Biology subjects

Backman, V.

Publications and source records attributed to Backman, V..

At least 19 recordsLinked to original sources

bioRxiv2026

Heterochromatin protein 1α acts as an essential stabilizer of chromatin packing domains

Background: The spatial organization of the human genome emerges from an enzyme-guided self-assembly process in which chromatin has a bidirectional relationship with transcription. Central to this architecture are chromatin packing domains, 50-200 nm structures exhibiting mass-scaling behavior. Nascent domains arise through transcription and cohesin/CTCF-mediated loop extrusion, while their maturation depends on conditions that permit differential access of epigenetic enzymes to their dense cores and continuously lower density periphery, resulting in the accumulation of heterochromatic marks in domain cores and euchromatic marks outside of the domain cores. In turn, packing domains support transcription by creating an "ideal zone" peripherally to their cores due to a nonmonotonic dependence of transcription rate on local chromatin density. Results: We investigated the role of heterochromatin protein 1 alpha (HP1), a key chromatin crosslinking factor with affinity to H3K9me3 marks, in maintaining packing domain organization. Paradoxically, short-term HP1 degradation resulted in overall downregulation of genes, contrasting with the canonical view that repression of heterochromatin allows chromatin to become more accessible for transcription. While some packing domains persisted, heterochromatic cores of dense packing domains were disrupted. When exposed to hypoxia, HP1-depleted cells exhibited a dampened transcriptional response, showcasing that when domain maturation is disrupted, stress response pathways are not effectively activated. Conclusions: This work highlights HP1 crosslinking as an essential component of domain maturation, as HP1 loss disrupts domain geometry and inhibits efficient transcriptional reprogramming. Our findings suggest an intriguing perspective on the potential relationship between chromatin architecture and gene regulation that could inform chromatin engineering-based treatments.

genomics↗

bioRxiv2026

Genome-Wide Mapping of Major Histone Modifications Reveals Distinct Epigenetic Regulatory States in a Reef-Building Coral

Reef-building corals exhibit extensive physiological and transcriptional plasticity, yet the chromatin-level regulation of coral gene expression remains poorly characterized. Here, we generated the first genome-wide maps of major histone modifications in a reef-building coral by performing chromatin immunoprecipitation sequencing (ChIP-seq) in adult Pocillopora damicornis maintained under ambient conditions. We profiled two active marks, H3K4me3 and H3K27ac, and two repressive marks, H3K27me3 and H3K9me3, and integrated these maps with RNA sequencing (RNA-seq) to relate chromatin state to transcriptional output. As in other eukaryotes, H3K4me3 and H3K27ac were enriched around transcriptional start sites and positively associated with gene expression, whereas H3K27me3 and H3K9me3 showed broader enrichment patterns and were negatively associated with transcription. Partitioning highly expressed genes by promoter chromatin state revealed that promoters with strong H3K4me3/H3K27ac signal carried YY1-family motifs and served core cellular functions, while a second group enriched for cell-surface receptors and proteolysis lacked these active promoter marks and motif repertoire, despite similar transcript abundance. Repressive marks also distinguished functional genomic states: H3K27me3 was enriched over lowly expressed receptor-like and developmental gene classes, whereas H3K9me3 was prominent at transposable element-like loci and broad non-genic regions. We further identified H3K27ac-enriched, H3K4me3-low candidate enhancer-like elements proximal to core promoters, many near genes with transcription factor-related functions. Together, these results define a baseline chromatin-state landscape in reef-building corals, offering a resource for validating targeted chromatin assays and a foundation for future studies of coral gene regulation across environmental, developmental, and symbiotic contexts.

genomics↗

bioRxiv2026

Bounded ecological memory in coral reefs: divergent demographic pathways under recurrent heatwaves and collapse under extreme thermal stress

