Bulletin of the American Physical Society
APS March Meeting 2023
Volume 68, Number 3
Las Vegas, Nevada (March 5-10)
Virtual (March 20-22); Time Zone: Pacific Time
Session F06: DNA Mechanics and Gene Expression IFocus
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Sponsoring Units: DBIO Chair: Shubham Tripathi, Yale University Room: Room 129 |
Tuesday, March 7, 2023 8:00AM - 8:36AM |
F06.00001: Engineering high-precision, dynamic genetic control systems via supercoiling-guided design Invited Speaker: Kate E Galloway Integrating synthetic circuitry into larger transcriptional networks to mediate predictable cellular behaviors remains a challenge within synthetic biology. Rational de novo design of synthetic circuits for cell engineering remains challenging. In particular, the stochastic nature of transcription makes coordinating expression across multiple genetic elements challenging. To address this challenge, my lab recently developed a theoretical framework for exploring how DNA supercoils—dynamic structures induced during transcription—influence transcription and gene expression in synthetic and native gene systems. We find that gene syntax—the relative ordering and orientation of genes—defines the expression profiles, variance, burst dynamics, and intergene correlation of two-gene systems. By applying our model to both a synthetic toggle switch and the endogenous zebrafish segmentation network, we find that supercoiling can enhance or weaken conventional biochemical regulatory strategies such as mRNA- and protein-mediated feedback loops. In cell culture, we confirmed that two-gene circuits qualitatively match the syntax-specific profiles predicted by our model. Our model integrates supercoiling-mediated biophysical feedback with classic gene regulation motifs such as transcriptional repressors that are well-studied in native and synthetic contexts. Our model provides an extensible framework for modeling an arbitrary number of genes and regulatory architectures. Our results suggest that supercoiling couples behavior between neighboring genes, representing a novel regulatory mechanism. Additionally, our predictions suggest why some circuit designs fail and provide a path to improving transgenic designs. Harnessing the insights from our model will enable enhanced transcriptional control, providing a robust method to tune expression levels, dynamics, and noise needed for the construction of transgene systems including synthetic gene circuits in primary cells and diverse cell engineering applications including cellular reprogramming. |
Tuesday, March 7, 2023 8:36AM - 8:48AM |
F06.00002: Using optogenetics to find the signal that maintains the DNA damage checkpoint Sahand Rahi, Marco Labagnara Cell cycle checkpoints arrest the cell division cycle when certain types of damage such as double-strand DNA breaks (DSBs) are detected. However, after prolonged arrests, many checkpoints are eventually overridden when damage persists. We recently studied the DNA damage checkpoint theoretically and experimentally in budding yeast and found that DDC override strikes a balance between speed and risk (Sadeghi et al., Nature Physics 2022). However, the nature of the signal maintaining the checkpoint has not been known. For the DDC, two different signals have been proposed in the past, emanating from the tips of broken chromosomes or from single-strand DNA (ssDNA) that is resected around the DNA break. To test which model is correct, we measured the rate of DNA resection using single-cell probes. We integrated light-activated promoter-fluorescent protein gene constructs at specific intervals from DNA break sites. By detecting when their activity was silenced, we could observe how much DNA had been resected. Using these measurements for wild-type and resection-defective strains, we found that a mixed model fit the data best: Both resected DNA as well as DNA break ends make distinct contributions to maintaining the DDC arrest. Furthermore, one DNA break affects resection of another, indicating interactions between DSBs. |
Tuesday, March 7, 2023 8:48AM - 9:00AM |
F06.00003: Rules of transcriptional bursting revealed by absolute quantification of nascent RNA in living fly embryos Po-Ta Chen, Benjamin Zoller, Michal Levo, Thomas Gregor Transcriptional bursting is a common regulatory strategy for controlling gene outputs in various organisms. Over the past 15 years, experiments that dissected transcriptional regulation into its smaller parts have helped us identify many molecular processes associated with burst modulation under various physiological conditions, such as enhancer regulation, transcription factor regulation, epigenetics modifications, etc. However, these reductionist insights have not yet been successful in adding up to a coherent biophysical model that links molecular mechanisms to empirical bursting dynamics. In this work, we provide a unified perspective on this problem by quantifying transcriptional activity in live Drosophila embryos with single RNA detection sensitivity. We reveal a common regulatory strategy that displays surprising constraints on transcriptional bursting under a whole variety of different experimental conditions, including cis- and trans- perturbations in mutant fly strains. We observe a massive data collapse across many conditions, hinting at general rules underlying the dynamics of transcriptional bursting that could be linked to evolutionarily conserved biophysical properties of the core eukaryotic transcriptional machinery. |
Tuesday, March 7, 2023 9:00AM - 9:12AM |
