Virtual NSF-Funded MIT Workshop
Genome Architecture and Dynamics
June 15-18, 2020
Aakash Basu, Johns Hopkins University
Title: Measuring DNA mechanics on the genome scale
Abstract: Mechanical deformations of DNA such as bending are ubiquitous and implicated in diverse cellular functions. However, the lack of high-throughput tools to measure the mechanical properties of DNA limits our understanding of whether and how DNA sequences modulate DNA mechanics and associated chromatin transactions genome-wide. We developed an assay called loop-seq to measure the intrinsic cyclizability of DNA – a proxy for DNA bendability – in high throughput. We measured the intrinsic cyclizabilities of 270,806 50 bp DNA fragments that span the entire length of S. cerevisiae chromosome V and other genomic regions, and also include random sequences. We discovered sequence-encoded regions of unusually low bendability upstream of Transcription Start Sites (TSSs). These regions disfavor the sharp DNA bending required for nucleosome formation and are co-centric with known Nucleosome Depleted Regions (NDRs). We show biochemically that low bendability of linker DNA about 40 bp away from a nucleosome edge inhibits nucleosome sliding into the linker by the chromatin remodeler INO80. It explains how INO80 can create promoter-proximal nucleosomal arrays in the absence of any other factors by reading the DNA mechanical landscape. At the chromosome scale, sequence-dependent mechanical modulations make DNA around nucleosomal dyads significantly more bendable than linker DNAs, and this contrast increases for nucleosomes deeper into gene bodies. It suggests that DNA mechanics plays a previously unappreciated role in organizing nucleosomes far from the influence of remodelers that operate near TSSs. Altering gene sequences by randomly selecting synonymous codons does not preserve this contrast, suggesting that evolutionary selection among synonymous codons has been impacted by the way sequence modulates mechanics. Finally, we provide evidence that transcription through the TSS-proximal nucleosomes is impacted by local DNA mechanics. Overall, this first genome-scale map of DNA mechanics hints at a ‘mechanical code’ with broad functional implications.
Sumitabha Brahmachari, Rice University
Title: Interchromosome organization via lengthwise compaction
Abstract: Reliable structuring of the three-dimensional architecture of chromosome polymers, regulating the randomizing effect of stochastic thermal fluctuations, is important for biological function. A minimal polymer model recapitulating the ensemble-averaged structure of chromosomes requires two ingredients: Lengthwise compaction and chromatin self-adhesion. Lengthwise compaction forces the polymer to compact along its contour thus increasing cis-chromosome interactions. While, self-adhesion among specific blocks of chromatin (e.g., heterochromatin) tends to form segregated chromatin globules that, in general, promote both cis- and trans-chromosome interactions. We find that lengthwise compaction can effectively screen trans-chromosome interactions, and thus inhibit segregation of trans-chromosomal chromatin. We propose that screening of trans-chromosome interactions via lengthwise compaction is a general principle for regulation of the genome architecture.
Christina Caragine, New York University
Title: Surface Fluctuations and Coalescence of Nucleolar Droplets in the Human Cell Nucleus
Abstract: The nucleolus is a membraneless organelle embedded in chromatin solution inside the cell nucleus. By analyzing the surface dynamics and fusion kinetics of nucleoli in live human cells, we find that the nucleolar surface exhibits subtle, but measurable, shape fluctuations and the radius of the neck connecting two fusing nucleoli grows as r(t)~t1/2 [1]. This is consistent with liquid droplets with low surface tension ~10-6 Nm-1 coalescing in a fluid of higher viscosity ~103 Pa s, i.e. chromatin solution. We find the neck velocity, dr/dt, is comparable to the velocity of chromatin solution [2]. Surprisingly, nucleolar coalescence occurs in an active fluid, yet can be described by coalescence theory for passive liquid droplets, suggesting the measured quantities might be effective quantities. Our study presents a noninvasive approach, using natural probes to investigate material properties of the cell as well as to understand the physical interactions between nucleoli and chromatin solution [1,3].
1. Caragine CM et al, Phys. Rev. Lett. (2018)
2. Zidovska A et al, Proc. Natl. Acad. Sci. (2013)
3. Caragine CM et al, eLife (2019) ”
Ryan Cheng, Rice University
Title: Exploring Chromosomal Structural Heterogeneity Across Multiple Cell Lines
Abstract: We study the structural ensembles of human chromosomes across different cell types. Using computer simulations, we generate cell-specific 3D chromosomal structures and compare them to recently published chromatin structures obtained through super-resolution microscopy. We demonstrate using a combination of machine learning and polymer physics simulations that epigenetic information can be used to predict the structural ensembles of multiple human cell lines. The chromosomal structures obtained in silico are quantitatively consistent with those obtained through super-resolution microscopy as well as DNA-DNA proximity ligation assays. Theory predicts that chromosome structures are fluid and can only be described by an ensemble, which is consistent with the observation that chromosomes exhibit no unique fold. Nevertheless, our analysis of both structures from simulation and super-resolution microscopy reveals that short segments of chromatin make transitions between a closed conformation and an open dumbbell conformation. This conformational transition appears to be consistent with a two-state process with an effective free energy cost of about four times the effective information theoretic temperature. Finally, we study the conformational changes associated with the switching of genomic compartments observed in human cell lines. Genetically identical but epigenetically distinct cell types appear to rearrange their respective structural ensembles to expose segments of transcriptionally active chromatin, belonging to the A genomic compartment, towards the surface of the chromosome, while inactive segments, belonging to the B compartment, move to the interior. The formation of genomic compartments resembles hydrophobic collapse in protein folding, with the aggregation of denser and predominantly inactive chromatin driving the positioning of active chromatin toward the surface of individual chromosomal territories.
