 200 year ago, Chevreul (1786-1889) discovered that the sweetness in the urine of diabetics comes from grape sugar or D-glucose. Glycation with D-glucose leads to the formation of advanced glycation end products (AGEs), which is an important biochemical abnormality that accompanies diabetes mellitus. As one of the most reactive glycation reagents, D-ribose, which can yield large amounts of AGEs in vivo and in vitro, has long been ignored in Diabetes studies. In this laboratory, we have observed abnormally high levels of D-ribose in the urine of diabetic patients, compared with those of agematched normal control. Our results suggested that diabetes mellitus is not only suffered from dysmetabolism of D-glucose, but also dysmetabolism of D-ribose. Here, we show that treatment with D-ribose in Chinese hamster ovary cells increased expression of cellular AGEs along with the increase of D-ribose concentration and administration time. Mass spectrum analysis of isolated AGE-modified proteins from cell lysates showed glucose-regulated protein 78 kD (GRP78) as a ribosylated protein. Co-immunoprecipitation assay further confirmed interaction between AGEs and GRP78. Immunofluorescence colocalization analysis manifested partial superimposition of AGEs and GRP78. As showed in Western blotting results, changes in GRP78 protein levels in cells exhibited in a time dependent manner in the D-ribose treatment. Inhibition of GRP78 expression with RNAi or function in D-ribose-treated cells both dramatically increased the level of AGEs. The findings from Chinese hamster ovary cells indicated that GRP78 is a major component response to ribosylation playing in protection role for cells in the presence of D-ribose. 1037-Pos Board B105 The Impact of IDPs on Circadian Rhythms Jennifer M. Hurley. Department of Biological Sciences, Rensselaer Polytechnical Institute, Troy, NY, USA. Through the ideal programming of behavior, it is believed that our circadian clock, our internal molecular time keeper, enhances fitness by ensuring that many organismal functions are optimally timed with the phase of the circadian day. As clock regulation is extensive, controlling everything from blood pressure levels to sleep, disruptions of the clock can be detrimental; chronic disturbance of the clock leads to an increased risk for heart attack, cancer, diabetes, and mental health disorders among a host of other maladies. My work has shown that the highly conserved mechanism that regulates circadian timing, a molecular transcription-translation negative feedback loop, is profoundly affected by IDPs. The core clock complex in the clock model organism Neurospora crassa consists of two sets of protein pairs, the positive arm (comprised of the White Collar 1/ White Collar 2 Complex or WCC) and the negative arm (comprised of the Frequency (FRQ)/Frequency-Interacting RNA Helicase (FRH) complex or FFC). The WCC drives the expression of the FFC, which then regulates its own transcription by inhibiting the activity of the WCC. The FFC is then targeted for degradation, allowing for the reactivation of the WCC, restarting the cycle. I have demonstrated that FRQ is an Intrinsically Disordered Protein (IDP); currently, the reason for the IDP nature of FRQ is unknown. In addition, FRH stabilizes FRQ and mediates the interactions between the core clock proteins using its own disordered N-terminus. This intrinsic disorder in core clock genes is conserved from Neurospora to higher eukaryotes and my labs seeks to identify the reason for the conservation of a high level of protein disorder in the circadian clock.
