Publications

All Publications


  • Mapping Transcriptome-Wide and Genome-Wide RNA-DNA Contacts with Chromatin-Associated RNA Sequencing (ChAR-seq). Methods in molecular biology (Clifton, N.J.) Limouse, C., Jukam, D., Smith, O. K., Fryer, K. A., Straight, A. F. 2020; 2161: 115–42

    Abstract

    RNAs play key roles in the cell as molecular intermediates for protein synthesis and as regulators of nuclear processes such as splicing, posttranscriptional regulation, or chromatin remodeling. Various classes of non-coding RNAs, including long non-coding RNAs (lncRNAs), can bind chromatin either directly or via interaction with chromatin binding proteins. It has been proposed that lncRNAs regulate cell-state-specific genes by coordinating the locus-dependent activity of chromatin-modifying complexes. Yet, the vast majority of lncRNAs have unknown functions, and we know little about the specific loci they regulate. A key step toward understanding chromatin regulation by RNAs is to map the genomic loci with which every nuclear RNA interacts and, reciprocally, to identify all RNAs that target a given locus. Our ability to generate such data has been limited, until recently, by the lack of methods to probe the genomic localization of more than a few RNAs at a time. Here, we describe a protocol for ChAR-seq, an RNA-DNA proximity ligation method that maps the binding loci for thousands of RNAs at once and without the need for specific RNA or DNA probe sequences. The ChAR-seq approach generates chimeric RNA-DNA molecules in situ and then converts those chimeras to DNA for next-generation sequencing. Using ChAR-seq we detect many types of chromatin-associated RNA, both coding and non-coding. Understanding the RNA-DNA interactome and its changes during differentiation or disease with ChAR-seq will likely provide key insights into chromatin and RNA biology.

    View details for DOI 10.1007/978-1-0716-0680-3_10

    View details for PubMedID 32681510

  • Chromatin-Associated RNA Sequencing (ChAR-seq). Current protocols in molecular biology Jukam, D., Limouse, C., Smith, O. K., Risca, V. I., Bell, J. C., Straight, A. F. 2019: e87

    Abstract

    RNA is a fundamental component of chromatin. Noncoding RNAs (ncRNAs) can associate with chromatin to influence gene expression and chromatin state; many also act at long distances from their transcriptional origin. Yet we know almost nothing about the functions or sites of action for most ncRNAs. Current methods to identify sites of RNA interaction with the genome are limited to the study of a single RNA at a time. Here we describe a protocol for ChAR-seq, a strategy to identify all chromatin-associated RNAs and map their DNA contacts genome-wide. In ChAR-seq, proximity ligation of RNA and DNA to a linker molecule is used to construct a chimeric RNA-DNA molecule that is converted to DNA for sequencing. In a single assay, ChAR-seq can discover de novo chromatin interactions of distinct RNAs, including nascent transcripts, splicing RNAs, and long noncoding RNAs (lncRNAs). Resulting "maps" of genome-bound RNAs should provide new insights into RNA biology. © 2019 by John Wiley & Sons, Inc.

    View details for PubMedID 30786161

  • Measurement of Mesoscale Conformational Dynamics of Freely Diffusing Molecules with Tracking FCS BIOPHYSICAL JOURNAL Limouse, C., Bell, J. C., Fuller, C. J., Straight, A. F., Mabuchi, H. 2018; 114 (7): 1539–50

    Abstract

    Few techniques are suited to probe the structure and dynamics of molecular complexes at the mesoscale level (∼100-1000 nm). We have developed a single-molecule technique that uses tracking fluorescence correlation spectroscopy (tFCS) to probe the conformation and dynamics of mesoscale molecular assemblies. tFCS measures the distance fluctuations between two fluorescently labeled sites within an untethered, freely diffusing biomolecule. To achieve subdiffraction spatial resolution, we developed a feedback scheme that allows us to maintain the molecule at an optimal position within the laser intensity gradient for fluorescence correlation spectroscopy. We characterized tFCS spatial sensitivity by measuring the Brownian end-to-end dynamics of DNA molecules as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted nucleosome arrays and can assay transient protein-mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the applicability of tFCS to a wide variety of biochemical processes involving mesoscale conformational dynamics.

    View details for PubMedID 29642025

    View details for PubMedCentralID PMC5954409

  • Dentate gyrus mossy cells control spontaneous convulsive seizures and spatial memory Science Bui, A., et al 2018: 787–90

    Abstract

    Temporal lobe epilepsy (TLE) is characterized by debilitating, recurring seizures and an increased risk for cognitive deficits. Mossy cells (MCs) are key neurons in the hippocampal excitatory circuit, and the partial loss of MCs is a major hallmark of TLE. We investigated how MCs contribute to spontaneous ictal activity and to spatial contextual memory in a mouse model of TLE with hippocampal sclerosis, using a combination of optogenetic, electrophysiological, and behavioral approaches. In chronically epileptic mice, real-time optogenetic modulation of MCs during spontaneous hippocampal seizures controlled the progression of activity from an electrographic to convulsive seizure. Decreased MC activity is sufficient to impede encoding of spatial context, recapitulating observed cognitive deficits in chronically epileptic mice.