Mateusz Chiliński, University of Warsaw
Title: ConsensuSV: consensus structural-variant caller for next generation sequencing for selected families from 1000 Genomes Project
Abstract: The talk presents the ongoing research results on the connection between 3D structure of human genome and its functional effects on the development of clinical phenotypes. The biological samples being analysed origin first from 1000 Genomes Project (three healthy families, parents with daughters), secondly two Polish families where the child developed diabetes type 1, and thirdly single family from Japan, where the child developed leukaemia. We use 12 gold-standard callers for obtaining accurate Structural Variants (SV) for each family. The results of the tools are merged using our novel algorithm ConsensuSV, which integrates the SV sets using machine learning by combining decision trees and neural networks trained and benchmarked on the high quality SVs from 1000 Genomes Project. Such approach allows us to create the sets of high-confidence Structural Variants for each analysed Trios. Further, we applied ConsensuSV to families of known phenotype. The resulting list of SVs are used for the identification of genes which expression is altered due to the changes in spatial chromatin organisation. Finally, the functional impact of observed SVs is validated by analysing the list of biological processes that involve those genes.
Basilio Cieza Huaman, Johns Hopkins University
Title: Nucleation and Non-Equilibrium Drives Phase Separation in A Cooperative Kinetic Reaction-Diffusion
Abstract: Model Eukaryotic cells need to organize their chemical reactions in space and time. They manage to do this through a membrane and a membraneless subcompartmentalization of their intracellular space. Plenty of studies support the view of membraneless subcompartmentalization assemble by intracellular Liquid-Liquid phase separation (LLPS). Because of its biological importance, there is an increasing interest in understanding the physical properties driving LLPS. Thus far, there is a consensus that multivalence is the critical parameter that determines if a protein will phase separate or not. However, there are not many studies about the equilibrium/nonequilibrium physics of LLPS or how cells use nucleation to regulate where and when LLPS occurs. Here we develop a new kinetic reaction-diffusion model to study LLPS. We propose that LLPS occurs mainly through two independent reactions. Nucleation and self-recruitment. Our model shows clustering just at nonequilibrium conditions, suggesting that nonequilibrium conditions are mandatory to see LLPS. Clusters also show different dynamics (release/recruitment of particles) and shape depending on the nucleation frequency and cooperativity strength. Finally, we observed nucleation to promote the transition from the diffuse state to the condense state suggesting that cells may use nucleation to regulate LLPS.
Vinicius Contessoto, Rice University
Title: Exploring the Energy Landscape of Chromosomes: Transitions Between Interphase and Mitotic Phase
Abstract:Understanding the genome architecture and how the chromosomes are organized during the different phases of the cell is a significant challenge that involves scientists from diverse areas of knowledge. Recent studies of synchronized HIC maps of the DT40 chicken cell line in different phases provide information of the genome organization during this transition. Here, using the maximum entropy approach in the coarse-grained chromosome model, we investigate the folding-unfolding dynamics of chromosomal structures. In other words, we studied the transition of chromosomes between the interphase to mitotic phase. Some preliminary results indicate that the genome structural organization during the mitotic phase might be related to the modulation of non-specific long-range interactions. This modulation leads the chromosome structure to lose some compartments associated with the long genomic distance contacts. These preliminaries’ findings suggest that the local (short genomic distances) interactions play an essential role in the condensation process.
Patrick Cramer, Max Planck Institute for Biophysical Chemistry
Title: Recent advances in understanding chromatin transcription
Abstract: Liquid-liquid phase separation (LLPS) driven by interactions of multivalent macromolecules has emerged as an important mechanism to organize the cell interior. Much of our previous work has focused on the role of LLPS in organizing signaling clusters at membranes. Recently our efforts have expanded in a new direction, to understand whether and how LLPS might regulate the organization of chromatin in the eukaryotic nucleus. In my talk I will discuss or finding that polynucleosome arrays, which mimic the multivalent chromatin polymer, have an intrinsic capacity to undergo LLPS. Moreover, may factors known to control chromatin organization in cells have parallel effects on phase separated chromatin droplets in vitro, including histone H1, internucleosome spacing and histone tail acetylation. Our data suggest a new framework, based on intrinsic phase separation of the chromatin polymer, to understand the organization and regulation of eukaryotic genomes.
Iris Dror, UCLA
Title: XIST mediates unique mechanism of X-chromosome dosage compensation in early human development
Abstract: Female placental mammals silence one of the two X-chromosomes through X-chromosome inactivation (XCI). This essential process is mediated by the lncRNA XIST that spreads along the X to mediate gene silencing. During female human pre-implantation development, a unique mechanism for dosage compensation takes place, where instead of silencing of one of the two X-chromosomes as in XCI, gene expression is reduced from both X-chromosomes in a process called X-chromosome dampening (XCD). Thus, X-linked gene dosage in humans is regulated first by XCD and upon implantation by XCI. Moreover, XIST accumulates on both dampened X chromosomes at this stage, but unable to induce complete gene silencing. Thus, X-linked gene dosage in humans is regulated first by XCD and upon implantation by XCI. To understand the unique mechanism of dosage compensation that takes place during human pre-implantation development, we are addressing the fundamental questions of how XCD is achieved mechanistically and why XIST is not inducing silencing during XCD. To study these questions, we are taking advantage of naïve human embryonic stem cells (hESCs) and somatic cells, which capture the XCD and XCI state, respectively.
To this end, we have explored XIST localization in female naïve hESCs and somatic cells and discovered that XIST spreads over the entire X, but displays striking differences in enrichment along the X between XCD and XCI, which are explained by differences in the 3D folding of the X. We also made the remarkable observation that XIST spreads to autosomal regions in naïve hESCs but not in somatic cells, consistent with XIST dispersal observed by imaging. Importantly, in naïve hESCs and blastocysts, X-linked and autosomal genes enriched for XIST are downregulated, demonstrating that XIST mediates XCD as well as autosomal repression. Additional data demonstrate that XIST fulfills its function in XCD and autosomal repression through SPEN, similar to its mechanism of action in XCI. Taken together, our data show that XIST mediates both XCD and XCI, uncover an unprecedented function of XIST in autosomes, and define new principles that govern the regulation of gene expression by lncRNAs.”
