RESUMEN
Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.
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Imagenología Tridimensional , Transcriptoma , Animales , Ratones , Humanos , Transcriptoma/genética , Imagenología Tridimensional/métodos , Programas Informáticos , Perfilación de la Expresión Génica/métodos , Ganglios Linfáticos/patología , Ganglios Linfáticos/metabolismo , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Encéfalo/metabolismo , Ratones Endogámicos C57BL , Metástasis Linfática , FemeninoRESUMEN
Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to overcome limitations posed by difficult-to-isolate cell types, an alternative approach aiming at recovering single nuclei instead of intact cells can be utilized for sequencing, making transcriptome profiling of individual cells universally applicable. These techniques have become a cornerstone in the study of brain organoids, establishing them as models of the developing human brain. Leveraging the potential of single-cell and single-nucleus transcriptomics in brain organoid research, this protocol presents a step-by-step guide encompassing key procedures such as organoid dissociation, single-cell or nuclei isolation, library preparation and sequencing. By implementing these alternative approaches, researchers can obtain high-quality datasets, enabling the identification of neuronal and non-neuronal cell types, gene expression profiles, and cell lineage trajectories. This facilitates comprehensive investigations into cellular processes and molecular mechanisms shaping brain development.
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Encéfalo , Transcriptoma , Humanos , Organoides , Perfilación de la Expresión Génica , Núcleo CelularRESUMEN
Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes in programmable spatiotemporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. Spatial and single-cell transcriptomic analyses showed that this local induction was sufficient to generate stereotypically patterned organoids and revealed new insights into SHH's contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.
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Proteínas Hedgehog , Optogenética , Humanos , Proteínas Hedgehog/metabolismo , Organoides/metabolismo , Diferenciación Celular , Expresión GénicaRESUMEN
RNA abundance is tightly regulated in eukaryotic cells by modulating the kinetic rates of RNA production, processing, and degradation. To date, little is known about timedependent kinetic rates during dynamic processes. Here, we present SLAMDropseq, a method that combines RNA metabolic labeling and alkylation of modified nucleotides in methanolfixed cells with dropletbased sequencing to detect newly synthesized and preexisting mRNAs in single cells. As a first application, we sequenced 7280 HEK293 cells and calculated genespecific kinetic rates during the cell cycle using the novel package Eskrate. Of the 377 robustcycling genes that we identified, only a minor fraction is regulated solely by either dynamic transcription or degradation (6 and 4%, respectively). By contrast, the vast majority (89%) exhibit dynamically regulated transcription and degradation rates during the cell cycle. Our study thus shows that temporally regulated mRNA degradation is fundamental for the correct expression of a majority of cycling genes. SLAMDropseq, combined with Eskrate, is a powerful approach to understanding the underlying mRNA kinetics of singlecell gene expression dynamics in continuous biological processes.
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Ciclo Celular , ARN Mensajero , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ciclo Celular/genética , Cinética , Análisis de Secuencia de ARN/métodos , HumanosRESUMEN
BACKGROUND: Acute kidney injury (AKI) occurs frequently in critically ill patients and is associated with adverse outcomes. Cellular mechanisms underlying AKI and kidney cell responses to injury remain incompletely understood. METHODS: We performed single-nuclei transcriptomics, bulk transcriptomics, molecular imaging studies, and conventional histology on kidney tissues from 8 individuals with severe AKI (stage 2 or 3 according to Kidney Disease: Improving Global Outcomes (KDIGO) criteria). Specimens were obtained within 1-2 h after individuals had succumbed to critical illness associated with respiratory infections, with 4 of 8 individuals diagnosed with COVID-19. Control kidney tissues were obtained post-mortem or after nephrectomy from individuals without AKI. RESULTS: High-depth single cell-resolved gene expression data of human kidneys affected by AKI revealed enrichment of novel injury-associated cell states within the major cell types of the tubular epithelium, in particular in proximal tubules, thick ascending limbs, and distal convoluted tubules. Four distinct, hierarchically interconnected injured cell states were distinguishable and characterized by transcriptome patterns associated with oxidative stress, hypoxia, interferon response, and epithelial-to-mesenchymal transition, respectively. Transcriptome differences between individuals with AKI were driven primarily by the cell type-specific abundance of these four injury subtypes rather than by private molecular responses. AKI-associated changes in gene expression between individuals with and without COVID-19 were similar. CONCLUSIONS: The study provides an extensive resource of the cell type-specific transcriptomic responses associated with critical illness-associated AKI in humans, highlighting recurrent disease-associated signatures and inter-individual heterogeneity. Personalized molecular disease assessment in human AKI may foster the development of tailored therapies.