    View details for DOI 10.1126/science.aan4074

  • Dentate gyrus mossy cells control spontaneous convulsive seizures and spatial memory. Science (New York, N.Y.) Bui, A. D., Nguyen, T. M., Limouse, C., Kim, H. K., Szabo, G. G., Felong, S., Maroso, M., Soltesz, I. 2018; 359 (6377): 787–90

    Abstract

    Temporal lobe epilepsy (TLE) is characterized by debilitating, recurring seizures and an increased risk for cognitive deficits. Mossy cells (MCs) are key neurons in the hippocampal excitatory circuit, and the partial loss of MCs is a major hallmark of TLE. We investigated how MCs contribute to spontaneous ictal activity and to spatial contextual memory in a mouse model of TLE with hippocampal sclerosis, using a combination of optogenetic, electrophysiological, and behavioral approaches. In chronically epileptic mice, real-time optogenetic modulation of MCs during spontaneous hippocampal seizures controlled the progression of activity from an electrographic to convulsive seizure. Decreased MC activity is sufficient to impede encoding of spatial context, recapitulating observed cognitive deficits in chronically epileptic mice.

    View details for PubMedID 29449490

  • Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bisaria, N., Greenfeld, M., Limouse, C., Mabuchi, H., Herschlag, D. 2017; 114 (37): E7688–E7696

    Abstract

    Decades of study of the architecture and function of structured RNAs have led to the perspective that RNA tertiary structure is modular, made of locally stable domains that retain their structure across RNAs. We formalize a hypothesis inspired by this modularity-that RNA folding thermodynamics and kinetics can be quantitatively predicted from separable energetic contributions of the individual components of a complex RNA. This reconstitution hypothesis considers RNA tertiary folding in terms of ΔGalign, the probability of aligning tertiary contact partners, and ΔGtert, the favorable energetic contribution from the formation of tertiary contacts in an aligned state. This hypothesis predicts that changes in the alignment of tertiary contacts from different connecting helices and junctions (ΔGHJH) or from changes in the electrostatic environment (ΔG+/-) will not affect the energetic perturbation from a mutation in a tertiary contact (ΔΔGtert). Consistent with these predictions, single-molecule FRET measurements of folding of model RNAs revealed constant ΔΔGtert values for mutations in a tertiary contact embedded in different structural contexts and under different electrostatic conditions. The kinetic effects of these mutations provide further support for modular behavior of RNA elements and suggest that tertiary mutations may be used to identify rate-limiting steps and dissect folding and assembly pathways for complex RNAs. Overall, our model and results are foundational for a predictive understanding of RNA folding that will allow manipulation of RNA folding thermodynamics and kinetics. Conversely, the approaches herein can identify cases where an independent, additive model cannot be applied and so require additional investigation.

    View details for PubMedID 28839094

  • Xenopus laevis M18BP1 Directly Binds Existing CENP-A Nucleosomes to Promote Centromeric Chromatin Assembly. Developmental cell French, B. T., Westhorpe, F. G., Limouse, C., Straight, A. F. 2017; 42 (2): 190–99.e10

    Abstract

    Vertebrate centromeres are epigenetically defined by nucleosomes containing the histone H3 variant, CENP-A. CENP-A nucleosome assembly requires the three-protein Mis18 complex (Mis18α, Mis18β, and M18BP1) that recruits the CENP-A chaperone HJURP to centromeres, but how the Mis18 complex recognizes centromeric chromatin is unknown. Using Xenopus egg extract, we show that direct, cell-cycle-regulated binding of M18BP1 to CENP-A nucleosomes recruits the Mis18 complex to interphase centromeres to promote new CENP-A nucleosome assembly. We demonstrate that Xenopus M18BP1 binds CENP-A nucleosomes using a motif that is widely conserved except in mammals. The M18BP1 motif resembles a CENP-A nucleosome binding motif in CENP-C, and we show that CENP-C competes with M18BP1 for CENP-A nucleosome binding at centromeres. We show that both CENP-C and M18BP1 recruit HJURP to centromeres for new CENP-A assembly. This study defines cellular mechanisms for recruiting CENP-A assembly factors to existing CENP-A nucleosomes for the epigenetic inheritance of centromeres.

