Quantitative single cell 5hmC sequencing reveals non-canonical gene regulation by non-CG hydroxymethylation

Oxidative modification of 5-methylcytosine (5mC) generates 5-hydroxymethylcytosine (5hmC), a DNA modification that exhibits unique epigenetic regulatory functions and impacts diverse biological processes. However, standard single-nucleus/cell bisulfite sequencing methods cannot resolve the base ambiguity between 5mC and 5hmC to accurately measure cell-type specific epigenomic patterns and gene regulatory functions of 5hmC or true 5mC. Here, we develop single-nucleus 5hmC sequencing (snhmC-seq) for quantitative and unbiased 5hmC profiling in single cells by harnessing differential deaminase activity of APOBEC3A towards 5mC and chemically protected 5hmC. We used snhmC-seq to profile single nuclei from cryopreserved mouse brain samples to reveal epigenetic heterogeneity of 5hmC at single-cell resolution and uncovered a non-canonical gene regulatory role of genic 5hmC in non-CG context.

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Ancient microRNA profiles of a 14,300-year-old canid are taxonomically informative and give glimpses into gene regulation from the Pleistocene

10.1101/2019.12.16.877761 ◽

2019 ◽

Author(s):

Bastian Fromm ◽

Marcel Tarbier ◽

Oliver Smith ◽

Love Dalén ◽

M. T. P. Gilbert ◽

...

Keyword(s):

Gene Regulation ◽

Messenger Rna ◽

Great Promise ◽

Biological Processes ◽

Cell Type ◽

Key Technology ◽

Rna Fragments ◽

Cell Type Specific ◽

Gene Regulatory ◽

Regulatory Functions

AbstractAncient DNA sequencing is the key technology for paleogenomic studies and today a routine method in many laboratories. Recent analyses have shown that, under favoring conditions, also RNA can be sequenced from historical and even ancient samples. We have re-analyzed ancient RNA data from a Pleistocene canid and find - in addition to the previously described messenger RNA fragments - intact microRNAs, which are short transcripts with important gene regulatory functions. With an extraordinary age of 14,300 years, the canid microRNA profiles are the oldest ever reported. Despite their age, we show that the microRNA profiles are conclusive of taxonomic origin, tissue identity with organ- and cell-type specific signatures, and that they yield glimpses into gene regulatory activity and biological processes from the Pleistocene. In summary, we here show that straightforward microRNA analyses hold great promise for deeper insights into gene regulation in extinct animals.

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Exosomic microRNAs as emerging key regulators of intercellular communication in the tumor microenvironment and beyond

microRNA Diagnostics and Therapeutics ◽

10.1515/micrnat-2016-0001 ◽

2016 ◽

Vol 2 (1) ◽

Author(s):

Mariam Murtadha ◽

Muller Fabbri

Keyword(s):

Tumor Microenvironment ◽

Cancer Patients ◽

Intercellular Communication ◽

Potential Role ◽

Biological Fluids ◽

Healthy Controls ◽

Gene Regulatory ◽

Non Coding Rnas ◽

Regulatory Functions

AbstractMicroRNAs (miRs) are small non-coding RNAs with key gene regulatory functions. Recent evidence has shown that miRs have a central role in shaping the biology of the Tumor Microenvironment (TME). The discovery that some exosomes contain high levels of miR cargo that shuttle between cells and mediate intercellular cross-talk has shifted the focus of miR research towards understanding the biological role of exosomic miRs. In this review, we highlight the emerging role of exosomic miRs in molding the tumor microenvironment towards pro-tumor conditions by altering intercellular communication. We briefly discuss some mechanisms of selective loading of miRs into exosomes, as well as emerging evidence that exosomic miRs are present in all biological fluids. Furthermore, we describe the differences in the exosomic miR signatures between cancer patients and healthy controls, and the potential role of exosomic miRs as diagnostic, prognostic, and therapeutic biomarkers.

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Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer

10.21203/rs.3.rs-257705/v2 ◽

2021 ◽

Author(s):

Inga-Maria Launonen ◽

Nuppu Lyytikäinen ◽

Julia Casado ◽

Ella Anttila ◽

Angéla Szabó ◽

...

