Biology subjects

El-Samad, H.

Publications and source records attributed to El-Samad, H..

At least 19 recordsLinked to original sources

bioRxiv2026

A Physics-based Model Reveals Mechanisms of Epigenetic Memory

In multi-cellular eukaryotic organisms, cell type and specific functional identity are defined by the epigenetic patterning of chemical modifications to DNA and chromatin that modulate the expression and silencing of specific genes. When a cell divides, histones containing important epigenetic marks are distributed between the two daughter strands leading to a temporary dilution of epigenetic information and cell identity. In this work we introduce a physics-based model of epigenetic memory that explains how cells restore and maintain H3K9me3 and H3K27me3 histone methylation patterning after cell division. We demonstrate that emergence and maintenance of the epigenetic program is driven by an evolved mechanism that makes use of the biophysics of polymers, phase condensates and enzymatic activity. We validate our model via genome-wide epigenetic time-course simulation and comparison to experimental epigenetic data from multiple donors, multiple cell types, and for multiple epigenetic marks. Finally, we use our model as a conceptual framework to understand cellular reprogramming by hypothesizing that these processes first contend with and later utilize somatic epigenetic maintenance programs.

biophysics↗

bioRxiv2025

Macroscopic Analyses of RNA-Seq Data to Reveal Chromatin Modifications in Aging and Disease

Regulation of gene expression is fundamental for proper cellular function, and is constrained by the local chromatin environment of each gene, which varies spatially along the chromosome and is shaped by epigenetic modifications. Epigenetic modifications induce changes in the local chromatin structure, which can influence gene expression, by affecting the accessibility of DNA to transcription factors. Such changes are particularly relevant in aging and genetic disorders like Hutchinson-Gilford Progeria Syndrome (HGPS) and Werner Syndrome (WRN), where altered chromatin structure contributes to disease pathology. In this study, we analyze RNA-seq data using macroscopic metrics designed to be explicitly sensitive to chromatin modifications. The first metric, intra-chromosomal gene correlation length, measures spatial correlations in gene expressions along the chromosome. The second metric employs an energy landscape model based on the Arrhenius equation to estimate the energetic barriers associated with chromatin state transitions. We apply these metrics to various aging-related datasets, demonstrating their sensitivity to changes in the chromatin structure and the interpretability of the resulting outputs. The intra-chromosomal gene correlation length is particularly effective in quantifying changes in RNA-seq profiles due to increased chromatin accessibility during aging (and conversely, reduced accessibility due to treatment). This metric not only accurately distinguishes cell states, but also provides insight into the direction of aging. For instance, our observations on the effects of anti-sense oligonucleotide (ASO) treatment align with the existing literature, demonstrating that ASO partially restores chromatin structure in diseased cells. They additionally quantify the more pronounced effects in HGPS compared to WRN. The barrier energy landscape further extends this capability by offering a framework for understanding the progressive degradation of the regulatory mechanisms. Together, these metrics provide robust screening tools that enhance our ability to exploit common measurements such as RNA-seq to derive new phenotypes such as chromatin dynamics on aging and disease, offering an alternative perspective that complements traditional analytical techniques and enriches our understanding of cellular states.

bioinformatics↗

bioRxiv2024

Small molecule and cell contact-inducible systems for controlling expression and differentiation in stem cells

Synthetic developmental biology uses engineering approaches to understand multicellularity with goals ranging from recapitulating development to building synthetic organisms. Current approaches include engineering multicellular patterning, controlling differentiation, and implementing cooperative cellular behaviors in model systems. Synthetic biology tools enable these pursuits with genetic circuits that drive customized responses to arbitrary stimuli, synthetic receptors that enable orthogonal signaling channels, and light- or drug-inducible systems that enable precise spatial and temporal control of cell function. Mouse embryonic stem cells (mESCs) offer a well-studied and genetically tractable pluripotent chassis for pursuing synthetic development questions however, there is minimal characterization of existing synthetic biology tools in mESCs and we lack genetic toolkits for rapid iterative engineering of synthetic development workflows. Here, we began to address this challenge by characterizing small molecule and cell contact-inducible systems for gene expression in and differentiation of mESCs. We show that small molecule and cell-contact inducible systems work reliably and efficiently for controlling expression of arbitrary genetic payloads. Furthermore, we show that these systems can drive direct differentiation of mESCs into neurons. Each of these systems can readily be used on their own or in combination, opening many possibilities for studying developmental principles with high precision.