Transcription 1038-Pos Board B106 Single-Cell, Single-mRNA Analysis of Ccnb1 Promoter Regulation Nidhi Vishnoi, Jie Yao. Cell Biology, Yale University School of Medicine, New Haven, CT, USA. Promoter activation controls gene transcriptional output. Although single cell imaging has enabled analyzing transcription kinetics at native genes, studying
promoter regulation in vivo requires a new approach to measure mRNA biogenesis as a consequence of promoter activities. In this study, we generated single-copy promoter transgenes integrated at an identified locus in cultured mouse C2C12 myoblast cells and performed a thorough single-cell analysis on Ccnb1 promoter regulation. Single molecule RNA FISH revealed that Ccnb1 promoter transgene expression was over one order of magnitude higher than that of the promoterless transgene integrated at the same locus. mRNA counts, RNA polymerase II (Pol II) density and Pol II firing rates of the Ccnb1 promoter transgene recapitulated those of the native Ccnb1 gene both among asynchronous cells and during the cell cycle, confirming that Ccnb1 promoter transgene expression predominantly reflects native Ccnb1 promoter activities. Interestingly, we observed distinct activation states of the Ccnb1 promoter among cells synchronized at G1 or G2/M, indicating that cell-to-cell variations in Ccnb1 promoter activities were not simply due to cell cycle stages. Expressing a dominant negative mutant of NF-YA, a key transcription activator of the Ccnb1 promoter, reduced its transcriptional output by increasing its ‘‘OFF’’/‘‘ON’’ time ratios of both the native Ccnb1 gene and the Ccnb1 promoter transgene and also by modestly reducing Pol II firing rates of the native Ccnb1 gene. Intriguingly, comparing histone modification levels at the Ccnb1 promoter transgenes and at the native Ccnb1 gene revealed that higher levels of active histone marks did not predispose higher transcriptional activities. In summary, we have developed a new method that enables studying the functions of transcription factors and cellular pathways in promoter regulation and bridges transcription imaging with molecular analysis. 1039-Pos Board B107 Visualization of Active Transcription Sites in Human Cardiomyocytes Supports the Concept of Burst-Like Transcription of MYH7 Kathrin Kowalski1, Ante Radocaj1, Cristobal G. dos Remedios2, Antonio Francino3, Francisco Navarro-Lo´pez4, Theresia Kraft1, Bernhard Brenner1. 1 Molecular- and Cell Physiology, Medizinische Hochschule Hannover, Hannover, Germany, 2Anatomy, Bosch Institute, Sydney, Australia, 3 Molecular- and Cell Physiology, Hospital Clı´nic (IDIBAPS), Barcelona, Spain, 4Hospital Clı´nic (IDIBAPS), Barcelona, Spain. Hypertrophic Cardiomyopathy (HCM) has a prevalence of 1 in 500 individuals. More than 1000 mutations in >20 different sarcomeric and some nonsarcomeric proteins have been identified in HCM-patients. In 30-40% the HCM-mutation is found in the b-myosin heavy chain (b-MyHC) gene (MYH7). Until now, a common mechanism triggering HCM is unknown. From previous work of our group on MYH7-mutations we hypothesized that the characteristic HCM-phenotype with hypertrophy, myocyte disarray, and interstitial fibrosis could be a consequence of cell-to-cell functional imbalance due to cell-to-cell variation in the expression of mutated vs. wildtype b-MyHC. Such cell-to-cell variability could result from stochastic, independent burstlike transcription of mutant and wildtype MYH7-alleles. To investigate whether the MYH7-alleles are indeed transcribed in a discontinuous, burst-like manner, we visualized active transcription sites of MYH7 in cardiomyocytes of a patient with b-MyHC-mutation R723G and of a nontransplanted donor heart using fluorescence in situ hybridization. To visualize pre-mRNA in active transcription sites we used two probes sets, each containing 48 single-labeled 20-bases-long oligonucleotides to probe intronic and exonic sequences of MYH7-pre-mRNA, while cytoplasmic MYH7-mRNA was labelled by the exonic probe set. Both probe sets were hybridized to their target sequences in 16 mm thick sections of cardiac samples. Active transcription sites were identified as bright spots inside the nuclei of cardiomyocytes showing co-localization of both probe sets. 26% of the nuclei had no active transcription sites while cytoplasmic MYH7-mRNA was revealed by the exonic probes. Absence of active transcription sites is inconsistent with continuous transcription of the two MYH7-alleles but expected for random burst-like transcription. Model calculations showed that independent, stochastic, burst-like transcription of mutant and wildtype MYH7-alleles can account for the experimentally observed cell-to-cell variability in mutant MYH7-mRNA and functional imbalances. 1040-Pos Board B108 Single Molecule Analysis of Transcription in Live Cells Reveals the Gene Regulatory Function of MYC In Vivo Simona Patange1,2, Michelle Girvan1, David Levens2, Daniel R. Larson2. 1 University of Maryland, College Park, MD, USA, 2National Institutes of Health, Bethesda, MD, USA. We seek to determine how the MYC oncogene modulates gene transcription kinetics with single-molecule imaging techniques.