Keyword(s):

Tumor Microenvironment ◽

Single Cell ◽

Single Cells ◽

High Grade ◽

Prognostic Role ◽

Immune Microenvironment ◽

Proteomic Data ◽

Tumor Immune Microenvironment ◽

Cellular Phenotypes

Abstract The majority of high-grade serous ovarian cancers (HGSCs) are deficient in homologous recombination (HR) DNA repair, most commonly due to mutations or hypermethylation of the BRCA1/2 genes. We aimed to discover how BRCA1/2 mutations shape the cellular phenotypes and spatial interactions of the tumor microenvironment. Using a highly multiplex immunofluorescence and image analysis we generated spatial proteomic data for 21 markers in 124,623 single cells from 112 tumor cores originating from 31 tumors with BRCA1/2 mutation (BRCA1/2mut), and from 13 tumors without alterations in HR genes (HRwt). We identified a phenotypically distinct tumor microenvironment in the BRCA1/2mut tumors with evidence of increased immunosurveillance. Importantly, we found an opposing prognostic role of a proliferative tumor-cell phenotypic subpopulation in the HR-genotypes, which associated with enhanced spatial tumor-immune interactions by the CD8+ and CD4+T-cells in BRCA1/2mut tumors. The single-cell spatial landscapes indicate distinct patterns of spatial immunosurveillance with the premise to improve immunotherapeutic strategies and patient stratification in HGSC.

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Single-cell transcriptomics allows novel insights into aging and circadian processes

Briefings in Functional Genomics ◽

10.1093/bfgp/elaa014 ◽

2020 ◽

Vol 19 (5-6) ◽

pp. 343-349

Author(s):

Sara S Fonseca Costa ◽

Marc Robinson-Rechavi ◽

Jürgen A Ripperger

Keyword(s):

Single Cell ◽

Single Cells ◽

Cellular Level ◽

Biological Processes ◽

New Methods ◽

New Findings ◽

Single Cell Rna Sequencing ◽

Aging And Circadian Rhythms ◽

Insight Into ◽

Different Time Scales

Abstract Aging and circadian rhythms are two biological processes that affect an organism, although at different time scales. Nevertheless, due to the overlap of their actions, it was speculated that both interfere or interact with each other. However, to address this question, a much deeper insight into these processes is necessary, especially at the cellular level. New methods such as single-cell RNA-sequencing (scRNA-Seq) have the potential to close this gap in our knowledge. In this review, we analyze applications of scRNA-Seq from the aging and circadian rhythm fields and highlight new findings emerging from the analysis of single cells, especially in humans or rodents. Furthermore, we judge the potential of scRNA-Seq to identify common traits of both processes. Overall, this method offers several advantages over more traditional methods analyzing gene expression and will become an important tool to unravel the link between these biological processes.

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Single-cell genomics reveals region-specific developmental trajectories underlying neuronal diversity in the prenatal human hypothalamus

10.1101/2021.07.20.453090 ◽

2021 ◽

Author(s):

Brian Herb ◽

Hannah J Glover ◽

Aparna Bhaduri ◽

Alex M Casella ◽

Tracy L Bale ◽

...

Keyword(s):

Developmental Trajectories ◽

Single Cells ◽

Neuronal Diversity ◽

Cell Type ◽

Human Hypothalamus ◽

Paired Samples ◽

Cell Type Specific ◽

Gene Regulatory ◽

Stem Cell Populations ◽

Temporal Trajectory

The hypothalamus is critically important for regulating most autonomic, metabolic, and behavioral functions essential for life and species propagation, yet a comprehensive understanding of neuronal subtypes and their development in the human brain is lacking. Here, we characterized the prenatal human hypothalamus by sequencing the transcriptomes of 45,574 single-cells from 12 embryos, spanning gestational weeks 4 through 25. These cells describe a temporal trajectory from proliferative stem cell populations to maturing neurons and glia, including 38 distinct excitatory and inhibitory neuronal subtypes. Merging these data with paired samples from the cortex and ganglionic eminences (GE) revealed two distinct neurogenesis pathways, one shared between GE and hypothalamus and a second unique to cortex. Gene regulatory network modeling predicted that these distinct maturation trajectories involve the activation of region- and cell type-specific transcription factor networks. These results provide the first comprehensive transcriptomic view of human hypothalamus development at cellular resolution.

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DeepDRIM: a deep neural network to reconstruct cell-type-specific gene regulatory network using single-cell RNA-Seq Data

10.1101/2021.02.03.429484 ◽

2021 ◽

Author(s):

Jiaxing Chen ◽

Chinwang Cheong ◽

Liang Lan ◽

Xin Zhou ◽

Jiming Liu ◽

...

Keyword(s):

Neural Network ◽

Single Cell ◽

Regulatory Networks ◽

Deep Neural Network ◽

Neighborhood Context ◽

Cellular Heterogeneity ◽

Specific Gene ◽

Rna Seq ◽

Cell Type Specific ◽

Gene Regulatory

AbstractSingle-cell RNA sequencing is used to capture cell-specific gene expression, thus allowing reconstruction of gene regulatory networks. The existing algorithms struggle to deal with dropouts and cellular heterogeneity, and commonly require pseudotime-ordered cells. Here, we describe DeepDRIM a supervised deep neural network that represents gene pair joint expression as images and considers the neighborhood context to eliminate the transitive interactions. Deep-DRIM yields significantly better performance than the other nine algorithms used on the eight cell lines tested, and can be used to successfully discriminate key functional modules between patients with mild and severe symptoms of coronavirus disease 2019 (COVID-19).