synthetic biology↗

bioRxiv2024

Degron-based bioPROTACs for controlling signaling in CAR T cells

Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in pre-clinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous, cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit is able to disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are a powerful tool for expanding the cell engineering toolbox for CAR T cells.

synthetic biology↗

bioRxiv2024

De novo-designed minibinders expand the synthetic biology sensing repertoire

Synthetic and chimeric receptors capable of recognizing and responding to user-defined antigens have enabled "smart" therapeutics based on engineered cells. These cell engineering tools depend on antigen sensors which are most often derived from antibodies. Advances in the de novo design of proteins have enabled the design of protein binders with the potential to target epitopes with unique properties and faster production timelines compared to antibodies. Building upon our previous work combining a de novo-designed minibinder of the Spike protein of SARS-CoV-2 with the synthetic receptor synNotch (SARSNotch), we investigated whether minibinders can be readily adapted to a diversity of cell engineering tools. We show that the Spike minibinder LCB1 easily generalizes to a next-generation proteolytic receptor SNIPR that performs similarly to our previously reported SARSNotch. LCB1-SNIPR successfully enables the detection of live SARS-CoV-2, an improvement over SARSNotch which can only detect cell-expressed Spike. To test the generalizability of minibinders to diverse applications, we tested LCB1 as an antigen sensor for a chimeric antigen receptor (CAR). LCB1-CAR enabled CD8+ T cells to cytotoxically target Spike-expressing cells. We further demonstrate that two other minibinders directed against the clinically relevant epidermal growth factor receptor are able to drive CAR-dependent cytotoxicity with efficacy similar to or better than an existing antibody-based CAR. Our findings suggest that minibinders represent a novel class of antigen sensors that have the potential to dramatically expand the sensing repertoire of cell engineering tools.

synthetic biology↗

bioRxiv2022

Optogenetic control of RelA reveals effect of transcription factor dynamics on downstream gene expression

Many transcription factors (TFs) translocate to the nucleus with varied dynamic patterns in response to different inputs. A notable example of such behavior is RelA, a subunit of NF-{kappa}B, which translocates to the nucleus with either pulsed or sustained dynamics, depending on the stimulus. Our understanding of how these dynamics are interpreted by downstream genes has remained incomplete, partly because ubiquitously used environmental inputs activate other transcriptional regulators in addition to RelA. Here, we use an optogenetic tool, CLASP (controllable light-activated shuttling and plasma membrane sequestration), to control RelA spatiotemporal dynamics in mouse fibroblasts and quantify their effect on downstream genes using RNA-seq. Using RelA-CLASP, we show for the first time that nuclear translocation of RelA, without post-translational modifications or activation of other transcriptional regulators, is sufficient to activate downstream genes. Furthermore, we find that TNF, a common endogenous input, regulates many genes independently of RelA, and that this gene regulation is different from that induced by RelA-CLASP. Genes responsive to RelA-CLASP show a wide range of dynamics in response to a constant RelA input. We use a simple promoter model to recapitulate these diverse dynamic responses, as well as data collected in response to a pulsed RelA-CLASP input, and extract features of many RelA-responsive promoters. We also pinpoint many genes for which more complex models, involving feedback or multi-step promoters, may be needed to explain their response to constant and pulsed TF inputs. This study introduces a new robust tool for studying mammalian transcriptional regulation and demonstrates the power of optogenetic tools in dissecting the quantitative features of important cellular pathways.

systems biology↗

bioRxiv2021

Sentinel cells enable genetic detection of SARS-CoV-2 Spike protein

The COVID-19 pandemic has demonstrated the need for exploring different diagnostic and therapeutic modalities to tackle future viral threats. In this vein, we propose the idea of sentinel cells, cellular biosensors capable of detecting viral antigens and responding to them with customizable responses. Using SARS-CoV-2 as a test case, we developed a live cell sensor (SARSNotch) using a de novo-designed protein binder against the SARS-CoV-2 Spike protein. SARSNotch is capable of driving custom genetically-encoded payloads in immortalized cell lines or in primary T lymphocytes in response to purified SARS-CoV-2 Spike or in the presence of Spike-expressing cells. Furthermore, SARSNotch is functional in a cellular system used in directed evolution platforms for development of better binders or therapeutics. In keeping with the rapid dissemination of scientific knowledge that has characterized the incredible scientific response to the ongoing pandemic, we extend an open invitation for others to make use of and improve SARSNotch sentinel cells in the hopes of unlocking the potential of the next generation of smart antiviral therapeutics.