William Earnshaw, University of Edinburgh
Title: Evolving DNA-protein transactions during mitotic chromosome formation
Abstract:
Itaru Samejima, Kumiko Samejima, Georg Kustatscher, William C. Earnshaw. University of Edinburgh, Edinburgh, United Kingdom
e-mail: bill.earnshaw@ed.ac.uk
The discovery of mitotic chromosomes by Flemming in 1878 established a major question in Cell Biology that remains unanswered all of these years later: How does the interphase nucleus transform itself into threadlike mitotic chromosomes that segregate the genome in mitosis? I will discuss an integrated approach combining chemical genetics, gene targeting by CRISPR/Cas9, proteomics and microscopy coupled with machine learning that we are developing to enable us to characterise the changes that occur as vertebrate cells make the transition from interphase into mitosis. We use chemical genetics with an analogsensitive mutant of the key cell cycle kinase CDK1 to accumulate cells at the G2/M border. Removal of the 1NM-PP1 inhibitor allows cells to enter mitosis within minutes in a synchronous wave. Published studies combining HiC and mathematical modelling led us to develop a model suggesting that mitotic chromosome formation involves two processes; chromatin compaction, and a functionally distinct architectural remodeling that disassembles the interphase nuclear structure leading to a remarkable transformation that shapes individual chromatids. Those chromatids are organised by a helical scaffold of condensin II that extrudes chromatin loops of up to ~400 kb within which are nested loops of about 80 kb extruded by condensin I. Those experiments, combined with our new studies of changes in protein association with DNA/chromatin as cells enter mitosis are enabling us to describe the ebb and flow as different classes of proteins move on and off DNA as cells enter mitosis. This is yielding insights into the coordinated behaviour of structural proteins such as of condensin I and II and cohesin during mitotic chromosome formation. Interestingly, the earliest events of prophase appear to involve detachment of the ribosomal RNA processing machinery from the chromosomes while the polymerase itself apparently remains active. These dramatic changes within the nucleolus occur long before there is any sign of nucleolar disassembly, which occurs much later in prophase.
Funded by Wellcome.
Jonah Eaton, New York University
Title: Structural and Dynamical Signatures of Local DNA Damage in Live Cells
Abstract: The dynamic organization of chromatin inside the cell nucleus plays a key role in gene regulation and maintaining genome integrity. While the static folded state of the genome has been studied before, the dynamical signatures of processes such as transcription or DNA repair are unknown. We investigate the interphase chromatin dynamics in human cells in response to local damage, DNA double strand breaks (DSBs), by monitoring the DSB dynamics and the compaction of the surrounding chromatin in live cells. We find DSBs to possess a unique chromatin compaction profile, while being more mobile when located in the nuclear interior as opposed to the periphery. We show that DSB motion is subdiffusive, ATP-dependent, and exhibits unique dynamical signatures compared to undamaged chromatin. We find that DSB mobility follows a universal relationship based on the local environment suggesting that the repair processes are robust and likely deterministic. Such knowledge may help in detection of DNA damage in live cells and aid our biophysical understanding of genome integrity in health and disease [Eaton & Zidovska, Biophys. J., 2020].
Lucas Farnung, Max Planck, Göttingen
Title: CHD chromatin remodellers influence local chromatin organization
Abstract: Chromatin remodelling plays important roles in modulating local chromatin organization. To investigate how different members of the CHD family of chromatin remodellers engage a nucleosome, we determined the cryo-EM structures of S. cerevisiae Chd1 and H. sapiens CHD4 in complex with a nucleosome to 4.8 Å and 3.1 Å, respectively.
Whereas Chd1 functions in euchromatic regions of the genome during active transcription, CHD4 plays a central role in the establishment and maintenance of repressive genome regions.
Consistent with these observations, we find that Chd1 induces unwrapping of two helical turns of nucleosomal DNA but CHD4 does not unravel nucleosomal DNA. It is likely that the evolution of auxiliary domains in different CHD subfamilies led to these different functionalities, which have a direct impact on the organization of local genome architecture.”
Taekjip Ha, Johns Hopkins University
Title: CRISPR and DNA Repair
Margaret Johnson, Johns Hopkins University
Title: Modeling nonequilibrium self-assembly in the cell, in the nucleus, and on the membrane
Abstract: For successful gene expression, a series of multi-domain proteins must recruit to genomic targets to activate remodeling and transcription. To quantify the global dynamics of pioneer proteins such as GAF as they partition across genomic targets, recruit downstream proteins, and activate transcription in response to temperature stress, our computational models must capture localized self-assembly of large protein populations, out-of-equilibrium. We have developed reaction-diffusion software, NERDSS, that enables this type of spatio-temporal simulations, with immediate comparison to time-dependent experimental results. We describe here the theoretical basis for this approach, its immediate applications, and its extensibility. We illustrate the application of NERDSS to developing a model of localized assembly on membranes for the clathrin coat protein, providing the first kinetic model of clathrin assembly that recapitulates time-dependent fluorescence data. Lastly, we discuss how varying the stoichiometries of assembly components or enzymatic activity can sensitively tune residence times and disassembly.
Sangjin Kim, University of Illinois Urbana-Champaign
Title: DNA supercoiling mediates long-distance interactions between RNA polymerases
Abstract: DNA genomes are continuously read by processive molecular motors, called RNA polymerases (RNAPs). For example, Escherichia coli, a bacterial model organism, has a genomic DNA of about four million bases, and a few hundreds of RNAPs can be detected somewhere on the genome at a given time, creating a situation like cars on the highway. While the dynamics of this molecular traffic is important in understanding the structure and function of the genome, we still lack a clear understanding of how even a few RNAPs work in the genomic context. Experimental results suggested that RNAPs can exhibit collective group dynamics by exploiting dynamic changes in DNA topology. In this talk, I will describe our current understanding of RNAP dynamics in connection with the dynamics of DNA topology. I will also present my lab’s theoretical efforts to make a complete model explaining the emergence of RNAP group dynamics in the genomic context.