209a
strength of the solution has been observed for a number of IDPs, TC has not been directly probed during the course of binding. To experimentally investigate the role of TC in fast binding, we employed single-molecule Fo¨rster resonance energy transfer (FRET) spectroscopy that monitors the conformational evolution of an IDP from the unbound state to the bound complex. We studied binding of the transactivation domain (TAD) of the tumor suppressor protein p53 (negatively charged, 10) and the nuclear co-activator binding domain (NCBD) of CBP (positively charged, þ6). The N- and C-termini of TAD were labeled with Alexa 647 and Alexa 488 site-specifically and molecules were immobilized on a glass surface using a biotin-neutravidin linkage to monitor binding to unlabeled NCBD in solution. Because of the fast binding kinetics, the bound state, TC, and unbound state could not be clearly resolved in binned trajectories. Instead of binning, we used the maximum likelihood method (Gopich and Szabo, JPCB(2009)) to analyze photon trajectories collected at high illumination intensity and to measure the binding kinetics (photon count rate of 50 - 100 ms1) and the lifetime of TC (photon count rate of 500 - 1000 ms1). We found that the association rate reaches ~ 1 x 109 M1s1, which is close to the diffusion-limited rate, and the lifetime of TC is relatively long (~100 microsecond) compared to the kinetics at low ionic strength (~ 0.04 M). More importantly, both quantities decrease significantly with the increasing ionic strength, indicating that the destabilization of TC results in the slow association. This result demonstrates that the fast association of an IDP can be achieved by stabilizing TC through native/non-native electrostatic interactions, which would allow longer time for an IDP to interact with a target protein to fold and bind. 1034-Pos Board B102 Therapeutic Potential of Peptides Derived from Intrinsically Disordered Protein Regions Stephen Beesley1, Efrosini Artikis2, Ewa Bienkiewicz1. 1 Florida State University, Tallahassee, FL, USA, 2Biophysics Program, University of Michigan, Ann Arbor, FL, USA. The conformational duality of the prion protein translates into strikingly distinct fates this protein can impart on the living systems. In one case, the outcome is the irrevocable neurodegeneration and fatal disease. In stark contrast, the cellular form of the prion protein (PrPc) partakes in lifesustaining cellular functions that include neuroprotection, signal transduction, and angiogenesis. Importantly, PrPc has been shown to be involved in a natural response to vascular injury, with the cell damage and death due to stroke being significantly increased in its absence. In vascular injury, including stroke, one of the key cell death-triggering events is the release of toxic levels of free hemin. A potential solution to this damage is a peptide therapeutic agent that would neutralize hemin, thus reducing deleterious effects of bleeding. Both the hemorrhagic and ischemic stroke (with ‘‘microbleeds’’ inflicting secondary damage) would benefit from this approach. Using biophysical methods, we have identified a peptide derived from the intrinsically disordered PrPc N-terminus and tested a hypothesis that this hemin-sequestering fragment could bind and neutralize hemin leading to reduction in brain damage due to bleeding. We employed fluorescence, surface plasmon resonance, and a well-established mouse model of intracerebral hemorrhagic stroke (ICH) to carry out these studies. Neurological/behavioral deficiencies and brain tissue damage caused by stroke were tested in the presence and absence of the peptide treatment. Our results indicate that the peptide derived from the intrinsically disordered prion domain binds hemin and reduces deleterious effects of stroke in-vivo, making it a strong candidate for further development as a novel therapeutic intervention in vascular injury events. 1035-Pos Board B103 Ensemble and Single Molecule Biophysical Studies of TDP43 Phoebe S. Tsoi, Kyoungjae Choi, Josephine C. Ferreon, Allan Chris M. Ferreon. Pharmacology, Baylor College of Medicine, Houston, TX, USA. Life expectancy has been significantly extended through innovation in modern medicine. Ironically, with longevity comes the greater threat of neurodegenerative diseases: chronic, debilitating illnesses with no cures and limited treatments. The World Health Organization has projected neurodegenerative diseases to overtake cancer in the number of annual disease-related deaths by 2040, second only to cardiovascular disease. TDP43 is a 43 kDa RNA splicing regulator, as well as one of the best biomarkers for several neurodegenerative diseases including Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). TDP43 aggregates have been found in 97% of ALS patients and up to 50% of AD patients. We have studied TDP43 using single molecule Fo¨rster resonance energy transfer (smFRET) combined with ensemble optical (UV Vis and far UV CD) and NMR spectroscopy to determine the structure of TDP43 domains and extrapolate the correlation between structure and pathology. The extent of order or disorder of the N terminal domain has been
