Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, ...we developed a single-cell multi-omics sequencing technology （single-cell COOL-seq） that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammali- an cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within 〈 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenie regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin acces- sibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA methylation dynamics at single-base resolution in early mouse embryos and provides new insights into the heterogeneous yet highly ordered features of epigenomic reprogramming during this process.
Recent studies have shown that meningeal lymphatic vessels (MLVs), which are located both dorsally and basally beneath the skull, provide a route for draining macromolecules and trafficking immune ...cells from the central nervous system (CNS) into cervical lymph nodes (CLNs), and thus represent a potential therapeutic target for treating neurodegenerative and neuroinflammatory diseases. However, the roles of MLVs in brain tumor drainage and immunity remain unexplored. Here we show that dorsal MLVs undergo extensive remodeling in mice with intracranial gliomas or metastatic melanomas. RNA-seq analysis of MLV endothelial cells revealed changes in the gene sets involved in lymphatic remodeling, fluid drainage, as well as inflammatory and immunological responses. Disruption of dorsal MLVs alone impaired intratumor fluid drainage and the dissemination of brain tumor cells to deep CLNs (dCLNs). Notably, the dendritic cell (DC) trafficking from intracranial tumor tissues to dCLNs decreased in mice with defective dorsal MLVs, and increased in mice with enhanced dorsal meningeal lymphangiogenesis. Strikingly, disruption of dorsal MLVs alone, without affecting basal MLVs or nasal LVs, significantly reduced the efficacy of combined anti-PD-1/CTLA-4 checkpoint therapy in striatal tumor models. Furthermore, mice bearing tumors overexpressing VEGF-C displayed a better response to anti-PD-1/CTLA-4 combination therapy, and this was abolished by CCL21/CCR7 blockade, suggesting that VEGF-C potentiates checkpoint therapy via the CCL21/CCR7 pathway. Together, the results of our study not only demonstrate the functional aspects of MLVs as classic lymphatic vasculature, but also highlight that they are essential in generating an efficient immune response against brain tumors.
Circular RNAs (circRNAs) are a new class of non-polyadenylated non-coding RNAs that may play important roles in many biological processes. Here we develop a single-cell universal poly(A)-independent ...RNA sequencing (SUPeR-seq) method to sequence both polyadenylated and non-polyadenylated RNAs from individual cells. This method exhibits robust sensitivity, precision and accuracy. We discover 2891 circRNAs and 913 novel linear transcripts in mouse preimplantation embryos and further analyze the abundance of circRNAs along development, the function of enriched genes, and sequence features of circRNAs. Our work is key to deciphering regulation mechanisms of circRNAs during mammalian early embryonic development.
DNA methylation is crucial for a wide variety of biological processes, yet no technique suitable for the methylome analysis of DNA methylation at single-cell resolution is available. Here, we ...describe a methylome analysis technique that enables single-cell and single-base resolution DNA methylation analysis based on reduced representation bisulfite sequencing (scRRBS). The technique is highly sensitive and can detect the methylation status of up to 1.5 million CpG sites within the genome of an individual mouse embryonic stem cell (mESC). Moreover, we show that the technique can detect the methylation status of individual CpG sites in a haploid sperm cell in a digitized manner as either unmethylated or fully methylated. Furthermore, we show that the demethylation dynamics of maternal and paternal genomes after fertilization can be traced within the individual pronuclei of mouse zygotes. The demethylation process of the genic regions is faster than that of the intergenic regions in both male and female pronuclei. Our method paves the way for the exploration of the dynamic methylome landscapes of individual cells at single-base resolution during physiological processes such as embryonic development, or during pathological processes such as tumorigenesis.
The mammalian prefrontal cortex comprises a set of highly specialized brain areas containing billions of cells and serves as the centre of the highest-order cognitive functions, such as memory, ...cognitive ability, decision-making and social behaviour. Although neural circuits are formed in the late stages of human embryonic development and even after birth, diverse classes of functional cells are generated and migrate to the appropriate locations earlier in development. Dysfunction of the prefrontal cortex contributes to cognitive deficits and the majority of neurodevelopmental disorders; there is therefore a need for detailed knowledge of the development of the prefrontal cortex. However, it is still difficult to identify cell types in the developing human prefrontal cortex and to distinguish their developmental features. Here we analyse more than 2,300 single cells in the developing human prefrontal cortex from gestational weeks 8 to 26 using RNA sequencing. We identify 35 subtypes of cells in six main classes and trace the developmental trajectories of these cells. Detailed analysis of neural progenitor cells highlights new marker genes and unique developmental features of intermediate progenitor cells. We also map the timeline of neurogenesis of excitatory neurons in the prefrontal cortex and detect the presence of interneuron progenitors in early developing prefrontal cortex. Moreover, we reveal the intrinsic development-dependent signals that regulate neuron generation and circuit formation using single-cell transcriptomic data analysis. Our screening and characterization approach provides a blueprint for understanding the development of the human prefrontal cortex in the early and mid-gestational stages in order to systematically dissect the cellular basis and molecular regulation of prefrontal cortex function in humans.