Megan King, Yale
Title: A nucleosome-constrained model for topologically associating domains
Abstract: Chromosome conformation capture techniques (e.g Hi-C) reveal that intermediate-scale chromatin organization is comprised of “topologically associating domains” (TADs) on the tens to thousands of kb scale. The loop extrusion factor (LEF) model provides a framework for how TADs arise: cohesin or condensin extrude DNA loops, until they encounter boundary elements. Despite recent in vitro studies demonstrating that cohesin and condensin can drive loop formation on (largely) naked DNA, evidence supporting the LEF model in living cells is lacking. Here, we combine experimental measurements of chromatin dynamics in vivo with simulations to further develop the LEF model. We show that the activity of the INO80 nucleosome remodeler enhances chromatin mobility, while cohesin and condensin restrain chromatin mobility. Motivated by these findings and the observations that cohesin is loaded preferentially at nucleosome-depleted transcriptional start sites and its efficient translocation requires nucleosome remodeling we propose a new LEF model in which LEF loading and loop extrusion direction depend on the underlying architecture of transcriptional units. Using solely genome annotation without imposing boundary elements, the model predicts TADs that reproduce experimental Hi-C data, including boundaries that are CTCF-poor. Furthermore, polymer simulations based on the model show that LEF-catalyzed loops reduce chromatin mobility, consistent with our experimental measurements. Overall, this work reveals new tenets for the origins of TADs in eukaryotes, driven by transcription-coupled nucleosome remodeling.
Dana Krepel Zussman, Rice University
Title: The Structures of SMC Motors: towards the mechanism of the extrusion complex
Abstract:
Dana Krepel1, Aram Davtyan1, Nicholas P. Schafer1 Peter G. Wolynes1 and José N. Onuchic1
1Center for Theoretical Biological Physics, Rice University, Houston, TX 77005
Protein assemblies consisting of structural maintenance of chromosomes (SMC) and kleisin subunits are essential for the process of chromosome segregation across all domains of life. While limited structural data exist for the proteins that comprise the (SMC)–kleisin complexes, the complete structures of the entire complexes remain unknown, making mechanistic studies difficult. Using an integrative approach that combines crystallographic structural information about the globular subdomains, along with coevolutionary information and an energy landscape optimized force field (AWSEM), we predict atomic-scale structures for several tripartite SMC−kleisin complexes, including prokaryotic condensin, eukaryotic cohesin, and eukaryotic condensin. The molecular dynamics simulations of the SMC−kleisin protein complexes suggest that these complexes exist as a broad conformational ensemble that is made up of different topological isomers. The simulations suggest a critical role for the SMC coiled-coil regions, where the coils intertwine with various linking numbers. The twist and writhe of these braided coils are coupled with the motion of the SMC head domains, suggesting that the complexes may function as topological motors. Opening, closing, and translation along the DNA of the SMC−kleisin protein complexes would allow these motors to couple to the topology of DNA when DNA is entwined with the braided coils. Our findings constitute the first step toward studying the structure–function relationship of the various molecular motors operating on DNA.
Daniel Lee, Princeton University
Title: Chromatin Mechanics Dictates Subdiffusion and Coarsening Dynamics of Embedded Condensates
Daniel S.W. Lee1,2,3, Ned S. Wingreen1,3, Clifford P. Brangwynne 1,2,41 Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, USA
2Department of Chemical and Biological Engineering, Princeton University3Department of Molecular Biology, Princeton University 4 The Howard Hughes Medical Institute
Abstract:
DNA is organized into chromatin, a complex polymeric material which stores information and controls gene expression. An emerging mechanism for biological organization, particularly within the crowded nucleus, is the biomolecular phase separation of condensed droplets of protein and nucleic acids. However, the way in which chromatin impacts the dynamics of phase separation and condensate formation is poorly understood. Here, we utilize a powerful optogenetic strategy to examine the interplay of droplet coarsening with the surrounding viscoelastic chromatin network. We demonstrate that droplet growth dynamics are directly inhibited by the chromatin-dense environment, which gives rise to an anomalously slow coarsening exponent, , contrasting with the classical prediction of. Using scaling arguments and simulations, we show how this arrested growth can arise due to subdiffusion of individual condensates, predicting , where is the anomalous diffusion exponent. Tracking the fluctuating motion of condensates within chromatin reveals a subdiffusive exponent, , which explains the anomalous coarsening behavior and is also consistent with Rouse-like dynamics arising from the entangled chromatin. Our findings have implications for the biophysical regulation of the size and shape of biological condensates and shed light on the intimate connection between their dynamics and the local mechanical environment within cells.
Yang Liu, Johns Hopkins University
Title: Very Fast CRISPR On Demand
Abstract: CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR, creates double-strand-breaks (DSBs) at submicron and seconds scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated over 100 kilobases per minute, reaching up to 30 megabases. Using single cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated with inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single allele resolution. Together, very fast CRISPR enables DNA repair studies at high resolution in space, time and genomic coordinates.
Zaida (Zan) Luthey-Schulten, University of Illinois Urbana-Champaign
Title: Macroeconomics of a Minimal Cell: Integration of Experiments, Theory, and Simulations
Abstract:
Using cryo-electron tomograms of the genetically minimal cell JCVI-syn3A (Hutchison et al.
2016, Breuer et al. 2018) from the lab of E. Villa (UCSD), we built a realistic spatial model of
the cell geometry and distributions of ribosomes and DNA. The reaction model for genetic
information integrates the kinetics of DNA replication, transcription of all 493 genes in JCVIsyn3A, translation and degradation of all 452 of the cell’s protein-coding mRNA. Genome-wide proteomics data are used as estimates of the initial protein abundance. To validate the kinetic parameters, the reactions are solved stochastically using the Gillespie algorithm over the ~2 hour cell cycle and then compared to the results of the spatial reaction-diffusion master equation description which includes the ribosome and DNA distributions. Introducing spatial heterogeneity, diffusion, and degradosomes produces a realistic estimate of mRNA degradation and protein distributions in qualitative agreement with proteomics data. I will also report on the challenges and preliminary results of coupling the genetic information processes with the metabolic networks to describe the overall economy of a minimal cell.