210a
Monday, February 13, 2017
controversial, but we have conclusive data that this domain is fully folded and plays a critical role in TDP43 oligomerization. The C terminal domain is an intrinsically disordered region that aggregates readily. Our studies show that this domain is fully unstructured, and we have been able to induce folding using the osmolyte/chemical chaperone trimethylamine N oxide (TMAO). 1036-Pos Board B104 GRP78 is a Major Ribosylated Protein in CHO Cells Rong_Qiao He1, Yang Lu2, Bei Bei Wu1, Le Xiang YU1, Chan Shuai Han1, Yan Wei1. 1 State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Science, Beijing, China, 2School of Life Science, University of Science and Technology of China, Hefei, Anhui, China. Over 200 year ago, Chevreul (1786-1889) discovered that the sweetness in the urine of diabetics comes from grape sugar or D-glucose. Glycation with D-glucose leads to the formation of advanced glycation end products (AGEs), which is an important biochemical abnormality that accompanies diabetes mellitus. As one of the most reactive glycation reagents, D-ribose, which can yield large amounts of AGEs in vivo and in vitro, has long been ignored in Diabetes studies. In this laboratory, we have observed abnormally high levels of D-ribose in the urine of diabetic patients, compared with those of agematched normal control. Our results suggested that diabetes mellitus is not only suffered from dysmetabolism of D-glucose, but also dysmetabolism of D-ribose. Here, we show that treatment with D-ribose in Chinese hamster ovary cells increased expression of cellular AGEs along with the increase of D-ribose concentration and administration time. Mass spectrum analysis of isolated AGE-modified proteins from cell lysates showed glucose-regulated protein 78 kD (GRP78) as a ribosylated protein. Co-immunoprecipitation assay further confirmed interaction between AGEs and GRP78. Immunofluorescence colocalization analysis manifested partial superimposition of AGEs and GRP78. As showed in Western blotting results, changes in GRP78 protein levels in cells exhibited in a time dependent manner in the D-ribose treatment. Inhibition of GRP78 expression with RNAi or function in D-ribose-treated cells both dramatically increased the level of AGEs. The findings from Chinese hamster ovary cells indicated that GRP78 is a major component response to ribosylation playing in protection role for cells in the presence of D-ribose. 1037-Pos Board B105 The Impact of IDPs on Circadian Rhythms Jennifer M. Hurley. Department of Biological Sciences, Rensselaer Polytechnical Institute, Troy, NY, USA. Through the ideal programming of behavior, it is believed that our circadian clock, our internal molecular time keeper, enhances fitness by ensuring that many organismal functions are optimally timed with the phase of the circadian day. As clock regulation is extensive, controlling everything from blood pressure levels to sleep, disruptions of the clock can be detrimental; chronic disturbance of the clock leads to an increased risk for heart attack, cancer, diabetes, and mental health disorders among a host of other maladies. My work has shown that the highly conserved mechanism that regulates circadian timing, a molecular transcription-translation negative feedback loop, is profoundly affected by IDPs. The core clock complex in the clock model organism Neurospora crassa consists of two sets of protein pairs, the positive arm (comprised of the White Collar 1/ White Collar 2 Complex or WCC) and the negative arm (comprised of the Frequency (FRQ)/Frequency-Interacting RNA Helicase (FRH) complex or FFC). The WCC drives the expression of the FFC, which then regulates its own transcription by inhibiting the activity of the WCC. The FFC is then targeted for degradation, allowing for the reactivation of the WCC, restarting the cycle. I have demonstrated that FRQ is an Intrinsically Disordered Protein (IDP); currently, the reason for the IDP nature of FRQ is unknown. In addition, FRH stabilizes FRQ and mediates the interactions between the core clock proteins using its own disordered N-terminus. This intrinsic disorder in core clock genes is conserved from Neurospora to higher eukaryotes and my labs seeks to identify the reason for the conservation of a high level of protein disorder in the circadian clock.