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Lesión Renal Aguda , COVID-19 , Lesión Renal Aguda/genética , COVID-19/genética , Enfermedad Crítica , Humanos , Riñón , TranscriptomaRESUMEN
Acute kidney injury (AKI) is a major health issue, the outcome of which depends primarily on damage and reparative processes of tubular epithelial cells. Mechanisms underlying AKI remain incompletely understood, specific therapies are lacking and monitoring the course of AKI in clinical routine is confined to measuring urine output and plasma levels of filtration markers. Here we demonstrate feasibility and potential of a novel approach to assess the cellular and molecular dynamics of AKI by establishing a robust urine-to-single cell RNA sequencing (scRNAseq) pipeline for excreted kidney cells via flow cytometry sorting. We analyzed 42,608 single cell transcriptomes of 40 urine samples from 32 patients with AKI and compared our data with reference material from human AKI post-mortem biopsies and published mouse data. We demonstrate that tubular epithelial cells transcriptomes mirror kidney pathology and reflect distinct injury and repair processes, including oxidative stress, inflammation, and tissue rearrangement. We also describe an AKI-specific abundant urinary excretion of adaptive progenitor-like cells. Thus, single cell transcriptomics of kidney cells excreted in urine provides noninvasive, unprecedented insight into cellular processes underlying AKI, thereby opening novel opportunities for target identification, AKI sub-categorization, and monitoring of natural disease course and interventions.
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Lesión Renal Aguda , Humanos , Ratones , Animales , Lesión Renal Aguda/patología , Riñón/patología , Biomarcadores/orina , Estrés Oxidativo , Células Epiteliales/patologíaRESUMEN
The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla. Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 °C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.
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Núcleo Celular , Transcriptoma , Animales , Riñón , Ratones , ARN , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: Single-cell transcriptomes from dissociated tissues provide insights into cell types and their gene expression and may harbor additional information on spatial position and the local microenvironment. The kidney's cells are embedded into a gradient of increasing tissue osmolality from the cortex to the medulla, which may alter their transcriptomes and provide cues for spatial reconstruction. METHODS: Single-cell or single-nuclei mRNA sequencing of dissociated mouse kidneys and of dissected cortex, outer, and inner medulla, to represent the corticomedullary axis, was performed. Computational approaches predicted the spatial ordering of cells along the corticomedullary axis and quantitated expression levels of osmo-responsive genes. In situ hybridization validated computational predictions of spatial gene-expression patterns. The strategy was used to compare single-cell transcriptomes from wild-type mice to those of mice with a collecting duct-specific knockout of the transcription factor grainyhead-like 2 (Grhl2CD-/-), which display reduced renal medullary osmolality. RESULTS: Single-cell transcriptomics from dissociated kidneys provided sufficient information to approximately reconstruct the spatial position of kidney tubule cells and to predict corticomedullary gene expression. Spatial gene expression in the kidney changes gradually and osmo-responsive genes follow the physiologic corticomedullary gradient of tissue osmolality. Single-nuclei transcriptomes from Grhl2CD-/- mice indicated a flattened expression gradient of osmo-responsive genes compared with control mice, consistent with their physiologic phenotype. CONCLUSIONS: Single-cell transcriptomics from dissociated kidneys facilitated the prediction of spatial gene expression along the corticomedullary axis and quantitation of osmotically regulated genes, allowing the prediction of a physiologic phenotype.
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Corteza Renal/metabolismo , Corteza Renal/patología , Médula Renal/metabolismo , Médula Renal/patología , Transcriptoma , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hibridación in Situ , Túbulos Renales/metabolismo , Túbulos Renales/patología , Ratones , Ratones Endogámicos C57BL , Concentración OsmolarRESUMEN
Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration.