Anthropogenic warming exposes coral reefs to recurrent marine heatwaves (MHWs), yet responses to comparable thermal stress remain highly variable. A central challenge is determining whether reduced bleaching reflects acclimatization-like persistence of existing colonies or mortality-driven filtering that produces demographically depleted assemblages. Using colony-level surveys across the Florida Reef Tract (2014-2023), we show bleaching sensitivity declined from 2014 through 2022, consistent with ecological memory. A spatial matched-event framework integrating bleaching severity and adult colony density resolved these reduced-bleaching outcomes into two distinct pathways: demographic persistence (stable/increasing density) and mortality-filtered tolerance (declining density). Persistence pathways were maintained under predictable exposure regimes characterized by lower interannual thermal variability. This persistence was driven by weedy life-history taxa (e.g., Porites spp.) replacing historical framework builders, indicating stability reflects ecological reassembly rather than uniform increases in thermal tolerance. Under the unprecedented extremes of the 2023 MHW, this acclimatization-like buffering broke down, causing widespread sensitization and collapse of previously persistent networks. These results support a bounded ecological memory framework; prior exposure reduces bleaching sensitivity under predictable thermal regimes but fails under extreme heat stress. Consequently, modern refugia are best defined by adult standing stock retention representing systems undergoing selective ecological reassembly rather than full functional recovery.

ecology↗

bioRxiv2026

One Chromatin, Many Structures: From Ensemble Contact Maps to Single-Cell 3D Organization

Understanding how chromatin folds in three dimensions remains challenging because most experimental assays capture low-dimensional projections of an underlying, highly heterogeneous polymer. Here we present an ensemble-based interpretive framework built on the previously introduced Self-Returning Excluded Volume (SR-EV) model, a minimal generator of nucleosome-resolution chromatin conformations based on stochastic return rules and excluded-volume geometry. Despite its simplicity, SR-EV reproduces key experimental signatures across scales: heterogeneous nanoscale packing domains resembling ChromEMT and ChromSTEM observations, sparse and highly variable single-configuration contact patterns analogous to single-cell chromosome conformation capture (Hi-C), and robust ensemble-level contact enrichment consistent with topologically associating domains (TADs). In this framework, Hi-C loop and TAD signatures are interpreted as ensemble-level statistical enrichments rather than invariant features of single-cell conformations. SR-EV is explicitly designed to generate large ensembles of complete three-dimensional chromatin configurations that can be projected consistently onto two-dimensional contact maps and one-dimensional genomic profiles. By introducing architectural-protein effects only through ensemble selection rather than explicit forces, SR-EV supports a separation between intrinsic polymer geometry and regulatory bias and suggests that TAD-like features can emerge as statistical enrichments rather than deterministic three-dimensional structures. Coordination number and probe-based accessibility computed directly from SR-EV provide a unified link between three-dimensional packing, two-dimensional contact maps, and one-dimensional genomic profiles. Together, these results establish SR-EV as a minimal and physically grounded reference framework for interpreting how heterogeneous chromatin ensembles give rise to multimodal experimental observables, while remaining consistent with the fact that chromatin organization is realized in individual cells. SIGNIFICANCEChromatin domains, boundaries, and contact enrichments are often interpreted as fixed structural entities, even though most experimental measurements average over large and heterogeneous cell populations. The SR-EV framework shows that many of these features can be understood as emerging from minimal geometric rules combined with ensemble-level bias, without requiring explicit molecular interactions or deterministic folding mechanisms. By distinguishing single-configuration heterogeneity from ensemble-level statistical organization - including the emergence of packing domains- SR-EV supports an interpretation in which chromatin organization is realized in individual cells but must be analyzed through ensembles. This perspective clarifies the probabilistic nature of genome architecture and provides a tractable reference framework for interpreting multimodal genomic and imaging data.