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Direct correlation between motile behavior and protein abundance in single cells

10.1101/067918 ◽

2016 ◽

Author(s):

Yann S Dufour ◽

Sébastien Gillet ◽

Nicholas W Frankel ◽

Douglas B Weibel ◽

Thierry Emonet

Keyword(s):

Protein Expression ◽

Single Cell ◽

Phenotypic Diversity ◽

Single Cells ◽

Selective Advantage ◽

Single Cell Protein ◽

Cell Protein ◽

Chemotaxis Proteins

AbstractUnderstanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage.

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Reconstructing A/B compartments as revealed by Hi-C using long-range correlations in epigenetic data

10.1101/019000 ◽

2015 ◽

Author(s):

Jean-Philippe Fortin ◽

Kasper D Hansen

Keyword(s):

Single Cell ◽

Long Range ◽

Bisulfite Sequencing ◽

Cell Types ◽

Cell Type ◽

Long Range Correlations ◽

Illumina 450K ◽

Genome Bisulfite Sequencing ◽

Cell Type Specific ◽

Dnase Hypersensitivity

Analysis of Hi-C data has shown that the genome can be divided into two compartments called A/B compartments. These compartments are cell-type specific and are associated with open and closed chromatin. We show that A/B compartments can be reliably estimated using epigenetic data from several different platforms, the Illumina 450k DNA methylation microarray, DNase hypersensitivity sequencing, single-cell ATAC sequencing and single-cell whole-genome bisulfite sequencing. We do this by exploiting the fact that the structure of long range correlations differs between open and closed compartments. This work makes A/B compartments readily available in a wide variety of cell types, including many human cancers.

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Single-cell atlas of progressive supranuclear palsy reveals a distinct hybrid glial cell population

10.1101/2021.04.11.439393 ◽

2021 ◽

Author(s):

Abhijeet Sharma ◽

Won-Min Song ◽

Kurt Farrell ◽

Kristen Whitney ◽

Bin Zhang ◽

...

Keyword(s):

Progressive Supranuclear Palsy ◽

Single Cell ◽

Neurodegenerative Disorder ◽

Hybrid Cell ◽

Unfolded Protein ◽

Homeostatic Function ◽

Single Nucleus ◽

Cell Type Specific ◽

The Unfolded Protein Response ◽

Protein Response

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder whose molecular complexity at a single cell level has not been evaluated. Here we analyzed 45,559 high quality nuclei from the subthalamic nucleus and associated basal ganglia regions from post-mortem human PSP brains with varying degrees of tau pathology compared to controls (n=3 per group). We identified novel astrocyte-oligodendrocyte hybrid cell populations that overexpress neurotropic factors in conjunction with suppression of the unfolded protein response pathway. Notably, trajectory analysis identified subpopulations of hybrid cells with distinct astrocytic, oligodendrocytic and hybrid molecular states that change from a neuroprotective hybrid cell to an astrocytic cell with impaired homeostatic function in PSP. Our single nucleus transcriptomic data provides insights into the cell-type-specific contributions to the disease for investigating the molecular and cellular basis of PSP

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Single cell and single nucleus RNA-Seq reveal cellular heterogeneity and homeostatic regulatory networks in adult mouse stria vascularis

10.1101/756635 ◽

2019 ◽

Author(s):

Soumya Korrapati ◽

Ian Taukulis ◽

Rafal Olszewski ◽

Madeline Pyle ◽

Shoujun Gu ◽

...

Keyword(s):

Single Cell ◽

Gene Regulatory Networks ◽

Regulatory Networks ◽

Molecular Mechanisms ◽

Stria Vascularis ◽

Cell Types ◽

Cellular Heterogeneity ◽

Genetic Mechanisms ◽

Single Nucleus ◽

Gene Regulatory

AbstractThe stria vascularis (SV) generates the endocochlear potential (EP) in the inner ear and is necessary for proper hair cell mechanotransduction and hearing. While channels belonging to SV cell types are known to play crucial roles in EP generation, relatively little is known about gene regulatory networks that underlie the ability of the SV to generate and maintain the EP. Using single cell and single nucleus RNA-sequencing, we identify and validate known and rare cell populations in the SV. Furthermore, we establish a basis for understanding molecular mechanisms underlying SV function by identifying potential gene regulatory networks as well as druggable gene targets. Finally, we associate known deafness genes with adult SV cell types. This work establishes a basis for dissecting the genetic mechanisms underlying the role of the SV in hearing and will serve as a basis for designing therapeutic approaches to hearing loss related to SV dysfunction.

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