F06.00004: DNA supercoiling-transcription interplay in the presence of nucleosomes Shubham Tripathi, Sumitabha Brahmachari, Jose N Onuchic, Herbert Levine During transcription elongation, RNA polymerases (RNAPs) positively supercoil (overtwist) the downstream DNA and negatively supercoil (untwist) the upstream DNA. The resulting torsional stress can slow down transcription elongation. In eukaryotes, steric hindrance by nucleosomes can present an additional barrier to RNAP translocation. The overall effect of how nucleosomes alter the DNA torsional response and the steric hindrance they present will dictate transcription dynamics in eukaryotes. Here, we calculate the DNA torsional response as a function of the nucleosome density and show that since nucleosomes store negative supercoiling in the relaxed configuration, they can act as buffers to lower the torsional stress from RNAP-generated DNA supercoiling. Consequently, the presence of nucleosomes can speed up transcription elongation, despite the steric hindrance. Just like prokaryotes, eukaryotic transcription is further sped-up at high RNAP recruitment rates via cooperation between co-transcribing RNAPs. RNAP-generated supercoiling also drives transcriptional bursting, and the supercoiling dynamics can dictate transcriptional noise. Overall, we present a mechanistic description of the supercoiling-transcription interplay in eukaryotes. |
Tuesday, March 7, 2023 9:12AM - 9:24AM |
F06.00005: Self-assembly of DNA binding proteins can help control transcription initiation. Mankun Sang, Margaret E Johnson Transcription pioneer factors such as GAF (GAGA factor) are proteins essential for exposing DNA from highly packed eukaryotic chromatin. Experiments have recently shown that the assembly of GAF into higher-order oligomers is necessary for its pioneer function; however, they lack enough spatial or temporal resolution to study how oligomer formation impacts DNA transcription. Importantly, the distributions of GAFs and their targets on chromatin are non-homogeneous; thus, a spatial model is necessary. Here we implemented a rigid-body reaction-diffusion model to quantify how interactions between GAF proteins and between GAF to DNA control residence times, as measured by single-particle tracking experiments. We show how clusters of specific vs non-specific binding sites on the DNA can control the degree of oligomerization between the GAFs. Further, we can establish when stable oligomers can change the apparent affinity of GAF for the DNA binding sites. We characterize how 1D sliding along the DNA can promote oligomerization, even when nucleosomes can act as local barriers. With support from single-particle tracking data, our models show how mutations to specific binding domains shift the distributions of GAF throughout the nucleus. Our methods provide molecular mechanisms for how clustering between proteins can help them target specific DNA regions within the nucleus. |
Tuesday, March 7, 2023 9:24AM - 10:00AM |
F06.00006: Discovery of an endogenous cellular pathway that regulates transcriptional noise to promote cell-fate specificationLeor WeinbergerUniv. of California San Francisco (UCSF); Gladstone Institutes Invited Speaker: Leor Weinberger Stochastic fluctuations in gene expression (‘noise’) are often considered detrimental but, in other fields, fluctuations are harnessed for benefit (e.g., ‘dither’ or amplification of thermal fluctuations to accelerate chemical reactions). We recently showed that DNA base-excision repair amplifies transcriptional noise to generate increased cellular plasticity and facilitate cellular reprogramming (Desai et al. Science 2021). Specifically, the DNA-repair protein Apex1, which recognizes both naturally occurring and unnatural base modifications, amplifies expression noise while homeostatically maintaining mean-expression levels. This amplified expression noise originates from shorter duration, higher intensity, transcriptional bursts generated by Apex1-mediated DNA supercoiling. The remodeling of DNA topology first impedes and then accelerates transcription to maintain mean levels. Strikingly, this homeostatic noise amplification mechanism, termed ‘Discordant Transcription through Repair’ (DiThR; pronounced /’dither’/), potentiates cellular reprogramming and differentiation. These data reveal a functional role for transcriptional fluctuations mediated by DNA base modifications in embryonic development and disease. |
Tuesday, March 7, 2023 10:00AM - 10:12AM |
F06.00007: Reconstructing Chromatin Structural Connectivity from Sparse Locus Tracking Experiments Using Polymer Dynamics Modeling Sayantan Dutta, Jude Lee, Liang-Fu Chen, Alistair N Boettiger, Andrew J Spakowitz Chromatin dynamics is key to understanding a number of biological processes such as transcriptional regulation that is linked to establishment of cellular identity. However, visualization of chromatin is mostly limited to live imaging of a few fluorescently labeled chromosomal segments (or loci) or high-resolution reconstruction of multiple loci from a single time frame. In this study, we present an exact analytical framework that provides a probabilistic description of the time evolution of a flexible polymer structure given its structure at an earlier time point. Using this framework, we propose an algorithm that tracks the polymer configuration from live images of chromatin marked with fluorescent markers. Our theory identifies the time resolution of microscopy for a given spacing of markers, where the algorithm will track the polymer with high confidence, and we demonstrate the feasibility of our algorithm using synthetic data generated from numerical simulations. We then leverage experimental locus-tracking measurements as a basis for interpreting the strength of our modeling approach. Altogether, this study provides a computational approach founded on polymer physics to describe the dynamic configuration of biopolymers generated with increasingly sophisticated and high-resolution experimental tools. |
Tuesday, March 7, 2023 10:12AM - 10:24AM Author not Attending |