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Moléculas de Adhesión Celular/fisiología , Lectinas Tipo C/fisiología , Músculo Esquelético/fisiología , Factor de Transcripción PAX7/genética , Regeneración , Células Satélite del Músculo Esquelético/fisiología , Síndrome Debilitante/genética , Animales , Biopsia , Preescolar , Consanguinidad , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Músculo Esquelético/citología , Músculo Esquelético/lesiones , Mutación , Factor de Transcripción PAX7/metabolismo , Cultivo Primario de Células , Células Satélite del Músculo Esquelético/trasplante , Análisis de la Célula Individual , Trasplante Heterólogo/métodos , Síndrome Debilitante/terapia , Secuenciación del ExomaRESUMEN
Herpesvirus infection initiates a range of perturbations in the host cell, which remain poorly understood at the level of individual cells. Here, we quantify the transcriptome of single human primary fibroblasts during the first hours of lytic infection with HSV-1. By applying a generalizable analysis scheme, we define a precise temporal order of early viral gene expression and propose a set-wise emergence of viral genes. We identify host cell genes and pathways relevant for infection by combining three different computational approaches: gene and pathway overdispersion analysis, prediction of cell-state transition probabilities, as well as future cell states. One transcriptional program, which correlates with increased resistance to infection, implicates the transcription factor NRF2. Consequently, Bardoxolone methyl and Sulforaphane, two known NRF2 agonists, impair virus production, suggesting that NRF2 activation restricts viral infection. Our study provides insights into early stages of HSV-1 infection and serves as a general blueprint for the investigation of heterogeneous cell states in virus infection.
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Fibroblastos/metabolismo , Herpes Simple/genética , Herpesvirus Humano 1 , Interacciones Huésped-Patógeno/genética , Factor 2 Relacionado con NF-E2/genética , Fibroblastos/virología , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Isotiocianatos/farmacología , Factor 2 Relacionado con NF-E2/agonistas , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacología , Cultivo Primario de Células , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Sulfóxidos , Replicación Viral/efectos de los fármacosRESUMEN
Background Three different cell types constitute the glomerular filter: mesangial cells, endothelial cells, and podocytes. However, to what extent cellular heterogeneity exists within healthy glomerular cell populations remains unknown.Methods We used nanodroplet-based highly parallel transcriptional profiling to characterize the cellular content of purified wild-type mouse glomeruli.Results Unsupervised clustering of nearly 13,000 single-cell transcriptomes identified the three known glomerular cell types. We provide a comprehensive online atlas of gene expression in glomerular cells that can be queried and visualized using an interactive and freely available database. Novel marker genes for all glomerular cell types were identified and supported by immunohistochemistry images obtained from the Human Protein Atlas. Subclustering of endothelial cells revealed a subset of endothelium that expressed marker genes related to endothelial proliferation. By comparison, the podocyte population appeared more homogeneous but contained three smaller, previously unknown subpopulations.Conclusions Our study comprehensively characterized gene expression in individual glomerular cells and sets the stage for the dissection of glomerular function at the single-cell level in health and disease.
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Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Glomérulos Renales/fisiología , Células Mesangiales/metabolismo , Podocitos/metabolismo , Análisis de Secuencia de ARN , Animales , Células Cultivadas , Regulación de la Expresión Génica , Glomérulos Renales/citología , Masculino , Ratones , Ratones Endogámicos , Valores de ReferenciaRESUMEN
Individual mRNA molecules can be imaged in fixed cells by hybridization with multiple, singly labeled oligonucleotide probes, followed by computational identification of fluorescent signals. This approach, called single-molecule RNA fluorescence in situ hybridization (smRNA FISH), allows subcellular localization and absolute quantification of RNA molecules in individual cells. Here, we describe a simple smRNA FISH protocol for two-color imaging of a circular RNA, CDR1as, simultaneously with an unrelated messenger RNA. The protocol can be adapted to circRNAs that coexist with overlapping, noncircular mRNA isoforms produced from the same genetic locus.