biophysics↗

bioRxiv2024

Leveraging chromatin packing domains to target chemoevasion in vivo

Cancer cells exhibit a remarkable resilience to cytotoxic stress, often adapting through transcriptional changes linked to alterations in chromatin structure. In several types of cancer, these adaptations involve epigenetic modifications and restructuring of topologically associating domains (TADs). However, the underlying principles by which chromatin architecture facilitates such adaptability across different cancers remain poorly understood. To investigate the role of chromatin in this process, we developed a physics-based mechanistic model that connects chromatin organization to cell fate decisions, specifically survival following chemotherapy. Our model builds on the observation that chromatin forms packing domains, which influence transcriptional efficiency through macromolecular crowding. The model accurately predicts chemoevasion in vitro, suggesting that changes in packing domains affect the likelihood of survival. Consistent results across diverse cancer types indicate that the model captures fundamental principles of chromatin-mediated adaptation, independent of the specific cancer or chemotherapy mechanisms involved. Based on these insights, we hypothesized that compounds capable of modulating packing domains, termed Transcriptional Plasticity Regulators (TPRs), could prevent cellular adaptation to chemotherapy. Using live-cell chromatin imaging, we conducted a compound screen that identified several TPRs which synergistically enhanced chemotherapyinduced cell death. The most effective TPR significantly improved therapeutic outcomes in a patient-derived xenograft (PDX) model of ovarian cancer. These findings underscore the central role of chromatin in cellular adaptation to cytotoxic stress and present a novel framework for enhancing cancer therapies, with broad potential across multiple cancer types.

biophysics↗

bioRxiv2024

The Impact of Charge Regulation and Ionic Intranuclear Environment on the Nucleosome Core Particle

We theoretically investigate how the intranuclear environment influences the charge of a nucleosome core particle (NCP) - the fundamental unit of chromatin consisting of DNA wrapped around a core of histone proteins. The molecular-based theory explicitly considers the size, shape, conformations, charges, and chemical states of all molecular species - thereby linking the structural state with the chemical/charged state of the system. We investigate how variations in monovalent and divalent salt concentrations, as well as pH, affect the charge distribution across different regions of an NCP and quantify the impact of charge regulation. The effective charge of an NCP emerges from a delicate and complex balance involving the chemical dissociation equilibrium of the amino acids and the DNA-phosphates, the electrostatic interaction between them, and the translational entropy of the mobile solution ions, i.e., counter ion release and ion condensation. From our results, we note the significant effect of divalent magnesium ions on the charge and electrostatic energy as well as the counterion cloud that surrounds an NCP, as a function of magnesium concentration, charge neutralization, and even charge inversion is predicted - in line with experimental observation of NCPs. The strong Mg-dependence of the nucleosome charge state arises from ion bridges between two DNA-phosphates and one Mg + ion. We demonstrate that to describe and predict the charged state of an NCP properly, it is essential to consider molecular details, such as DNA-phosphate ion condensation and the acid-base equilibrium of the amino acids that comprise the core histone proteins.

biophysics↗

bioRxiv2024

mRNA expression is co-regulated by non-nucleolar RNA polymerase I

The relationship between gene transcription and chromatin organization is an area of active study. Due to its role in mRNA synthesis, many studies have focused on the regulaton of RNA polymerase II (Pol-II) function by supranucleosomal structure and vice-versa. In contrast, there is little work on the function of RNA polymerase I (Pol-I) in non-nucleolar chromatin. Prior work has shown that Pol-I engages with components of Pol-II on rDNA, but its role in global transcription and chromatin structure beyond the nucleolus has largely been ignored. By pairing auxin-inducible degron technology with nanoscopic imaging, RNA-Seq, and Hi-C, we found that Pol-I and Pol-II co-regulate conformationally defined chromatin domains and mRNA synthesis. Mechanistically, Pol-I maintains the positioning of intronic and intergenic chromatin within domains for the proper expression of exon elements. Consequently, Pol-I loss disrupts genome connectivity, in situ chromatin domains, and the expression of mRNA, genome-wide.

genomics↗

bioRxiv2024

Three-color single-molecule localization microscopy in chromatin

Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales, from nanometers to micrometers. Single molecule localization microscopy (SMLM) methods, such as STORM, are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization. Multi-label imaging of chromatin is necessary to unpack its structural complexity. However, these efforts are challenged by the high-density nuclear environment, which can affect antibody binding affinities, diffusivity and non-specific interactions. Optimizing buffer conditions, fluorophore stability, and antibody specificity is crucial for achieving effective antibody conjugates. Here, we demonstrate a sequential immunolabeling protocol that reliably enables three-label studies within the dense nuclear environment. This protocol couples multiplexed localization datasets with a robust analysis algorithm, which utilizes localizations from one target as seed points for distance, density and multi-label joint affinity measurements to explore complex organization of all three targets. Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories, but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters. This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.