References:
Hutchison, CA., et al. “Design and synthesis of a minimal bacterial genome.” Science 351.6280 (2016).
Breuer, M, et al. “Essential metabolism for a minimal cell.” eLife 8 (2019): e36842.
Thornburg, ZR, et al. “Kinetic Modeling of the Genetic Information Processes in a Minimal Cell.” Frontiers in Molecular Biosciences 6 (2019).
Cecile Mathieu, St Jude Children’s Research Hospital
Title: A conformational switch regulates G3BP-RNA phase separation and biomolecular condensates formation in cells
Abstract: Liquid-liquid phase separation participates to the formation of a broad variety of biomolecular condensates in cells that have liquid-like properties and exchange of components with their surrounding environment. One main class of condensates are ribonucleoprotein (RNP) granules. They are found in both the nucleus and cytoplasm and are composed of hundreds of proteins and RNAs. Interestingly, despite similar material properties and constituents, these RNP granules harbor distinct identities. To understand how these condensates establish and maintain their identity, we focused on the mechanism of assembly of stress granules (SGs), cytoplasmic RNP granules that forms in response to stress. We focused on G3BP1/2, two paralogues essential for SGs formation during oxidative stress. We put in evidence that SGs formation involves G3BP1 phase separation with RNA. This process relies on specific valences that are encoded in both the N-terminal NTF2L domain, responsible for dimerization, and the RNA-binding domain (RBD), via RNA-binding. Interestingly, although the protein contains large intrinsically disordered regions (IDRs), these regions are not essential for SGs formation in cells nor for phase separation of the purified protein. Instead, we found that G3BP1 IDRs regulate the formation of SGs via a conformational switch, allowing the transition between a closed and open conformation. This conformational switch relies on electrostatic interactions between the negatively and positively charged IDRs, regulating RNA-binding and the subsequent phase separation of the protein as well as the assembly of SGs in cells.
Bhavya Mishra, Johns Hopkins University
Title: First Passage Times in multi-protein self-assembly and the role of dimensionality reduction
Abstract: Recognition of specific binding sites and the recruitment of other proteins, by a protein, in a fast and precise manner, are vital steps in various cellular processes such as transcription, translation, and clathrin-mediated endocytosis. In general, proteins diffuse in a 3D solution. But they can also localize to a low dimensional surface like DNA chain or membrane surface.
In a recent study, we quantified how localization of proteins on a low dimensional surface from solution reduces their search space, and proteins can exploit this dimensionality reduction to trigger the search process. Here, in this work, we further develop an analytical theory to approximate the role of localization in slowing or accelerating the overall binding process. We formulate the self-assembly of protein binding pairs as first-passage problems and calculate the meantime to approach the thermodynamic equilibrium.
Claudia Mimoso, Harvard Medical School
Title:Rapid and Efficient Co-Transcriptional Splicing Enhances Mammalian Gene
Expression
Abstract:
Kirsten A. Reimer, Claudia Mimoso, Karen Adelman, Karla M. Neugebauer
The spliceosome is a macromolecular ribonucleoprotein complex responsible for the cotranscriptional removal of introns from pre-mRNAs. Previous work on co-transcriptional splicing has supported the idea that RNA processing events are coordinated with each other and with transcription, however the spatial and temporal relationship between them remains elusive. To determine the spatial and temporal window for splicing and its relationship to RNAPII elongation in mammals, we performed long read sequencing (LRS) of nascent RNA and PRO-seq during mouse erythropoiesis. We find rapid and efficient co-transcriptional splicing by the mammalian spliceosome. Through LRS, we observe that 50% of introns are spliced within 141nt of transcribing the 3’ splice site (ss). PRO-seq signal around 5’ and 3’ ss show no evidence of pausing and no significant changes in RNA Polymerase II (RNAPII) elongation. The lack of altered elongation rates across splice junctions indicates that RNAPII elongation is not generally impacted by the transcription of splice sites and confirms that mammalian spliceosomes can act rapidly. Further, we do not observe changes in RNAPII elongation rate around introns defined by LRS to be inefficiently spliced or where the spliceosome frequently stalls after the first catalytic step. Lastly, the single molecule resolution of nascent RNAs provided by LRS revealed that individual transcripts that have not undergone splicing display impaired RNA cleavage at canonical gene ends as seen at the globin genes. Introducing a patient-derived mutation in β-globin improved splicing efficiency and 3’end cleavage, demonstrating that co-transcriptional splicing efficiency is a determinant of productive gene output.
Geeta Narlikar, University of California, San Francisco
Title: Can Phase-separation Explain Heterochromatin Properties?
Abstract:
Madeline Keenen1, Geeta J. Narlikar1, Sy Redding1
1Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA 94158, USA.
Heterochromatin forms compact genomic territories, whose nuclear localization is stably maintained in interphase. At the same time, the core heterochromatin protein, HP1 has been shown to come on and off from heterochromatin on the order of seconds. This raises the question of how the highly dynamic and weakly binding HP1 proteins can generate stably compacted chromatin states. A few years ago, we found that the heterochromatin protein HP1 promotes chromatin self-association and liquid droplet formation. Building on these results we find that phase-separated droplets formed by the human HP1 protein HP1a with DNA mimic stable DNA territories in vitro. While HP1a molecules rapidly mix upon fusion of in vitro reconstituted HP1a-DNA droplets, the DNA remains constrained within its territory for a substantially longer time. We further find that the HP1 paralog, HP1β dissolves the HP1a-DNA condensates. I will present these and other results and discuss whether the stereotypical properties of heterochromatin observed in vivo could arise from the phase-separation based properties observed in vitro.