F06.00008: A multicolour polymer model for the prediction of 3D structure and transcription in human chromatin Giuseppe Negro, Massimiliano Semeraro, Antonio Suma, Giuseppe Gonnella, Peter R Cook, Davide Marenduzzo Within each human cell three types of RNA polymerases and a panoply of different transcription factors bind chromatin to simultaneously determine 3D chromosome structure and cells transcriptional program. In some cases different types of proteins segregate to form specialised transcription factories, while in others they mix and gather together, binding the same chromatin regions. We introduce a polymer model which accounts for multiple types -- or ``colours'' -- of DNA-binding proteins. Molecular dynamics simulations shows a good agreement with experimental data, and the appearance of stable segregated/specialised and mixed clusters suggesting a transition between the two types based on their size. |
Tuesday, March 7, 2023 10:24AM - 10:36AM |
F06.00009: Structural Integrity and Relaxation Dynamics of Axially Stressed Chromosomes Sumitabha Brahmachari, Benjamin S Ruben, Vinicius Contessoto, Ryan R Cheng, Antonio B Oliviera Jr., Michele Di Pierro, Jose N Onuchic Micromechanical studies of mitotic chromosomes have revealed them to be remarkably extensible objects and informed early models of mitotic chromosome organization. We use a data-driven, coarse-grained polymer modeling approach, capable of generating ensembles of chromosome structures that are quantitatively consistent with experiments, to explore the relationship between the spatial organization of individual chromosomes and their emergent mechanical properties. In particular, we investigate the mechanical properties of our model chromosomes by axially stretching them. Simulated stretching led to a linear force-extension curve for small strain, with mitotic chromosomes behaving about ten-fold stiffer than interphase chromosomes. Studying the relaxation dynamics we found that chromosomes are viscoelastic solids, with a highly liquid-like, viscous behavior in interphase that becomes solid-like in mitosis. This emergent mechanical stiffness in our model originates from lengthwise compaction, an effective potential capturing the activity of loop-extruding SMC complexes. Chromosomes denature under large strains via unraveling, which is characterized by the opening of large-scale folding patterns. By quantitatively exploring the mechanical properties of chromosome structural features responsible for various patterns observed in ensemble-averaged contact (HiC) maps, our model provides a nuanced understanding of in vivo mechanics of chromosomes. |
Tuesday, March 7, 2023 10:36AM - 10:48AM |
F06.00010: Understanding the physical processes behind DNA-DNA proximity ligation assays Bernardo J Zubillaga Herrera, Amit Das, Ailun Wang, Michele Di Pierro In the last decade, DNA-DNA proximity ligation assays opened up powerful new ways to study the 3D organization of genomes, and have rapidly become a mainstay experimental technology. Yet many aspects of these experiments are still poorly understood. We study the inner workings of DNA-DNA proximity ligation assays by means of numerical experiments and theoretical modeling. Chromosomes are modeled at nucleosome resolution and evolved in time via molecular dynamics. A virtual Hi-C experiment is performed, in silico, reproducing the different steps of the Hi-C protocol. The virtual Hi-C protocol includes crosslinking of chromatin to an underlying proteic matrix, the action of restriction enzymes cutting DNA, and the subsequent ligation of DNA open ends which are found in proximity. The protocol is performed on ensembles of different structures as well as individual structures. Simulations enable the construction of ligation maps for the virtual Hi-C experiments and the calculation of ligation probabilities as functions of both genomic and Euclidean distance, as well as statistics of self-ligations or cyclization. Our results allow close examination of the features and pitfalls of the Hi-C methodology, as well as the ensuing data processing. Additionally, the methods developed allow characterizing how the many variables present in the experimental protocol affect the quality of results. |
Tuesday, March 7, 2023 10:48AM - 11:00AM |
F06.00011: Engineering CRISPR/dCas based toggle switches in Escherichia coli Yasu Xu Synthetic biology is a field that leverages genetic engineering methods to create modular components and novel biological interactions inside microorganisms. An example of this is the creation of bistable genetic circuits called toggle switches inside E. coli bacteria which can effectively store 1 bit of information in a living organism. While current implementations of genetic toggle switches are typically based on a LacI and TetR repressor pairs, these promoter-repressor pairs suffer from low orthogonality and limited programmability. Recent advances in synthetic biology have highlighted the advantages of using of catalytically 'dead' version of Cas proteins that can selectively bind to specific DNA sequences to create logic ON/OFF switches (Specht et al. 2020, Rouches et al. 2022). In this work, we first develop a thermodynamic model to investigate parameters that affects the bistability of toggle switches based on CRISPR binding. We later use a massively parallel assay called X-seq to efficiently characterize these parameters for hundreds of toggle-switch constructs. Additionally, to visualize the kinetic features of our CRISPR-based toggle switch constructs, we also took advantage of single-cell imaging technique and a microfulidic chip platform to demonstrate the actuation dynamics of a toggle switch in real-time. Our results also uncover different classes of activity that result from modulating the growth rate of the bacteria by supplying minimal and rich media. |
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