bioengineering↗

bioRxiv2024

Deep Learning-driven Automatic Nuclei Segmentation of Label-free Live Cell Chromatin-sensitive Partial Wave Spectroscopic Microscopy Imaging

Chromatin-sensitive Partial Wave Spectroscopic (csPWS) microscopy offers a non-invasive glimpse into the mass density distribution of cellular structures at the nanoscale, leveraging the spectroscopic information. Such capability allows us to analyze the chromatin structure and organization and the global transcriptional state of the cell nuclei for the study of its role in carcinogenesis. Accurate segmentation of the nuclei in csPWS microscopy images is an essential step in isolating them for further analysis. However, manual segmentation is error-prone, biased, time-consuming, and laborious, resulting in disrupted nuclear boundaries with partial or over-segmentation. Here, we present an innovative deep-learning-driven approach to automate the accurate nuclei segmentation of label-free live cell csPWS microscopy imaging data. Our approach, csPWS-seg, harnesses the Convolutional Neural Networks-based U-Net model with an attention mechanism to automate the accurate cell nuclei segmentation of csPWS microscopy images. We leveraged the structural, physical, and biological differences between the cytoplasm, nucleus, and nuclear periphery to construct three distinct csPWS feature images for nucleus segmentation. Using these images of HCT116 cells, csPWS-seg achieved superior performance with a median Intersection over Union (IoU) of 0.80 and a Dice Similarity Coefficient (DSC) score of 0.88. The csPWS-seg overcame the segmentation performance over the baseline U-Net model and another attention-based model, SE-U-Net, marking a significant improvement in segmentation accuracy. Further, we analyzed the performance of our proposed model with four loss functions: binary cross-entropy loss, focal loss, dice loss, and Jaccard loss. The csPWS-seg with focal loss provided the best results compared to other loss functions. The automatic and accurate nuclei segmentation offered by the csPWS-seg not only automates, accelerates, and streamlines csPWS data analysis but also enhances the reliability of subsequent chromatin analysis research, paving the way for more accurate diagnostics, treatment, and understanding of cellular mechanisms for carcinogenesis.

bioengineering↗

bioRxiv2024

Nuclear blebs are associated with destabilized chromatin packing domains

Disrupted nuclear shape is associated with multiple pathological processes including premature aging disorders, cancer-relevant chromosomal rearrangements, and DNA damage. Nuclear blebs (i.e., herniations of the nuclear envelope) have been induced by (1) nuclear compression, (2) nuclear migration (e.g., cancer metastasis), (3) actin contraction, (4) lamin mutation or depletion, and (5) heterochromatin enzyme inhibition. Recent work has shown that chromatin transformation is a hallmark of bleb formation, but the transformation of higher-order structures in blebs is not well understood. As higher-order chromatin has been shown to assemble into nanoscopic packing domains, we investigated if (1) packing domain organization is altered within nuclear blebs and (2) if alteration in packing domain structure contributed to bleb formation. Using Dual-Partial Wave Spectroscopic microscopy, we show that chromatin packing domains within blebs are transformed both by B-type lamin depletion and the inhibition of heterochromatin enzymes compared to the nuclear body. Pairing these results with single-molecule localization microscopy of constitutive heterochromatin, we show fragmentation of nanoscopic heterochromatin domains within bleb domains. Overall, these findings indicate that translocation into blebs results in a fragmented higher-order chromatin structure. SUMMARY STATEMENTNuclear blebs are linked to various pathologies, including cancer and premature aging disorders. We investigate alterations in higher-order chromatin structure within blebs, revealing fragmentation of nanoscopic heterochromatin domains.

bioengineering↗

bioRxiv2024

Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.

biophysics↗

bioRxiv2024

Chromatin packing domains persist after RAD21 depletion in 3D.

