Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions

SUMMARYDeciphering mechanisms in cell fate decisions requires single-cell holistic reconstructions of multi-dimensional epigenome in transcriptional regulation. Here we develop CoTECH, a combinatorial barcoding method allowing for high-throughput single-cell joint detection of chromatin occupancy and transcriptome. First, we used CoTECH to examine bivalent histone marks (H3K4me3 and H3K27me3) with transcription from naïve to primed mouse embryonic stem cells. Concurrent bivalent marks in pseudo-single cells linked via transcriptome were computationally derived, resolving pseudotemporal bivalency trajectories and disentangling a context-specific interplay between H3K4me3/H3K27me3 and transcription level. Next, CoTECH with H3K27ac, an active enhancer marker, revealed the regulatory basis of endothelial-to-hematopoietic transition in two waves of hematopoietic cells and distinctive enhancer-gene linking schemes guiding hemogenic endothelial cell (HEC) emergence, indicating a unique epigenetic control of transcriptional regulation for hematopoietic stem cell priming. Together, CoTECH provides an efficient framework for single-cell co-assay of chromatin occupancy and transcription, thus, enabling higher-dimensional epigenomic reconstructions.

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Post-transcriptional regulation in hematopoiesis: RNA binding proteins take control

Biochemistry and Cell Biology ◽

10.1139/bcb-2017-0310 ◽

2019 ◽

Vol 97 (1) ◽

pp. 10-20 ◽

Cited By ~ 7

Author(s):

Laura P.M.H. de Rooij ◽

Derek C.H. Chan ◽

Ava Keyvani Chahi ◽

Kristin J. Hope

Keyword(s):

Transcriptional Regulation ◽

Stem Cell ◽

Cell Fate ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Control Of Gene Expression ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Post Transcriptional Regulation

Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP–RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.

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Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate

10.1101/427237 ◽

2018 ◽

Author(s):

Maximilian W. Fries ◽

Kalina T. Haas ◽

Suzan Ber ◽

John Saganty ◽

Emma K. Richardson ◽

...

Keyword(s):

Cell Death ◽

Single Cell ◽

Cell Fate ◽

Genetic Heterogeneity ◽

Cellular Response ◽

Single Cells ◽

Caspase Activation ◽

Cell Fate Decisions ◽

Network Activation ◽

Biochemical Imaging

The biochemical activities underlying cell-fate decisions vary profoundly even in genetically identical cells. But such non-genetic heterogeneity remains refractory to current imaging methods, because their capacity to monitor multiple biochemical activities in single living cells over time remains limited1. Here, we deploy a family of newly designed GFP-like sensors (NyxBits) with fast photon-counting electronics and bespoke analytics (NyxSense) in multiplexed biochemical imaging, to define a network determining the fate of single cells exposed to the DNA-damaging drug cisplatin. By simultaneously imaging a tri-nodal network comprising the cell-death proteases Caspase-2, -3 and -92, we reveal unrecognized single-cell heterogeneities in the dynamics and amplitude of caspase activation that signify survival versus cell death via necrosis or apoptosis. Non-genetic heterogeneity in the pattern of caspase activation recapitulates traits of therapy resistance previously ascribed solely to genetic causes3,4. Chemical inhibitors that alter these patterns can modulate in a predictable manner the phenotypic landscape of the cellular response to cisplatin. Thus, multiplexed biochemical imaging reveals cellular populations and biochemical states, invisible to other methods, underlying therapeutic responses to an anticancer drug. Our work develops widely applicable tools to monitor the dynamic activation of biochemical networks at single-cell resolution. It highlights the necessity to resolve patterns of network activation in single cells, rather than the average state of individual nodes, to define, and potentially control, mechanisms underlying cellular decisions in health and disease.

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CoTECH for single-cell joint detection of transcriptome and chromatin occupancy

10.21203/rs.3.pex-1300/v1 ◽

2021 ◽

Author(s):

Yingjie Luo ◽

Haiqing Xiong ◽

Qianhao Wang ◽

Xianhong Yu ◽

Aibin He

Keyword(s):

Gene Regulation ◽

Single Cell ◽

High Throughput ◽

Single Cells ◽

Cellular Heterogeneity ◽

Joint Detection ◽

Biological Processes ◽

Experimental Procedure ◽

Higher Dimensional

Abstract Here we present CoTECH, a high-throughput co-aasay that measures chromatin occupancy and transcriptome in single cells. The CoTECH method adopts a combinatorial indexing strategy to enrich chromatin fragments of interest as reported in CoBATCH in combination with a modified Smart-seq2 procedure to simultaneously capture the 3’ mRNA profiles in the same single cells. The whole experimental procedure can be handled within three days.The CoTECH acquires data quality of 1000-9000 unique mapped reads (DNA partition) and 1500-4000 expressed genes (RNA partition) per cell. Experimentally linking chromatin occupancy to transcriptional outputs and inferred molecular association between multimodal omics datasets made possible by CoTECH enables reconstructions of higher dimensional epigenomic landscape, providing new insights into epigenome-centric gene regulation and cellular heterogeneity in many biological processes. This step-by-step protocol is related to the publication “Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions” in Nature Methods.