Rohit Pappu, Washington University in St. Louis
Title: Stickers-and-spacers framework for describing the physics of intracellular phase transitions
Abstract: A general stickers-and-spacers framework, derived from the physics of associative polymers has emerged for describing the phase behavior of proteins (and RNA molecules) that drive the formation of biomolecular condensates. This talk will highlight recent insights that have emerged from application of the stickers-and-spacers framework to predict phase behavior, explain experimental data regarding phase behavior, and generate insights regarding the functions of biomolecular condensates. The work to be highlighted showcases the emerging molecular grammar written into protein and RNA sequences and also sheds light on evolutionary aspects of this grammar.
Amy Pandya-Jones, UCLA
Title: A protein assembly mediates Xist localization and gene silencing
Abstract: Nuclear compartments play diverse roles in regulating gene expression, yet the molecular forces and components driving compartment formation remain largely unclear. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus and binding diverse diffusible proteins to achieve X-chromosome inactivation (XCI). The XCI-process therefore serves as paradigm for understanding how the RNA-mediated recruitment of diffusible proteins induces a functional compartment. Interestingly, the characteristics of the inactive X (Xi)-compartment change over time because upon initial Xist spreading and transcriptional shutoff a state is reached where gene silencing remains stable even if Xist is turned off. Here, we show that the Xist RNA-binding-proteins (RBPs) PTBP1, MATR3, TDP43, and CELF1 are required for gene silencing and anchor Xist within the Xi through multivalent association with Xist’s E-repeat-element. Upon binding by Xist, these RBPs form a condensate in the Xi using self-aggregation and heterotypic protein-protein interactions that can be sustained in the absence of Xist. Notably, these RBPs only become essential coincident with the transition to the Xist-independent XCI-phase4 indicating that the protein condensate seeded by the E-repeat of Xist underlies developmental switch from Xist-dependence to Xist-independence. Taken together, our data demonstrate that Xist forms the Xi-compartment by seeding a heteromeric condensate consisting of ubiquitous RBPs and uncover an unanticipated mechanism for epigenetic inheritance.
Dariusz Plewczynski, University of Warsaw
Title: Spatial chromatin architecture alteration by structural variations in human genomes
Abstract: We assess the tendency of structural variants to accumulate in spatially interacting genomic segments and design an algorithm to model chromatin conformational changes caused by structural variations. We show that differential gene transcription is closely linked to the variation in chromatin interaction networks mediated by RNA polymerase II. We also demonstrate that CTCF-mediated interactions are well conserved across populations, but enriched with disease-associated SNPs. Moreover, we find boundaries of topological domains as relatively frequent targets of duplications, which suggest that these duplications can be an important evolutionary mechanism of genome spatial organization.
Yifeng Qi, MIT
Title: Data-driven polymer model for mechanistic exploration of diploid genome organization Abstract: Chromosomes are positioned non-randomly inside the nucleus to coordinate with their transcriptional activity. The molecular mechanisms that dictate the global genome organization and the nuclear localization of individual chromosomes are not fully understood. We introduce a polymer model to study the organization of the diploid human genome: it is data-driven as all parameters can be derived from Hi-C data; it is also a mechanistic model since the energy function is explicitly written out based on a few biologically motivated hypotheses. These two features distinguish the model from existing approaches and make it useful both for reconstructing genome structures and for exploring the principles of genome organization. We carried out extensive validations to show that simulated genome structures reproduce a wide variety of experimental measurements, including chromosome radial positions and spatial distances between homologous pairs. Detailed mechanistic investigations support the importance of both specific inter-chromosomal interactions and centromere clustering for chromosome positioning. We anticipate the polymer model, when combined with Hi-C experiments, to be a powerful tool for investigating large scale rearrangements in genome structure upon cell differentiation and tumor progression.
Kevin Rhine, Johns Hopkins University
Title: ALS/FTLD-linked mutations in glycine residues of FUS lead to immiscibility with wild-type FUS
Abstract: The formation of pathogenic inclusions of RNA-binding proteins in neurons is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). One prominent protein in these inclusions is Fused in sarcoma (FUS), and over 70 mutations in Fus are linked to ALS/FTLD. In patients, all Fus mutations are heterozygous, indicating that the mutant drives pathology despite the presence of wild-type FUS. Here, we demonstrate that ALS FUS mutations in glycine (G) strikingly drive formation of droplets that are immiscible with wild-type FUS whereas arginine (R) mutants form miscible condensates with wild-type FUS. Remarkably, immiscibility between wild-type and G mutants begins at the earliest stages of FUS nucleation. In contrast to G mutants, the miscible R mutants physically interact with the wild-type FUS such that wild-type FUS rescues the mutant defects by reducing droplet size and recovering dynamic interactions with RNA. This result suggests disparate molecular mechanisms underlying pathogenesis and differing rescue potential depending on the type of mutation.
Michael Rosen, University of Texas, Southwestern
Title: Organization of Chromatin by Intrinsic and Regulated Phase Separation
Abstract: Liquid-liquid phase separation (LLPS) driven by interactions of multivalent macromolecules has emerged as an important mechanism to organize the cell interior. Much of our previous work has focused on the role of LLPS in organizing signaling clusters at membranes. Recently our efforts have expanded in a new direction, to understand whether and how LLPS might regulate the organization of chromatin in the eukaryotic nucleus. In my talk I will discuss or finding that polynucleosome arrays, which mimic the multivalent chromatin polymer, have an intrinsic capacity to undergo LLPS. Moreover, may factors known to control chromatin organization in cells have parallel effects on phase separated chromatin droplets in vitro, including histone H1, internucleosome spacing and histone tail acetylation. Our data suggest a new framework, based on intrinsic phase separation of the chromatin polymer, to understand the organization and regulation of eukaryotic genomes.
Guang Shi, University of Texas at Austin
Title: From Hi-C Contact Map to Three-dimensional Organization of Interphase Human Chromosomes
Abstract: The probabilities that two loci in chromosomes that are separated by a certain genome length can be inferred using Hi-C experiments. How to go from such maps to an ensemble of three dimensional structures, which is an important step in understanding the way nature has solved the packaging of the hundreds of million base pair chromosomes in tight spaces, is an open problem. We created a theory based on polymer physics and maximum entropy principle, leading to the HIPPS (Hi-C-Polymer Physics-Structures) method allows us to go from contact maps to 3D structures. We reconstruct the joint distribution of spatial positions of the loci, which creates an ensemble of structures for the 23 chromosomes from lymphoblastoid cells. The HIPPS method shows that the conformations of a given chromosome are highly heterogeneous even in a single cell type. Nevertheless, the differences in the heterogeneity of the same chromosome in different cells types (normal as well as cancerous cells) can be quantitatively determined using our theory.