Understanding chromatin organization requires integrating measurements of genome connectivity and physical structure. Prior work demonstrates that RAD21 depletion results in the complete loss of topologically associated and loop domains on Hi-C, but the corresponding change in physical structure has not been studied using electron microscopy. Pairing chromatin scanning transmission electron tomography with Hi-C, we study the role of cohesin in regulating the spatially resolved, conformationally defined chromatin packing domains. We find that only 20% of packing domains are lost on electron microscopy upon RAD21 depletion with the effect primarily on small, poorly packed (nascent) domains. Overall, this contrasts with the prevailing understanding of genome regulation, indicating that while cohesin influences domain formation, non-cohesin mediated mechanisms predominantly regulate the 3D genomic physical structure.

genomics↗

bioRxiv2024

DNA density is a better indicator of a nuclear bleb than lamin B loss

Nuclear blebs are herniations of the nucleus that occur in diseased nuclei that cause nuclear rupture leading to cellular dysfunction. Chromatin and lamins are two of the major structural components of the nucleus that maintain its shape and function, but their relative roles in nuclear blebbing remain elusive. Lamin B is reported to be lost in blebs by qualitative data while quantitative studies reveal a spectrum of lamin B levels in nuclear blebs dependent on perturbation and cell type. Chromatin has been reported to be decreased or de-compacted in nuclear blebs, but again the data are not conclusive. To determine the composition of nuclear blebs, we compared the immunofluorescence intensity of lamin B and DNA in the main nucleus body and nuclear bleb across cell types and perturbations. Lamin B nuclear bleb levels varied drastically across MEF wild type and chromatin or lamins perturbations, HCT116 lamin B1-GFP imaging, and human disease model cells of progeria and prostate cancer. However, DNA concentration was consistently decreased to about half that of the main nucleus body across all measured conditions. Using Partial Wave Spectroscopic (PWS) microscopy to measure chromatin density in the nuclear bleb vs body we find similar results that DNA is consistently less dense in nuclear blebs. Thus, our data spanning many different cell types and perturbations supports that decreased DNA is a better marker of a nuclear bleb than lamin B levels that vary widely.

cell biology↗

bioRxiv2023

Depletion of lamins B1 and B2 alters chromatin mobility and induces differential gene expression by a mesoscale-motion dependent mechanism

BACKGROUNDB-type lamins are critical nuclear envelope proteins that interact with the 3D genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron (AID) technology. RESULTSPaired with a suite of novel technologies, live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, in situ Hi-C, and CRISPR-Sirius, we demonstrate that lamin B1 and lamin B2 depletion transforms chromatin mobility, heterochromatin positioning, gene expression, and loci-positioning with minimal disruption to mesoscale chromatin folding. Using the AID system, we show that the disruption of B-lamins alters gene expression both within and outside lamin associated domains, with distinct mechanistic patterns depending on their localization. Critically, we demonstrate that chromatin dynamics, positioning of constitutive and facultative heterochromatic markers, and chromosome positioning near the nuclear periphery are significantly altered, indicating that the mechanism of action of B-type lamins is derived from their role in maintaining chromatin dynamics and spatial positioning. CONCLUSIONSOur findings suggest that the mechanistic role of B-type lamins is stabilization of heterochromatin and chromosomal positioning along the nuclear periphery. We conclude that degrading lamin B1 and lamin B2 has several functional consequences related to both structural disease and cancer.

bioengineering↗

bioRxiv2023

Active Transcription and Epigenetic Reactions Synergistically RegulateMeso-Scale Genomic Organization

In interphase nuclei, chromatin is organized into interspersed dense domains with characteristic sizes, both in the nuclear interior and periphery. However, the quantitative impact of transcription and histone modifications on the size and distribution of these domains remains unclear. Here, we introduce a mesoscale theoretical model that investigates the relationship between heterochromatic domain sizes and loop extrusion rates from these domains. The model considers chromatin-chromatin and chromatin-lamina interactions, methylation and acetylation kinetics, and diffusion of epigenetic marks and nucleoplasm. Our model generates testable predictions that help reveal the biophysics underlying chromatin organization in the presence of transcription-driven loop extrusion. This process is kinetically captured through the conversion of heterochromatin to euchromatin in response to RNAPII activity. We discovered that a balance between diffusive and reactive fluxes governs the steady-state sizes of heterochromatin domains. Using theory and simulations, we predicted that a loss of transcription results in increased chromatin compaction and larger heterochromatin domain sizes. To validate our predictions, we employed complementary super-resolution and nano-imaging techniques on five different cell lines with impaired transcription. We quantitatively assessed how domain sizes scale with loop extrusion rates at the hetero-euchromatin interfaces. Our analysis of previously obtained super-resolution images of nuclei revealed that excessive loop extrusion leads to smaller heterochromatin domains. The model successfully recapitulated these observations, explaining how transcription loss can counteract the effects of cohesin overloading. As the general biophysical mechanisms regulating heterochromatin domain sizes are independent of cell type, our findings have significant implications for understanding the role of transcription in global genome organization.