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Regulación de la Expresión Génica , Hibridación Fluorescente in Situ/métodos , ARN Largo no Codificante/genética , ARN/genética , Análisis de la Célula Individual/métodos , Humanos , ARN CircularRESUMEN
BACKGROUND: Herpesviruses can infect a wide range of animal species. Herpes simplex virus 1 (HSV-1) is one of the eight herpesviruses that can infect humans and is prevalent worldwide. Herpesviruses have evolved multiple ways to adapt the infected cells to their needs, but knowledge about these transcriptional and post-transcriptional modifications is sparse. RESULTS: Here, we show that HSV-1 induces the expression of about 1000 antisense transcripts from the human host cell genome. A subset of these is also activated by the closely related varicella zoster virus. Antisense transcripts originate either at gene promoters or within the gene body, and they show different susceptibility to the inhibition of early and immediate early viral gene expression. Overexpression of the major viral transcription factor ICP4 is sufficient to turn on a subset of antisense transcripts. Histone marks around transcription start sites of HSV-1-induced and constitutively transcribed antisense transcripts are highly similar, indicating that the genetic loci are already poised to transcribe these novel RNAs. Furthermore, an antisense transcript overlapping with the BBC3 gene (also known as PUMA) transcriptionally silences this potent inducer of apoptosis in cis. CONCLUSIONS: We show for the first time that a virus induces widespread antisense transcription of the host cell genome. We provide evidence that HSV-1 uses this to downregulate a strong inducer of apoptosis. Our findings open new perspectives on global and specific alterations of host cell transcription by viruses.
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Genoma Humano , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno/genética , ARN sin Sentido/genética , Transcripción Genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Regulación Viral de la Expresión Génica/efectos de los fármacos , Células HeLa , Código de Histonas , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN sin Sentido/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Transcripción Genética/efectos de los fármacos , Proteínas Virales/metabolismoRESUMEN
By the onset of morphogenesis, Drosophila embryos consist of about 6000 cells that express distinct gene combinations. Here, we used single-cell sequencing of precisely staged embryos and devised DistMap, a computational mapping strategy to reconstruct the embryo and to predict spatial gene expression approaching single-cell resolution. We produced a virtual embryo with about 8000 expressed genes per cell. Our interactive Drosophila Virtual Expression eXplorer (DVEX) database generates three-dimensional virtual in situ hybridizations and computes gene expression gradients. We used DVEX to uncover patterned expression of transcription factors and long noncoding RNAs, as well as signaling pathway components. Spatial regulation of Hippo signaling during early embryogenesis suggests a mechanism for establishing asynchronous cell proliferation. Our approach is suitable to generate transcriptomic blueprints for other complex tissues.
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Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/citología , Análisis de la Célula Individual/métodos , Transcriptoma , Animales , Comunicación Celular , Proteínas de Drosophila/genética , Hibridación in Situ , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Recent developments in droplet-based microfluidics allow the transcriptional profiling of thousands of individual cells in a quantitative, highly parallel and cost-effective way. A critical, often limiting step is the preparation of cells in an unperturbed state, not altered by stress or ageing. Other challenges are rare cells that need to be collected over several days or samples prepared at different times or locations. METHODS: Here, we used chemical fixation to address these problems. Methanol fixation allowed us to stabilise and preserve dissociated cells for weeks without compromising single-cell RNA sequencing data. RESULTS: By using mixtures of fixed, cultured human and mouse cells, we first showed that individual transcriptomes could be confidently assigned to one of the two species. Single-cell gene expression from live and fixed samples correlated well with bulk mRNA-seq data. We then applied methanol fixation to transcriptionally profile primary cells from dissociated, complex tissues. Low RNA content cells from Drosophila embryos, as well as mouse hindbrain and cerebellum cells prepared by fluorescence-activated cell sorting, were successfully analysed after fixation, storage and single-cell droplet RNA-seq. We were able to identify diverse cell populations, including neuronal subtypes. As an additional resource, we provide 'dropbead', an R package for exploratory data analysis, visualization and filtering of Drop-seq data. CONCLUSIONS: We expect that the availability of a simple cell fixation method will open up many new opportunities in diverse biological contexts to analyse transcriptional dynamics at single-cell resolution.