Sucheol Shin, University of Texas at Austin
Title: Genome structure–function relationship revealed by an active Chromosome Copolymer Model
Abstract: A mammalian genome functions with specificity while exhibiting heterogeneous spatial organization. It is unclear how the structural and functional aspects of such complex genomes are related with each other. In this presentation, we highlight the interactive relationship between genome structure and function using coarse-grained polymer simulations under non-equilibrium conditions. We model a human interphase chromosome as an active Chromosome Copolymer Model (CCM) polymer5 where active force is applied to genetically open (euchromatin) loci to mimic transcription. We demonstrate that although the organizational features at large length scale such as compartments and TADs are preserved, active force can preferentially enhance the chromatin packing at small length scale (at the sub-TAD level), which stabilizes the interactions between cis-regulatory elements, and hence the transcription process of individual genes. We illustrate that this effect of transcriptional activity varies greatly depending on the chromosome conformation within a given cell as well as the loop formation at each CTCF binding site. Our study elucidates new quantitative insights into how genome structure and function influence each other for gene regulation.
Andrew Spakowitz, Stanford University
Title: Homolog locus pairing is a transient, diffusion-mediated process in meiotic prophase
Abstract:
Trent Neuman, Bruno Beltran, Sean Burgess, Andrew Spakowitz
The pairing of homologous chromosomes in meiosis I is essential for sexual reproduction, yet the dynamic processes that contribute to pairing at individual homologous loci remain largely uncharacterized. We track individual homologous locus pairs using a fluorescent reporter-operator system (FROS) at various stages of meiosis. We observe mean squared change in displacements (MSCDs) that exhibit the $t^{1/2}$ power-law scaling behavior characteristic of polymer diffusion followed by a plateau-from which we can infer that homologous loci are weakly tethered to each other. We develop a theory for Rouse polymers with “homologous” linkages, and show that the plateau behavior is consistent with a handful of randomly-spaced linkages per chromosome forming over the course of meiosis. While ensemble averaged MSCDs appear to suggest a strongly viscoelastic environment ($t^\alpha$, with $\alpha \ll 1/2$), we show how this artifact can arise from regular diffusion due to the heterogeneous confinement (tethering) radii imposed by random linkages. These linkages are also sufficient to reproduce the global increase in pairing during mid-to-late prophase, even though the tagged loci end up hundreds of kilobases from the nearest linkage site on average. Brownian dynamics simulations of our linked polymers quantitatively reproduce the search times and pairing lifetimes observed at each successive stage of meiosis. A \textit{spo11$\Delta$} mutant deficient in double strand break (DSB) formation can also be reproduced by the same model with the linkages removed. Together, these results suggest that homolog pairing is driven purely by chromatin diffusion, and that the DSB-dependence of homolog pairing comes not from the tagged loci experiencing DSBs, but instead from distal homologous linkages along the chromosome.
Chad Stein, Harvard Medical School
Title: Exploring how the Integrator Complex Attenuates Transcription in Mammalian Cells
Abstract: Proper regulation of gene expression is essential for development, cellular homeostasis, and responding to the environment. Transcription by RNA Polymerase II (RNAPII) was previously thought to be mainly regulated at the level of initiation. In recent decades, however, key post-initiation regulatory mechanisms been uncovered, including promoter-proximal pausing of RNAPII. The fate of paused polymerase has been a longstanding question in the field, and a growing body of literature suggests that promoter-proximal termination at protein-coding genes is a common phenomenon. Importantly, this termination short-circuits the transcription cycle as no full-length mRNA is made despite RNAPII initiation.
Our group and others recently described the role of the Integrator complex in driving this premature transcription termination via its RNA endonuclease, Integrator Subunit 11 (INTS11). Many questions about the function and regulation of this essential complex remain to be addressed. Here, using mouse embryonic stem cells, I aim to characterize Integrator’s role in the regulation of mammalian gene expression. I find that over one thousand protein-coding genes are upregulated upon INTS11 knockdown, highlighting its role as a transcriptional attenuator. I have also endogenously Halo-Tagged INTS11, allowing me to perform rapid protein depletion using a HaloTag Proteolysis Targeting Chimera (HaloPROTAC). Notably, this will facilitate the sensitive definition of Integrator target genes, which has been difficult using long-term depletion strategies. Together, this work will provide fundamental insights into the function of this understudied but important complex.
Stoyno Stoynov, Bulgarian Academy of Sciences
Title: Dia2 on the crossroad between DNA replication and cell’s size homeostasis
Abstract: Controlling variation of the cell size requires coordination between cell growth and the cell cycle. However, the mechanisms that harmonize cell growth with cell division are not fully understood. It is known that Dia2 is involved in regulation of DNA replication and pseudohyphal growth but whether there is a connection between its role in these two processes is still unclear. Our results show that Dia2 is required for the coordination between cell growth and the cell cycle and for correct nuclear positioning in order to control cell size variation and to prevent multinucleation. However, improper completion of replication of the dia2 deletion strain detected by the activated checkpoint control at the extended G2/M-phase is necessary to trigger abnormal cell size and morphology.