biophysics↗

bioRxiv2022

H3.1Cys96 oxidation by mitochondrial ROS promotes chromatin remodeling, breast cancer progression to metastasis and multi-drug resistance

Transcription stability enforces cellular identity and is tightly controlled by restrictions imposed on both transcription factor function and target gene accessibility. Progression of cancer to metastasis and multi-drug resistance requires fluid transcriptional programs that can explore different genomic landscapes to enable clonal expansion of aggressive and treatment resistant phenotypes. Here, we show that increased levels of H2O2 produced in mitochondria leads to H3.1 oxidation at Cys96, a distinctive redox sensitive amino acid residue restricted to this histone variant, in the nucleus. The oxidation of Cys96 promotes the eviction of H3.1 from chromatin and its exchange with H3.3, thereby opening silenced portions of the chromatin. Mutation of Cys96 by an oxidation-resistant serine residue or quenching nuclear H2O2 reversed chemotherapy resistance and drove established metastatic disease into remission. Together, these results show that increased mitochondrial H2O2 production, characteristic of metabolic dysfunction, promotes transcriptional plasticity by removing structural chromatin restrictions imposed by the redox sensitive histone variant H3.1. We suggest that this new regulatory nexus between cancer metabolism and chromatin remodeling controls chromatin states that enable cancer progression and drug resistance acquisition.

cancer biology↗

bioRxiv2021

Chromatin forms nanoscale three-dimensional packing domains with fractal-like scaling behavior in vitro

Chromatin organization over a wide range of length scales plays a critical role in the regulation of transcription and deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein, we introduce ChromSTEM, a method that utilizes high angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and the statistical packing behavior of the chromatin polymer. Using chromatin mass and density analysis, we observed that chromatin forms spatially well-defined higher-order domains which are around 100 nm in radius, with a radially decreasing mass-density from the center to the periphery. Although the morphological properties of the domains vary within the same cell line, they seem to exhibit greater heterogeneity across cell lines, underlying a potential role of statistical chromatin packing in regulating cell-type-specific gene expression.

genomics↗

bioRxiv2021

Spike-in normalization for single-cell RNA-seq reveals dynamic global transcriptional activity mediating anti-cancer drug response

The transcriptional plasticity of cancer cells promotes intercellular heterogeneity in response to anti-cancer drugs and facilitates the generation of subpopulation surviving cells. Characterizing single-cell transcriptional heterogeneity after drug treatments can provide mechanistic insights into drug efficacy. Here we used single-cell RNA-seq to examine transcriptomic profiles of cancer cells treated with paclitaxel, celecoxib, and the combination of the two drugs. By normalizing the expression of endogenous genes to spike-in molecules, we found that celluar mRNA abundance shows dynamic regulation after drug treatment. Using a random forest model, we identified gene signatures classifying single cells into three states: transcriptional repression, amplification, and control-like. Treatment with paclitaxel or celecoxib alone generally repressed gene transcription across single cells. Interestingly, the drug combination resulted in transcriptional amplification and hyperactivation of mitochondrial oxidative phosphorylation pathway linking to enhanced cell killing efficiency. Finally, we identified a regulatory module enriched with metabolism and inflammation-related genes activated in a subpopulation of paclitaxel-treated cells, the expression of which predicted paclitaxel efficacy across cancer cell lines and in vivo patient samples. Our study highlights the dynamic global transcriptional activity driving single-cell heterogeneity during drug response and emphasizes the importance of adding spike-in molecules to study gene expression regulation using single-cell RNA-seq.

genomics↗