synthetic biology↗

bioRxiv2020

CoRa -A general approach for quantifying biological feedback control

Feedback control is a fundamental underpinning of life, underlying homeostasis of biological processes at every scale of organization, from cells to ecosystems. The ability to evaluate the contribution and limitations of feedback control mechanisms operating in cells is a critical step for understanding and ultimately designing feedback control systems with biological molecules. Here, we introduce CoRa -or Control Ratio-, a general framework that quantifies the contribution of a biological feedback control mechanism to adaptation using a mathematically controlled comparison to an identical system that does not contain the feedback. CoRa provides a simple and intuitive metric with broad applicability to biological feedback systems.

systems biology↗

bioRxiv2020

Accurate prediction of genetic circuit behavior requires multidimensional characterization of parts

Mathematical models can aid the design of genetic circuits, but may yield inaccurate results if individual parts are not modeled at the appropriate resolution. To illustrate the importance of this concept, we study transcriptional cascades consisting of two inducible synthetic transcription factors connected in series. Despite the simplicity of this design, we find that accurate prediction of circuit behavior requires mapping the dose responses of each circuit component along the dimensions of both its expression level and its inducer concentration. With such multidimensional characterizations, we were able to computationally explore the behavior of 16 different circuit designs. We experimentally verified a subset of these predictions and found substantial agreement. This method of biological part characterization enables the use of models to identify (un)desired circuit behaviors prior to experimental implementation, thus shortening the design-build-test cycle for more complex circuits.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsiSynTFinducible synthetic transcription factorYFPyellow fluorescent proteinGEMGal4 DNA binding domain, estradiol ligand binding domain, Msn2 activating domainZ3PMZ3 DNA binding domain, progesterone ligand binding domain, Msn2 activating domainZ4EMZ4 DNA binding domain, estradiol ligand binding domain, Msn2 activating domainView Full Text

synthetic biology↗

bioRxiv2020

TuNR: Orthogonal Control of Mean and Variability of Endogenous Genes in a Human Cell Line

Stochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineered a synthetic circuit to independently modulate mean expression and heterogeneity of transgenes and endogenous human genes. The circuit, a Tunable Noise Rheostat (TuNR), consists of a transcriptional cascade of two inducible transcriptional activators, where the output mean and variance can be modulated by two orthogonal small molecule inputs. In this fashion, different combinations of the inputs can achieve the same mean but with different population variability. With TuNR, we achieve low basal expression, over 1000-fold expression of a transgene product, and up to 7-fold induction of the endogenous gene NGFR. Importantly, for the same mean expression level, we are able to establish varying degrees of heterogeneity in expression within an isogenic population, thereby decoupling gene expression noise from its mean. TuNR is therefore a modular tool that can be used in mammalian cells to enable direct interrogation of the implications of cell-to-cell variability.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

Stress-Induced Transient Cell Cycle Arrest Coordinates Metabolic Resource Allocation to Balance Adaptive Tradeoffs

The ability of a cell to mount a robust response to an environmental perturbation is paramount to its survival. While cells deploy a spectrum of specialized counter-measures to deal with stress, a near constant feature of these responses is a down regulation or arrest of the cell cycle. It has been widely assumed that this modulation of the cell cycle is instrumental in facilitating a timely response towards cellular adaptation. Here, we investigate the role of cell cycle arrest in the hyperosmotic shock response of the model organism S. cerevisiae by deleting the osmoshock-stabilized cell cycle inhibitor Sic1, thus enabling concurrent stress response activation and cell cycle progression. Contrary to expectation, we found that removal of stress-induced cell cycle arrest accelerated the adaptive response to osmotic shock instead of delaying it. Using a combination of time-lapse microscopy, genetic perturbations and quantitative mass spectrometry, we discovered that unabated cell cycle progression during stress enables the liquidation of internal glycogen stores, which are then shunted into the osmotic shock response to fuel a faster adaptation. Therefore, osmo-adaptation in wild type cells is delayed because cell cycle arrest diminishes the ability of the cell to tap its glycogen stores. However, acceleration of osmo-adaptation in mutant cells that do not arrest comes at the cost of acute sensitivity to a subsequent osmo-stress. This indicates that despite the ostensible advantage faster adaptation poses, there is a trade-off between the short-term benefit of faster adaptation and the vulnerability it poses to subsequent insults. We suggest that cell cycle arrest acts as a carbon flux valve to regulate the amount of material that is devoted to osmotic shock, balancing short term adaptation with long-term robustness.