Dave Thirumalai, University of Texas at Austin
Title: Conformational Heterogeneity and Glassy Dynamics in Interphase Chromosomes
Abstract:
Recent experiments [1], simulations, and analytical models [2] have shown that
conformations of chromosomes are massively heterogeneous. The large-scale
variations are masked when only the average values of contact probabilities are
analyzed [3]. Hints of heterogeneity came to the fore when it was discovered that
there is discordance between the results from Hi-C and imaging data [4,5]. In some
cases, contacts between loci with high contact probability (Hi-C data) resulted in
larger spatial distance (imaging data) compared to contacts with low contact
probability, which was referred to as the Hi-C-FISH paradox [5]. Using precisely
solvable Generalized Rouse Model for Chromosomes (GRMC) [6] we solved the
paradox [2] by taking into account both population heterogeneity (a given contact is
present only in a fraction of cells) and conformational heterogeneity, describing
large variations in structures within a single subpopulation of cells. The theory
allowed us to quantitatively extract the distribution of populations successfully by
analyzing the data for 212 loci pairs [2]. There are large cell-to-cell variations in the
contact maps of a given chromosome [7], in accord with both single cell Hi-C
experiments [8] and imaging data [9]. A consequence of the extensive heterogeneity
is that the underlying dynamics of interphase chromosomes is glassy [7,10]. By
creating a Chromosome Copolymer Model (CCM) [7], which reproduces the
organization on mega base scale (compartments) and TADs on about 500 kilo bases,
we showed that the dynamics exhibits many aspects of glass-like dynamics. In
particular, some loci move fast while others are sluggish. We discovered that
chromosome organization is hierarchical involving the formation of TAD-sized
chromosome droplets (CDs) on short genomic scale followed by coalescence of the
CDs, reminiscent of Ostwald ripening. This mechanism, we speculate, could be
operative post mitosis, and would involve several Structural Maintenance
Complexes. Glassy landscapes for the condensed active chromosomes might provide
a balance between genomic conformational stability and biological functions.
References:
[1] Finn, E. H., et al. Cell 176.6 (2019): 1502-1515.
[2] G. Shi and D. Thirumalai, Nat. Comm. 10: 3894 (2019).
[3] J. Dekker, M. A. Marti-Renon, and L. Mirny, Nat. Rev. Genet. 14: 390 (2013).
[4] W. A. Bickmore and B. van Steensel. Cell 152: 1270-1284 (2013).
[5] G. Fudenberg and M. Imakaev. Nature methods 14: 67 (2017).
[6] J. D. Bryngelson and D. Thirumalai, Phys. Rev. Lett. 76: 542 (1996).
[7] G. Shi, L. Lei, C. Hyeon, and D. Thirumalai, Nat. Comm. 9:3161 (2018).
[8] T. Nagano et. al., Nature, 502: 59 (2013).
[9] S. Wang et. al., Science 353: 598 (2016).
[10] H. Kang,Y.G. Yoon, D. Thirumalai, and C. Hyeon, Phys. Rev. Lett. 115: 198102
(2015).
Wenjun Xie, MIT
Title: Characterizing chromatin folding coordinate and landscape with deep learning
Abstract:Genome organization is critical for setting up the spatial environment of gene transcription, and substantial progress has been made towards its high-resolution characterization. The underlying molecular mechanism for its establishment is much less understood. We applied a deep-learning approach, variational autoencoder (VAE), to analyze the fluctuation and heterogeneity of chromatin structures revealed by single-cell imaging and to identify a reaction coordinate for chromatin folding. This coordinate connects the seemingly random structures observed in individual cohesin-depleted cells as intermediate states along a folding pathway that leads to the formation of topologically associating domains (TAD). We showed that folding into wild-type-like structures remain energetically favorable in cohesin-depleted cells, potentially as a result of the phase separation between the two chromatin segments with active and repressive histone marks. The energetic stabilization, however, is not strong enough to overcome the entropic penalty, leading to the formation of only partially folded structures and the disappearance of TADs from contact maps upon averaging. Our study suggests that machine learning techniques, when combined with rigorous statistical mechanical analysis, are powerful tools for analyzing structural ensembles of chromatin.
Hao Yan, Yale University
Title: Dynamics of loop extruding polymers
Abstract: Chromatin organization is inextricably linked to its dynamics. The loop extrusion factor (LEF) model provides a framework for how topologically associating domains (TADs) arise. However, a characteristic subdiffusive behavior of MSD is observed in fission yeast on the seconds timescale and experiments show that cohesin or condensin largely constrains chromatin motion. We incorporated loop extrusion activity into the Rouse-chain model and performed polymer simulations to explore how loop formation changes polymer dynamics. The results show that the DNA-looping by cohesin and condensin largely reduces chromatin mobility, which is in agreement with experiments
Alexandra Zidovska, NYU
Title: Dynamics, flows and rheology of the human genome
Abstract: Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood. In interphase, time between two cell divisions, chromatin fills the cell nucleus in its minimally condensed polymeric state. Chromatin serves as substrate to a number of biological processes, e.g. gene expression and DNA replication, which require it to become locally restructured. These are energy-consuming processes giving rise to non-equilibrium dynamics. Chromatin dynamics has been traditionally studied by imaging of fluorescently labeled nuclear proteins and single DNA-sites, thus focusing only on a small number of tracer particles. Recently, we developed an approach, displacement correlation spectroscopy (DCS) based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells [1]. DCS revealed that chromatin movement was coherent across large regions (4–5μm) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes [1]. These large-scale, coupled motions were ATP-dependent and unidirectional for several seconds. Following these observations, we developed a hydrodynamic theory [2] as well as a microscopic model [3] of active chromatin dynamics. In this work we investigate the chromatin interactions with the nuclear envelope and compare the surface dynamics of the chromatin globule with its bulk dynamics [4], which we also explore using naturally present cellular probes [5].
[1] Zidovska A, Weitz DA, Mitchison TJ, PNAS, 110 (39), 15555-15560, 2013
[2] Bruinsma R, Grosberg AY, Rabin Y, Zidovska A, Biophys. J., 106 (9), 1871-1881, 2014
[3] Saintillan D, Shelley MJ, Zidovska A, PNAS, 115 (45) 11442-11447, 2018
[4] Chu F, Haley SC, Zidovska A, PNAS, 114 (39), 10338-10343, 2017
[5] Caragine CM, Haley SC, Zidovska A, PRL, 121, 148101, 2018