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Células Cultivadas/citología , Citometría de Flujo/métodos , Perfilación de la Expresión Génica/métodos , Análisis de la Célula Individual/métodos , Animales , Cerebelo/citología , Drosophila/citología , Embrión no Mamífero/citología , Citometría de Flujo/instrumentación , Perfilación de la Expresión Génica/instrumentación , Humanos , Metanol/química , Ratones , ARN Mensajero/análisis , Rombencéfalo/citología , Análisis de Secuencia de ARN , Análisis de la Célula Individual/instrumentación , Programas InformáticosRESUMEN
The incorporation of CENP-A into centromeric chromatin is an essential prerequisite for kinetochore formation. Yet, the molecular mechanisms governing this process are surprisingly divergent in different organisms. While CENP-A loading mechanisms have been studied in some detail in mammals, there are still large gaps to our understanding of CENP-A/Cid loading pathways in Drosophila. Here, we report on the characterization and delineation of at least three different CENP-A preloading complexes in Drosophila. Two complexes contain the CENP-A chaperones CAL1, FACT and/or Caf1/Rbap48. Notably, we identified a novel complex consisting of the histone acetyltransferase Hat1, Caf1 and CENP-A/H4. We show that Hat1 is required for proper CENP-A loading into chromatin, since knock-down in S2 cells leads to reduced incorporation of newly synthesized CENP-A. In addition, we demonstrate that CENP-A/Cid interacts with the HAT1 complex via an N-terminal region, which is acetylated in cytoplasmic but not in nuclear CENP-A. Since Hat1 is not responsible for acetylation of CENP-A/Cid, these results suggest a histone acetyltransferase activity-independent escort function for Hat1. Thus, our results point toward intriguing analogies between the complex processing pathways of newly synthesized CENP-A and canonical histones.
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Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Histona Acetiltransferasas/genética , Histonas/genética , Cinetocoros/metabolismo , Acetilación , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Proteína A Centromérica , Cromatina/ultraestructura , Citoplasma/metabolismo , Citoplasma/ultraestructura , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Regulación de la Expresión Génica , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Cinetocoros/ultraestructura , Datos de Secuencia Molecular , Plásmidos/química , Plásmidos/metabolismo , Proteína 4 de Unión a Retinoblastoma/genética , Proteína 4 de Unión a Retinoblastoma/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Eukaryotic nucleosomes consists of an (H3-H4)2 tetramer and two H2A-H2B dimers, around which 147 bp of DNA are wrapped in 1.7 left-handed helical turns. During chromatin assembly, the (H3-H4)2 tetramer binds first, forming a tetrasome that likely constitutes an important intermediate during ongoing transcription. We recently showed that (H3-H4)2 tetrasomes spontaneously switch between a left- and right-handed wrapped state of the DNA, a phenomenon that may serve to buffer changes in DNA torque induced by RNA polymerase in transcription. Within nucleosomes of actively transcribed genes, however, canonical H3 is progressively replaced by its variant H3.3. Consequently, one may ask if and how the DNA chirality dynamics of tetrasomes is altered by H3.3. Recent findings that H3.3-containing nucleosomes result in less stable and less condensed chromatin further underline the need to study the microscopic underpinnings of H3.3-containing tetrasomes and nucleosomes. Here we report real-time single-molecule studies of (H3.3-H4)2 tetrasome dynamics using Freely Orbiting Magnetic Tweezers and Electromagnetic Torque Tweezers. We find that the assembly of H3.3-containing tetrasomes and nucleosomes by the histone chaperone Nucleosome Assembly Protein 1 (NAP1) occurs in an identical manner to that of H3-containing tetrasomes and nucleosomes. Likewise, the flipping behavior of DNA handedness in tetrasomes is not impacted by the presence of H3.3. We also examine the effect of free NAP1, H3.3, and H4 in solution on flipping behavior and conclude that the probability for a tetrasome to occupy the left-handed state is only slightly enhanced by the presence of free protein. These data demonstrate that the incorporation of H3.3 does not alter the structural dynamics of tetrasomes, and hence that the preferred incorporation of this histone variant in transcriptionally active regions does not result from its enhanced ability to accommodate torsional stress, but rather may be linked to specific chaperone or remodeler requirements or communication with the nuclear environment.