systems biology↗

bioRxiv2019

Paralogs in the PKA regulon traveled different evolutionary routes to divergent expression in budding yeast.

Functional divergence of duplicate genes, or paralogs, is an important driver of novelty in evolution. In the model yeast Saccharomyces cerevisiae, there are 547 paralog gene pairs that survive from an interspecies Whole Genome Hybridization (WGH) that occurred [~]100MYA. Many WGH paralogs (or ohnologs) are known to have differential expression during the yeast Environmental Stress Response (ESR), of which Protein Kinase A (PKA) is a major regulator. While investigating the transcriptional response to PKA inhibition in S. cerevisiae, we discovered that approximately 1/6th (91) of all ohnolog pairs were differentially expressed with a striking pattern. One member of each pair tended to have low basal expression that increased upon PKA inhibition, while the other tended to have high but unchanging expression. Examination of PKA inhibition data in the pre-WGH species K. lactis and PKA-related stresses in other budding yeasts indicated that unchanging expression in response to PKA inhibition is likely to be the ancestral phenotype prior to duplication. Analysis of promoter sequences of orthologs of gene pairs that are differentially expressed in S. cerevisiae further revealed that the emergence of PKA-dependence took different evolutionary routes. In some examples, regulation by PKA and differential expression appears to have arisen following the WGH, while in others, regulation by PKA appears to have arisen in one of the two parental lineages prior to the WGH. More broadly, our results illustrate the unique opportunities presented by a WGH event for generating functional divergence by bringing together two parental lineages with separately evolved regulation into one species. We propose that functional divergence of two ohnologs can be facilitated through such regulatory divergence, which can persist even when functional differences are erased by gene conversion.

evolutionary biology↗

bioRxiv2019

Biphasic Response of Protein Kinase A to Cyclic Adenosine Monophosphate Triggers Distinct Epithelial Phenotypes

Despite the large diversity of the proteins involved in cellular signaling, many intracellular signaling pathways converge onto one of only dozens of small molecule second messengers. Cyclic adenosine monophosphate (cAMP), one of these second messengers, is known to regulate activity of both Protein Kinase A (PKA) and the Extracellular Regulated Kinase (ERK), among other signaling pathways. In its role as an important cellular signaling hub, intracellular cAMP concentration has long been assumed to monotonically regulate its known effectors. Using an optogenetictool that can introduce precise amounts of cAMP in MDCKI cells, we identify genes whose expression changes biphasically with monotonically increasing cAMP levels. By examining the behavior of PKA and ERK1/2 in the same dose regime, we find that these kinases also respond biphasically to increasing cAMP levels, with opposite phases. We reveal that this behavior results from an elaborate integration by PKA of many cellular signals triggered by cAMP. In addition to the direct activation of PKA, cAMP also modulates the activity of p38 and ERK, which then converge to inhibit PKA. These interactions and their ensuing biphasic PKA profile have important physiological repercussions, influencing the ability of MDCKI cells to proliferate and form acini. Our data, supported by computational modeling, synthesize a set of network interconnections involving PKA and other important signaling pathways into a model that demonstrates how cells can capitalize on signal integration to create a diverse set of responses to cAMP concentration and produce complex input-output relationships.

systems biology↗

bioRxiv2019

Optogenetic control reveals differential promoter interpretation of transcription factor nuclear translocation dynamics