Transcription 1038-Pos Board B106 Single-Cell, Single-mRNA Analysis of Ccnb1 Promoter Regulation Nidhi Vishnoi, Jie Yao. Cell Biology, Yale University School of Medicine, New Haven, CT, USA. Promoter activation controls gene transcriptional output. Although single cell imaging has enabled analyzing transcription kinetics at native genes, studying
promoter regulation in vivo requires a new approach to measure mRNA biogenesis as a consequence of promoter activities. In this study, we generated single-copy promoter transgenes integrated at an identified locus in cultured mouse C2C12 myoblast cells and performed a thorough single-cell analysis on Ccnb1 promoter regulation. Single molecule RNA FISH revealed that Ccnb1 promoter transgene expression was over one order of magnitude higher than that of the promoterless transgene integrated at the same locus. mRNA counts, RNA polymerase II (Pol II) density and Pol II firing rates of the Ccnb1 promoter transgene recapitulated those of the native Ccnb1 gene both among asynchronous cells and during the cell cycle, confirming that Ccnb1 promoter transgene expression predominantly reflects native Ccnb1 promoter activities. Interestingly, we observed distinct activation states of the Ccnb1 promoter among cells synchronized at G1 or G2/M, indicating that cell-to-cell variations in Ccnb1 promoter activities were not simply due to cell cycle stages. Expressing a dominant negative mutant of NF-YA, a key transcription activator of the Ccnb1 promoter, reduced its transcriptional output by increasing its ‘‘OFF’’/‘‘ON’’ time ratios of both the native Ccnb1 gene and the Ccnb1 promoter transgene and also by modestly reducing Pol II firing rates of the native Ccnb1 gene. Intriguingly, comparing histone modification levels at the Ccnb1 promoter transgenes and at the native Ccnb1 gene revealed that higher levels of active histone marks did not predispose higher transcriptional activities. In summary, we have developed a new method that enables studying the functions of transcription factors and cellular pathways in promoter regulation and bridges transcription imaging with molecular analysis. 1039-Pos Board B107 Visualization of Active Transcription Sites in Human Cardiomyocytes Supports the Concept of Burst-Like Transcription of MYH7 Kathrin Kowalski1, Ante Radocaj1, Cristobal G. dos Remedios2, Antonio Francino3, Francisco Navarro-Lo´pez4, Theresia Kraft1, Bernhard Brenner1. 1 Molecular- and Cell Physiology, Medizinische Hochschule Hannover, Hannover, Germany, 2Anatomy, Bosch Institute, Sydney, Australia, 3 Molecular- and Cell Physiology, Hospital Clı´nic (IDIBAPS), Barcelona, Spain, 4Hospital Clı´nic (IDIBAPS), Barcelona, Spain. Hypertrophic Cardiomyopathy (HCM) has a prevalence of 1 in 500 individuals. More than 1000 mutations in >20 different sarcomeric and some nonsarcomeric proteins have been identified in HCM-patients. In 30-40% the HCM-mutation is found in the b-myosin heavy chain (b-MyHC) gene (MYH7). Until now, a common mechanism triggering HCM is unknown. From previous work of our group on MYH7-mutations we hypothesized that the characteristic HCM-phenotype with hypertrophy, myocyte disarray, and interstitial fibrosis could be a consequence of cell-to-cell functional imbalance due to cell-to-cell variation in the expression of mutated vs. wildtype b-MyHC. Such cell-to-cell variability could result from stochastic, independent burstlike transcription of mutant and wildtype MYH7-alleles. To investigate whether the MYH7-alleles are indeed transcribed in a discontinuous, burst-like manner, we visualized active transcription sites of MYH7 in cardiomyocytes of a patient with b-MyHC-mutation R723G and of a nontransplanted donor heart using fluorescence in situ hybridization. To visualize pre-mRNA in active transcription sites we used two probes sets, each containing 48 single-labeled 20-bases-long oligonucleotides to probe intronic and exonic sequences of MYH7-pre-mRNA, while cytoplasmic MYH7-mRNA was labelled by the exonic probe set. Both probe sets were hybridized to their target sequences in 16 mm thick sections of cardiac samples. Active transcription sites were identified as bright spots inside the nuclei of cardiomyocytes showing co-localization of both probe sets. 26% of the nuclei had no active transcription sites while cytoplasmic MYH7-mRNA was revealed by the exonic probes. Absence of active transcription sites is inconsistent with continuous transcription of the two MYH7-alleles but expected for random burst-like transcription. Model calculations showed that independent, stochastic, burst-like transcription of mutant and wildtype MYH7-alleles can account for the experimentally observed cell-to-cell variability in mutant MYH7-mRNA and functional imbalances. 1040-Pos Board B108 Single Molecule Analysis of Transcription in Live Cells Reveals the Gene Regulatory Function of MYC In Vivo Simona Patange1,2, Michelle Girvan1, David Levens2, Daniel R. Larson2. 1 University of Maryland, College Park, MD, USA, 2National Institutes of Health, Bethesda, MD, USA. We seek to determine how the MYC oncogene modulates gene transcription kinetics with single-molecule imaging techniques.