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Single-cell molecular profiling provides a high-resolution map of basophil and mast cell differentiation

10.1101/848358 ◽

2019 ◽

Author(s):

Fiona K. Hamey ◽

Winnie W.Y. Lau ◽

Iwo Kucinski ◽

Xiaonan Wang ◽

Evangelia Diamanti ◽

...

Keyword(s):

Flow Cytometry ◽

Mast Cell ◽

Mast Cells ◽

Single Cell ◽

Cell Fate ◽

Single Cells ◽

Molecular Profiling ◽

Multicolor Flow Cytometry ◽

High Coverage ◽

Hematopoietic Stem

AbstractDifferentiation of hematopoietic stem and progenitor cells ensure a continuous supply of mature blood cells. Recent models of differentiation are represented as a landscape, in which individual progenitors traverse a continuum of multipotent cell states before reaching an entry point that marks lineage commitment. Basophils and mast cells have received little attention in these models and their differentiation trajectories are yet to be explored. Here, we have performed multicolor flow cytometry and high-coverage single-cell RNA sequencing analyses to chart the differentiation of hematopoietic progenitors into basophils and mast cells in mouse. Analysis of flow cytometry data reconstructed a detailed map of the differentiation, including a bifurcation of progenitors into two specific trajectories. Molecular profiling and pseudotime ordering of the single cells revealed gene expression changes during differentiation, with temporally separated regulation of mast cell protease genes. We validate that basophil and mast cell signature genes increased along the trajectories into their respective lineage, and we demonstrate how genes critical for each respective lineage are upregulated during the formation of the mature cells. Cell fate assays showed that multicolor flow cytometry and transcriptional profiling successfully predict the bipotent phenotype of a previously uncharacterized population of basophil-mast cell progenitor-like cells in mouse peritoneum. Taken together, we provide a detailed roadmap of basophil and mast cell development through a combination of molecular and functional profiling.

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Highly multiplexed spatially resolved gene expression profiling of mouse organogenesis

10.1101/2020.11.20.391896 ◽

2020 ◽

Author(s):

T. Lohoff ◽

S. Ghazanfar ◽

A. Missarova ◽

N. Koulena ◽

N. Pierson ◽

...

Keyword(s):

Gene Expression ◽

High Resolution ◽

Single Cell ◽

Cell Fate ◽

Target Genes ◽

Single Cells ◽

Spatial Context ◽

Sequencing Data ◽

Cell Fate Decisions ◽

Spatially Resolved

AbstractTranscriptional and epigenetic profiling of single-cells has advanced our knowledge of the molecular bases of gastrulation and early organogenesis. However, current approaches rely on dissociating cells from tissues, thereby losing the crucial spatial context that is necessary for understanding cell and tissue interactions during development. Here, we apply an image-based single-cell transcriptomics method, seqFISH, to simultaneously and precisely detect mRNA molecules for 387 selected target genes in 8-12 somite stage mouse embryo tissue sections. By integrating spatial context and highly multiplexed transcriptional measurements with two single-cell transcriptome atlases we accurately characterize cell types across the embryo and demonstrate how spatially-resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain-hindbrain boundary and the developing gut tube. Our spatial atlas uncovers axes of resolution that are not apparent from single-cell RNA sequencing data – for example, in the gut tube we observe early dorsal-ventral separation of esophageal and tracheal progenitor populations. In sum, by computationally integrating high-resolution spatially-resolved gene expression maps with single-cell genomics data, we provide a powerful new approach for studying how and when cell fate decisions are made during early mammalian development.

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Gene Regulatory Networks Governing Hematopoietic Stem Cell Development and Identity

Blood ◽

10.1182/blood.v118.21.sci-30.sci-30 ◽

2011 ◽

Vol 118 (21) ◽

pp. SCI-30-SCI-30 ◽

Cited By ~ 1

Author(s):