The dynamic translocation of transcription factors (TFs) in and out of the nucleus is thought to encode information, such as the identity of a stimulus. A corollary is the idea that gene promoters can decode different dynamic TF translocation patterns. Testing this TF encoding/promoter decoding hypothesis requires tools that allow direct control of TF dynamics without the pleiotropic effects associated with general perturbations. In this work, we present CLASP (Controllable Light Activated Shuttling and Plasma membrane sequestration), a tool that enables precise, modular, and reversible control of TF localization using a combination of two optimized LOV2 optogenetic constructs. The first sequesters the cargo in the dark at the plasma membrane and releases it upon exposure to blue light, while light exposure of the second reveals a nuclear localization sequence that shuttles the released cargo to the nucleus. CLASP achieves minute-level resolution, reversible translocation of many TF cargos, large dynamic range, and tunable target gene expression. Using CLASP, we investigate the relationship between Crz1, a naturally pulsatile TF, and its cognate promoters. We establish that some Crz1 target genes respond more efficiently to pulsatile TF inputs than to continuous inputs, while others exhibit the opposite behavior. We show using computational modeling that efficient gene expression in response to short pulsing requires fast promoter activation and slow inactivation and that the opposite phenotype can ensue from a multi-stage promoter activation, where a transition in the first stage is thresholded. These data directly demonstrate differential interpretation of TF pulsing dynamics by different genes, and provide plausible models that can achieve these phenotypes.

systems biology↗

bioRxiv2019

A dataset to explore kinase control of environmental stress responsive transcription

Cells respond to changes in environmental conditions by activating signal transduction pathways and gene expression programs. Here we present a dataset to explore the relationship between environmental stresses, kinases, and global gene expression in yeast. We subjected 28 drug-sensitive kinase mutants to 10 environmental conditions in the presence of inhibitor and performed mRNA deep sequencing. With these data, we reconstructed canonical stress pathways and identified examples of crosstalk among pathways. The data also implicated numerous kinases in novel environment-specific roles. However, rather than regulating dedicated sets of target genes, individual kinases tuned the magnitude of induction of the environmental stress response (ESR) - a gene expression signature shared across the set of perturbations - in environment-specific ways. This suggests that the ESR integrates inputs from multiple sensory kinases to modulate gene expression and growth control. As an example, we provide experimental evidence that the high osmolarity glycerol pathway is a constitutive negative regulator of protein kinase A, a known inhibitor of the ESR. These results elaborate the central axis of cellular stress response signaling.

systems biology↗

bioRxiv2018

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods that permit the assembly of DNA circuits in mammalian cells are laborious, slow, expensive and mostly not permissive of rapid prototyping of constructs. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be easily mixed and matched to combinatorially assemble one transcriptional unit with different characteristics, or a hierarchy of transcriptional units weaved into complex circuits. MTK renders many cell engineering operations facile, as showcased by our ability to use the toolkit to generate single-integration landing pads, to create and deliver libraries of protein variants and sgRNAs, and to iterate through Cas9-based prototype circuits. As a biological proof of concept, we used the MTK to successfully design and rapidly construct in mammalian cells a challenging multicistronic circuit encoding the Ebola virus (EBOV) replication complex. This construct provides a non-infectious biosafety level 2 (BSL2) cellular assay for exploring the transcription and replication steps of the EBOV viral life cycle in its host. Its construction also demonstrates how the MTK can enable important and time sensitive applications such as the rapid testing of pharmacological inhibitors of emerging BSL4 viruses that pose a major threat to human health.

synthetic biology↗

bioRxiv2018

Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.

synthetic biology↗

bioRxiv2018

Design and analysis of a Proportional-Integral-Derivative controller with biological molecules

The ability of cells to regulate their function through feedback control is a fundamental underpinning of life. The capability to engineer de novo feedback control with biological molecules is ushering in an era of robust functionality for many applications in biotechnology and medicine. To fulfill their potential, feedback control strategies implemented with biological molecules need to be generalizable, modular and operationally predictable. Proportional-Integral-Derivative (PID) control fulfills this role for technological systems and is a commonly used strategy in engineering. Integral feedback control allows a system to return to an invariant steady-state value after step disturbances, hence enabling its robust operation. Proportional and derivative feedback control used with integral control help sculpt the dynamics of the return to steady-state following perturbation. Recently, a biomolecular implementation of integral control was proposed based on an antithetic motif in which two molecules interact stoichiometrically to annihilate each others function. In this work, we report how proportional and derivative implementations can be layered on top of this integral architecture to achieve a biochemical PID control design. We illustrate through computational and analytical treatments that the addition of proportional and derivative control improves performance, and discuss practical biomolecular implementations of these control strategies.

synthetic biology↗