Single-cell nascent RNA sequencing unveils coordinated global transcription.

Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers1,2. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations3. However, fundamental questions about the temporal regulation of transcription and enhancer-gene coordination remain unanswered, primarily because of the absence of a single-cell perspective on active transcription. In this study, we present scGRO-seq-a new single-cell nascent RNA sequencing assay that uses click chemistry-and unveil coordinated transcription throughout the genome. We demonstrate the episodic nature of transcription and the co-transcription of functionally related genes. scGRO-seq can estimate burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells and can leverage replication-dependent non-polyadenylated histone gene transcription to elucidate cell cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO-seq enables the identification of networks of enhancers and genes. Our results suggest that the bursting of transcription at super-enhancers precedes bursting from associated genes. By imparting insights into the dynamic nature of global transcription and the origin and propagation of transcription signals, we demonstrate the ability of scGRO-seq to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression.

Single-cell nascent RNA sequencing using click-chemistry unveils coordinated transcription.

Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers1-5. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations6-9. However, fundamental questions in the temporal regulation of transcription and enhancer-gene synchrony remain unanswered primarily due to the absence of a single-cell perspective on active transcription. In this study, we present scGRO-seq - a novel single-cell nascent RNA sequencing assay using click-chemistry - and unveil the coordinated transcription throughout the genome. scGRO-seq demonstrates the episodic nature of transcription, and estimates burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells. It reveals the co-transcription of functionally related genes and leverages the replication-dependent non-polyadenylated histone genes transcription to elucidate cell-cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO-seq identifies networks of enhancers and genes and indicates that the bursting of transcription at super-enhancers precedes the burst from associated genes. By imparting insights into the dynamic nature of transcription and the origin and propagation of transcription signals, scGRO-seq demonstrates its unique ability to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression.

Type I interferon activates MHC class I-dressed CD11b+ conventional dendritic cells to promote protective anti-tumor CD8+ T cell immunity.

Tumor-infiltrating dendritic cells (DCs) assume varied functional states that impact anti-tumor immunity. To delineate the DC states associated with productive anti-tumor T cell immunity, we compared spontaneously regressing and progressing tumors. Tumor-reactive CD8+ T cell responses in Batf3-/- mice lacking type 1 DCs (DC1s) were lost in progressor tumors but preserved in regressor tumors. Transcriptional profiling of intra-tumoral DCs within regressor tumors revealed an activation state of CD11b+ conventional DCs (DC2s) characterized by expression of interferon (IFN)-stimulated genes (ISGs) (ISG+ DCs). ISG+ DC-activated CD8+ T cells ex vivo comparably to DC1. Unlike cross-presenting DC1, ISG+ DCs acquired and presented intact tumor-derived peptide-major histocompatibility complex class I (MHC class I) complexes. Constitutive type I IFN production by regressor tumors drove the ISG+ DC state, and activation of MHC class I-dressed ISG+ DCs by exogenous IFN-ß rescued anti-tumor immunity against progressor tumors in Batf3-/- mice. The ISG+ DC gene signature is detectable in human tumors. Engaging this functional DC state may present an approach for the treatment of human disease.

Antigen dominance hierarchies shape TCF1+ progenitor CD8 T cell phenotypes in tumors.

CD8 T cell responses against different tumor neoantigens occur simultaneously, yet little is known about the interplay between responses and its impact on T cell function and tumor control. In mouse lung adenocarcinoma, we found that immunodominance is established in tumors, wherein CD8 T cell expansion is predominantly driven by the antigen that most stably binds MHC. T cells responding to subdominant antigens were enriched for a TCF1+ progenitor phenotype correlated with response to immune checkpoint blockade (ICB) therapy. However, the subdominant T cell response did not preferentially benefit from ICB due to a dysfunctional subset of TCF1+ cells marked by CCR6 and Tc17 differentiation. Analysis of human samples and sequencing datasets revealed that CCR6+ TCF1+ cells exist across human cancers and are not correlated with ICB response. Vaccination eliminated CCR6+ TCF1+ cells and dramatically improved the subdominant response, highlighting a strategy to optimally engage concurrent neoantigen responses against tumors.

Characterizing the Macrophage Response to Immunomodulatory Biomaterials Through Gene Set Analyses.

The primary regulators of the innate immune response to implanted biomaterials are macrophages, which change phenotype over time to regulate multiple phases of the tissue repair process. Immunomodulatory biomaterials that target macrophage phenotype are a promising approach for promoting tissue repair. Although expression of multiple markers has been widely used to characterize macrophage phenotype, the complexity of the macrophage response to biomaterials makes interpretation difficult. The aim of this study was to put forth an objective method to characterize macrophage phenotype with respect to specific biological processes or standard phenotypes of interest. We investigated the utility of gene set analyses to analyze macrophages as they respond to model biomaterials in comparison to "reference" M1 and M2a macrophage phenotypes. Primary human macrophages were seeded onto crosslinked collagen scaffolds with or without adsorption of the proinflammatory cytokine interferon-gamma (IFNg). Gene expression of a custom-curated panel of 48 genes, representing the M1 and M2a gene signatures as well as other genes important for angiogenesis and tissue repair, was quantified using NanoString on days 3, 5, and 8 of culture. A dataset of phenotype controls, consisting of M0, M1, and M2a macrophages, was used as a source of comparison and to validate the methods of characterization. Gene expression of M1 and M2a markers showed mixed upregulation and downregulation by macrophages seeded on collagen and IFNg-adsorbed collagen scaffolds, highlighting the need for more holistic analyses. Euclidean distance measurements to the reference phenotypes were unable to resolve differences between groups. In contrast, rotation gene set testing with and without gene weighting based on the genes' ability to differentiate between M1, M2a, and M0 controls, followed by gene set variation analysis, showed that collagen scaffolds inhibited the classic M1 phenotype without promoting a classic M2a phenotype, and that IFNg-adsorbed collagen scaffolds promoted the M1 phenotype and inhibited the M2a phenotype. In summary, this work demonstrates a powerful, objective methodology for characterizing the macrophage response to biomaterials in comparison to reference macrophage phenotypes. With the addition of more macrophage phenotypes with defined gene expression signatures, this method could prove beneficial for characterizing complex hybrid phenotypes.

Dynamic mechanical response of polyvinyl alcohol-gelatin theta-gels for nucleus pulposus tissue replacement.

Intervertebral disk degeneration is one of the most significant contributors to low back pain. Thus, there is significant interest in designing new treatments and nucleus pulposus (NP) tissue replacements. Herein, the authors propose a biosynthetic material, comprised of a polyvinyl alcohol (PVA) and gelatin theta-gel, as an acellular NP tissue replacement. Theta-gels form during the solidification of PVA and gelatin (phase I), and the phase separation of a disklike short-chain polyethylene glycol (PEG, phase II). The PVA concentration and weight ratio of PVA to PEG were optimized, in order to achieve mechanical properties resembling NP tissue. Mechanical and material properties were analyzed for the PVA-gelatin theta-gels under static and dynamic conditions. Cyclic stress-strain testing demonstrated the theta-gels' ability to relax and perform properly under dynamic loading. Altering the molecular weight and concentration of the theta-gel constituents allows for a tunable material that can match a variety of native tissue properties.