Our understanding of how aging affects the cellular and molecular components of the vasculature and contributes to cardiovascular diseases is still limited. Here we report a single-cell ...transcriptomic survey of aortas and coronary arteries in young and old cynomolgus monkeys. Our data define the molecular signatures of specialized arteries and identify eight markers discriminating aortic and coronary vasculatures. Gene network analyses characterize transcriptional landmarks that regulate vascular senility and position FOXO3A, a longevity-associated transcription factor, as a master regulator gene that is downregulated in six subtypes of monkey vascular cells during aging. Targeted inactivation of FOXO3A in human vascular endothelial cells recapitulates the major phenotypic defects observed in aged monkey arteries, verifying FOXO3A loss as a key driver for arterial endothelial aging. Our study provides a critical resource for understanding the principles underlying primate arterial aging and contributes important clues to future treatment of age-associated vascular disorders.
Mammalian oocyte maturation depends on the translational activation of stored maternal mRNAs upon meiotic resumption. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) is a key oocyte ...factor that regulates maternal mRNA translation. However, the signal that triggers CPEB1 activation at the onset of mammalian oocyte maturation is not known. We provide evidence that a mitogen-activated protein kinase (MAPK) cascade couples maternal mRNA translation to meiotic cell cycle progression in mouse oocytes by triggering CPEB1 phosphorylation and degradation. Mutations of the phosphorylation sites or ubiquitin E3 ligase binding sites in CPEB1 have a dominant-negative effect in oocytes, and mimic the phenotype of ERK1/2 knockout, by impairing spindle assembly and mRNA translation. Overexpression of the CPEB1 downstream translation activator DAZL in ERK1/2-deficient oocytes partially rescued the meiotic defects, indicating that ERK1/2 is essential for spindle assembly, metaphase II arrest and maternal-zygotic transition (MZT) primarily by triggering the translation of key maternal mRNAs. Taken together, ERK1/2-mediated CPEB1 phosphorylation/degradation is a major mechanism of maternal mRNA translational activation, and is crucial for mouse oocyte maturation and MZT.
How transplanted haematopoietic stem cells (HSCs) behave soon after they reside in a preconditioned host has not been studied due to technical limitations. Here, using single-cell RNA sequencing, we ...first obtained the transcriptome-based classifications of 28 haematopoietic cell types. We then applied them in conjunction with functional assays to track the dynamic changes of immunophenotypically purified HSCs in irradiated recipients within the first week after transplantation. Based on our transcriptional classifications, most homed HSCs in bone marrow and spleen became multipotent progenitors and, occasionally, some HSCs gave rise to megakaryocytic-erythroid or myeloid precursors. Parallel in vitro and in vivo functional experiments supported the paradigm of robust differentiation without substantial HSC expansion during the first week. Therefore, this study uncovers the previously inaccessible kinetics and fate choices of transplanted HSCs in myeloablated recipients at early stage, with implications for clinical applications of HSCs and other stem cells.
Understanding the molecular mechanisms underlying human cartilage degeneration and regeneration is helpful for improving therapeutic strategies for treating osteoarthritis (OA). Here, we report the ...molecular programmes and lineage progression patterns controlling human OA pathogenesis using single-cell RNA sequencing (scRNA-seq).
We performed unbiased transcriptome-wide scRNA-seq analysis, computational analysis and histological assays on 1464 chondrocytes from 10 patients with OA undergoing knee arthroplasty surgery. We investigated the relationship between transcriptional programmes of the OA landscape and clinical outcome using severity index and correspondence analysis.
We identified seven molecularly defined populations of chondrocytes in the human OA cartilage, including three novel phenotypes with distinct functions. We presented gene expression profiles at different OA stages at single-cell resolution. We found a potential transition among proliferative chondrocytes, prehypertrophic chondrocytes and hypertrophic chondrocytes (HTCs) and defined a new subdivision within HTCs. We revealed novel markers for cartilage progenitor cells (CPCs) and demonstrated a relationship between CPCs and fibrocartilage chondrocytes using computational analysis. Notably, we derived predictive targets with respect to clinical outcomes and clarified the role of different cell types for the early diagnosis and treatment of OA.
Our results provide new insights into chondrocyte taxonomy and present potential clues for effective and functional manipulation of human OA cartilage regeneration that could lead to improved health.
Although genomic instability, epigenetic abnormality, and gene expression dysregulation are hallmarks of colorectal cancer, these features have not been simultaneously analyzed at single-cell ...resolution. Using optimized single-cell multiomics sequencing together with multiregional sampling of the primary tumor and lymphatic and distant metastases, we developed insights beyond intratumoral heterogeneity. Genome-wide DNA methylation levels were relatively consistent within a single genetic sublineage. The genome-wide DNA demethylation patterns of cancer cells were consistent in all 10 patients whose DNA we sequenced. The cancer cells' DNA demethylation degrees clearly correlated with the densities of the heterochromatin-associated histone modification H3K9me3 of normal tissue and those of repetitive element long interspersed nuclear element 1. Our work demonstrates the feasibility of reconstructing genetic lineages and tracing their epigenomic and transcriptomic dynamics with single-cell multiomics sequencing.
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