    View details for PubMedID 28743005

    View details for PubMedCentralID PMC5544353

  • Single-Molecule Fluorescence Reveals Commonalities and Distinctions among Natural and in Vitro-Selected RNA Tertiary Motifs in a Multistep Folding Pathway. Journal of the American Chemical Society Bonilla, S., Limouse, C., Bisaria, N., Gebala, M., Mabuchi, H., Herschlag, D. 2017; 139 (51): 18576–89

    Abstract

    Decades of study of the RNA folding problem have revealed that diverse and complex structured RNAs are built from a common set of recurring structural motifs, leading to the perspective that a generalizable model of RNA folding may be developed from understanding of the folding properties of individual structural motifs. We used single-molecule fluorescence to dissect the kinetic and thermodynamic properties of a set of variants of a common tertiary structural motif, the tetraloop/tetraloop-receptor (TL/TLR). Our results revealed a multistep TL/TLR folding pathway in which preorganization of the ubiquitous AA-platform submotif precedes the formation of the docking transition state and tertiary A-minor hydrogen bond interactions form after the docking transition state. Differences in ion dependences between TL/TLR variants indicated the occurrence of sequence-dependent conformational rearrangements prior to and after the formation of the docking transition state. Nevertheless, varying the junction connecting the TL/TLR produced a common kinetic and ionic effect for all variants, suggesting that the global conformational search and compaction electrostatics are energetically independent from the formation of the tertiary motif contacts. We also found that in vitro-selected variants, despite their similar stability at high Mg2+ concentrations, are considerably less stable than natural variants under near-physiological ionic conditions, and the occurrence of the TL/TLR sequence variants in Nature correlates with their thermodynamic stability in isolation. Overall, our findings are consistent with modular but complex energetic properties of RNA structural motifs and will aid in the eventual quantitative description of RNA folding from its secondary and tertiary structural elements.

    View details for PubMedID 29185740

  • Single-Molecule Fluorescence Reveals Commonalities and Distinctions among Natural and in Vitro-Selected RNA Tertiary Motifs in a Multistep Folding Pathway Journal of the American Chemical Society Bonilla, S., Limouse, C., Bisaria, N., Gebala, M., Mabuchi, H., Herschlag, D. 2017: 18576-18589

    Abstract

    Decades of study of the RNA folding problem have revealed that diverse and complex structured RNAs are built from a common set of recurring structural motifs, leading to the perspective that a generalizable model of RNA folding may be developed from understanding of the folding properties of individual structural motifs. We used single-molecule fluorescence to dissect the kinetic and thermodynamic properties of a set of variants of a common tertiary structural motif, the tetraloop/tetraloop-receptor (TL/TLR). Our results revealed a multistep TL/TLR folding pathway in which preorganization of the ubiquitous AA-platform submotif precedes the formation of the docking transition state and tertiary A-minor hydrogen bond interactions form after the docking transition state. Differences in ion dependences between TL/TLR variants indicated the occurrence of sequence-dependent conformational rearrangements prior to and after the formation of the docking transition state. Nevertheless, varying the junction connecting the TL/TLR produced a common kinetic and ionic effect for all variants, suggesting that the global conformational search and compaction electrostatics are energetically independent from the formation of the tertiary motif contacts. We also found that in vitro-selected variants, despite their similar stability at high Mg2+ concentrations, are considerably less stable than natural variants under near-physiological ionic conditions, and the occurrence of the TL/TLR sequence variants in Nature correlates with their thermodynamic stability in isolation. Overall, our findings are consistent with modular but complex energetic properties of RNA structural motifs and will aid in the eventual quantitative description of RNA folding from its secondary and tertiary structural elements.

    View details for DOI 10.1021/jacs.7b08870

    View details for PubMedCentralID PMC5748328

  • Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway. Proceedings of the National Academy of Sciences of the United States of America Bisaria, N., Greenfeld, M., Limouse, C., Pavlichin, D. S., Mabuchi, H., Herschlag, D. 2016; 113 (34): E4956-65

    Abstract

    The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations.

    View details for DOI 10.1073/pnas.1525082113

    View details for PubMedID 27493222

    View details for PubMedCentralID PMC5003260

  • Protein flexibility is required for vesicle tethering at the Golgi. eLife Cheung, P. P., Limouse, C., Mabuchi, H., Pfeffer, S. R. 2015; 4

    Abstract

    The Golgi is decorated with coiled-coil proteins that may extend long distances to help vesicles find their targets. GCC185 is a trans Golgi-associated protein that captures vesicles inbound from late endosomes. Although predicted to be relatively rigid and highly extended, we show that flexibility in a central region is required for GCC185's ability to function in a vesicle tethering cycle. Proximity ligation experiments show that that GCC185's N-and C-termini are within.

    View details for DOI 10.7554/eLife.12790

    View details for PubMedID 26653856

    View details for PubMedCentralID PMC4721967

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