Tariq Enver

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Single Cells ◽

Transcriptional Regulators ◽

Gata Factor ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Genome Wide ◽

A Genome ◽

Custom Made

Abstract Abstract SCI-30 Several studies have addressed questions about transcriptional regulation within particular hematopoietic cell compartments. Few, however, have attempted to capture the transcriptional changes that occur during the dynamic transition from one compartment to another. We have profiled gene expression as multipotential progenitors underwent commitment and differentiation to two alternative lineages, focusing on the first 3 days of differentiation when the majority of decisions about cell fate are made. We have combined this with genome-wide identification of the targets of three key transcription factors before and after differentiation; GATA-2, usually associated with the stem/progenitor compartment; GATA-1 (erythroid); and PU.1 (myeloid). These data have been compiled into a custom-made queryable database, designed to be intuitive to use and to provide tools to interrogate the data on many levels. We used correlation analyses to associate transcription factor binding with particular modules of co-expressed genes, alongside detailed sequence analysis of bound regions. These approaches have informed our understanding of GATA factor switching, and highlighted novel roles for both GATA-2 and Pu.1 in erythroid cells. Overall, the data reveal greater degree of complexity in the interplay between these three factors in regulating hematopoiesis than has hitherto been described, and highlights the importance of a genome-wide approach to understanding complex regulatory systems. A significant challenge in the field is how to relate these types of population-based data to the action of transcriptional regulators within single cells where cell fate decisions ultimately are affected. As a step toward this, we have generated single cell profiles of gene expression for a limited set of transcriptional regulators in self-renewing and committed blood cells and used these data to build a stochastic computational model, which affords exploration of commitment scenarios in silico. Disclosures: No relevant conflicts of interest to declare.

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Ultra-multiplexed analysis of single-cell dynamics reveals logic rules in differentiation

Science Advances ◽

10.1126/sciadv.aav7959 ◽

2019 ◽

Vol 5 (4) ◽

pp. eaav7959 ◽

Cited By ~ 19

Author(s):

Ce Zhang ◽

Hsiung-Lin Tu ◽

Gengjie Jia ◽

Tanzila Mukhtar ◽

Verdon Taylor ◽

...

Keyword(s):

Stem Cell ◽

Cell Differentiation ◽

Single Cell ◽

Cell Fate ◽

Single Cell Analysis ◽

Single Cells ◽

Critical Role ◽

Stem Cell Differentiation ◽

Cell Fate Decisions ◽

Cellular Microenvironments

Dynamical control of cellular microenvironments is highly desirable to study complex processes such as stem cell differentiation and immune signaling. We present an ultra-multiplexed microfluidic system for high-throughput single-cell analysis in precisely defined dynamic signaling environments. Our system delivers combinatorial and time-varying signals to 1500 independently programmable culture chambers in week-long live-cell experiments by performing nearly 106 pipetting steps, where single cells, two-dimensional (2D) populations, or 3D neurospheres are chemically stimulated and tracked. Using our system and statistical analysis, we investigated the signaling landscape of neural stem cell differentiation and discovered “cellular logic rules” that revealed the critical role of signal timing and sequence in cell fate decisions. We find synergistic and antagonistic signal interactions and show that differentiation pathways are highly redundant. Our system allows dissection of hidden aspects of cellular dynamics and enables accelerated biological discovery.

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Comprehensive analysis of single-cell RNA sequencing data from healthy human marrow hematopoietic cells

BMC Research Notes ◽

10.1186/s13104-020-05357-y ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Xin Zhao ◽

Shouguo Gao ◽

Sachiko Kajigaya ◽

Qingguo Liu ◽

Zhijie Wu ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Cell Fate ◽

Noncoding Rna ◽

Single Cells ◽

Regulation Of Gene Expression ◽

Sequencing Data ◽

Healthy Human ◽

Hematopoietic Stem ◽

Single Cell Rna Sequencing

Abstract Objective Single cell methodology enables detection and quantification of transcriptional changes and unravelling dynamic aspects of the transcriptional heterogeneity not accessible using bulk sequencing approaches. We have applied single-cell RNA-sequencing (scRNA-seq) to fresh human bone marrow CD34+ cells and profiled 391 single hematopoietic stem/progenitor cells (HSPCs) from healthy donors to characterize lineage- and stage-specific transcription during hematopoiesis. Results Cells clustered into six distinct groups, which could be assigned to known HSPC subpopulations based on lineage specific genes. Reconstruction of differentiation trajectories in single cells revealed four committed lineages derived from HSCs, as well as dynamic expression changes underlying cell fate during early erythroid-megakaryocytic, lymphoid, and granulocyte-monocyte differentiation. A similar non-hierarchical pattern of hematopoiesis could be derived from analysis of published single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), consistent with a sequential relationship between chromatin dynamics and regulation of gene expression during lineage commitment (first, altered chromatin conformation, then mRNA transcription). Computationally, we have reconstructed molecular trajectories connecting HSCs directly to four hematopoietic lineages. Integration of long noncoding RNA (lncRNA) expression from the same cells demonstrated mRNA transcriptome, lncRNA, and the epigenome were highly homologous in their pattern of gene activation and suppression during hematopoietic cell differentiation.

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Integration of spatial and single-cell transcriptomic data elucidates mouse organogenesis

Nature Biotechnology ◽

10.1038/s41587-021-01006-2 ◽

2021 ◽

Author(s):

T. Lohoff ◽

S. Ghazanfar ◽

A. Missarova ◽

N. Koulena ◽

N. Pierson ◽

...

Keyword(s):

Single Cell ◽

Cell Fate ◽

Target Genes ◽

Single Cells ◽

Cell Types ◽

Spatial Context ◽

Cell Fate Decisions ◽

Spatially Resolved ◽

Regulatory Processes ◽

Cell Transcriptome

AbstractMolecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8–12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain–hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal–ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development.

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