scholarly journals Single-Cell Protein Atlas of Transcription Factors Reveals the Combinatorial Code for Spatiotemporal Patterning the C. elegans Embryo

2020 ◽  
Author(s):  
Xuehua Ma ◽  
Zhiguang Zhao ◽  
Long Xiao ◽  
Weina Xu ◽  
Yangyang Wang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Single Cell Analysis ◽  
The Body ◽  
Cell Protein ◽  
Regulatory State ◽  
Protein Fusion ◽  
C Elegans ◽  
Time Specific ◽  
Combinatorial Code

SUMMARYA high-resolution protein atlas is essential for understanding the molecular basis of biological processes. Using protein-fusion reporters and imaging-based single-cell analyses, we present a protein expression atlas of C. elegans embryogenesis encompassing 266 transcription factors (TFs) in nearly all (90%) lineage-resolved cells. Single-cell analysis reveals a combinatorial code and cascade that elucidate the regulatory hierarchy between a large number of lineage-, tissue-, and time-specific TFs in spatiotemporal fate patterning. Guided by expression, we identify essential functions of CEH-43/DLX, a lineage-specific TF, and ELT-1/GATA3, a well-known skin fate specifier, in neuronal specification; and M03D4.4 as a pan-muscle TF in converging muscle differentiation in the body wall and pharynx. Finally, systems-level analysis of TF regulatory state uncovers lineage- and time-specific kinetics of fate progression and widespread detours of the trajectories of cell differentiation. Collectively, our work reveals a single-cell molecular atlas and general principles underlying the spatiotemporal patterning of a metazoan embryo.

scholarly journals In silico analysis of the transcriptional regulatory logic of neuronal identity specification throughout the C. elegans nervous system

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Lori Glenwinkel ◽  
Seth R Taylor ◽  
Kasper Langebeck-Jensen ◽  
Laura Pereira ◽  
Molly B Reilly ◽  
...  
Keyword(s):  
Nervous System ◽  
Transcription Factors ◽  
Single Cell ◽  
Expression Profiles ◽  
Neuronal Cell ◽  
In Silico Analysis ◽  
Specific Gene ◽  
Neuron Type ◽  
C Elegans ◽  
Neuronal Identity

The generation of the enormous diversity of neuronal cell types in a differentiating nervous system entails the activation of neuron type-specific gene batteries. To examine the regulatory logic that controls the expression of neuron type-specific gene batteries, we interrogate single cell expression profiles of all 118 neuron classes of the Caenorhabditis elegans nervous system for the presence of DNA binding motifs of 136 neuronally expressed C. elegans transcription factors. Using a phylogenetic footprinting pipeline, we identify cis-regulatory motif enrichments among neuron class-specific gene batteries and we identify cognate transcription factors for 117 of the 118 neuron classes. In addition to predicting novel regulators of neuronal identities, our nervous system-wide analysis at single cell resolution supports the hypothesis that many transcription factors directly co-regulate the cohort of effector genes that define a neuron type, thereby corroborating the concept of so-called terminal selectors of neuronal identity. Our analysis provides a blueprint for how individual components of an entire nervous system are genetically specified.

scholarly journals Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Harrison Specht ◽  
Edward Emmott ◽  
Aleksandra A. Petelski ◽  
R. Gray Huffman ◽  
David H. Perlman ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Single Cell Protein ◽  
Cellular Heterogeneity ◽  
Cell Protein ◽  
Alternatively Activated Macrophages ◽  
Monocytes And Macrophages ◽  
Molecular Phenotypes ◽  
Post Transcriptional Regulation

Abstract Background Macrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of the limitations of quantitative single-cell protein analysis. Results To overcome this limitation, we develop SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantifies over 3042 proteins in 1490 single monocytes and macrophages in 10 days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. Parallel measurements of transcripts by 10× Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus, SCoPE2 supports quantification with improved count statistics. This allowed exploring regulatory interactions, such as interactions between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53. Conclusions Even in a homogeneous environment, macrophage proteomes are heterogeneous. This heterogeneity correlates to the inflammatory axis of classically and alternatively activated macrophages. Our methodology lays the foundation for automated and quantitative single-cell analysis of proteins by mass spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.

scholarly journals HOX13-dependent chromatin accessibility modulates the target repertoires of the HOX factors

2019 ◽  
Author(s):  
Ines Desanlis ◽  
Yacine Kherdjemil ◽  
Alexandre Mayran ◽  
Yasser Bouklouch ◽  
Claudia Gentile ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell Analysis ◽  
Binding Specificity ◽  
Chromatin Accessibility ◽  
The Body ◽  
Binding Motifs ◽  
Dna Binding Motifs ◽  
Cellular Context ◽  
Pioneer Factors ◽  
Chromatin Opening

ABSTRACTHox genes encode essential transcription factors that control patterning during embryonic development. Distinct combinations of nested Hox expression domains establish cell and tissue identities1–3. Consequently, spatial or temporal de-regulation of Hox genes can cause severe alterations of the body plan3. While HOX factors have very similar DNA binding motifs, their binding specificity is, in part, mediated by co-factors4–6. Yet, the interplay between HOX binding specificities and the cellular context remains largely elusive. To gain insight into this question, we took advantage of developing limbs for which the differential expression of Hox genes is well-characterized7. We show that the transcription factors HOXA13 and HOXD13 (hereafter referred as HOX13) allow another HOX factor, HOXA11, to bind loci initially assumed to be HOX13-specific. Importantly, HOXA11 is unable to bind these loci in distal limbs lacking HOX13 function indicating that HOX13 modulates HOXA11 target repertoire. In addition, we find that the HOX13 factors implement the distal limb developmental program by triggering chromatin opening, a defining property of pioneer factors8,9. Finally, single cell analysis of chromatin accessibility reveals that HOX13 factors pioneer chromatin opening in a lineage specific manner. Together, our data uncover a new mechanism underlying HOX binding specificity, whereby tissue-specific variations in the target repertoire of HOX factors rely, at least in part, on HOX13-dependent chromatin accessibility.

scholarly journals Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity

2019 ◽  
Author(s):  
Harrison Specht ◽  
Edward Emmott ◽  
Aleksandra A. Petelski ◽  
R. Gray Huffman ◽  
David H. Perlman ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Single Cell Protein ◽  
Cellular Heterogeneity ◽  
Cell Protein ◽  
Alternatively Activated Macrophages ◽  
Monocytes And Macrophages ◽  
Molecular Phenotypes ◽  
Post Transcriptional Regulation

AbstractMacrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of limitations of quantitative single-cell protein analysis. To overcome this limitation, we developed SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantified over 3,042 proteins in 1,490 single monocytes and macrophages in ten days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. This gradient correlates to the inflammatory axis of classically and alternatively activated macrophages. Parallel measurements of transcripts by 10x Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus SCoPE2 supports quantification with improved count statistics. The joint distributions of proteins and transcripts allowed exploring regulatory interactions, such as between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53. Our methodology lays the foundation for quantitative single-cell analysis of proteins by mass-spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.Abstract Figure

scholarly journals Combined mRNA and protein single cell analysis in a dynamic cellular system using SPARC

2019 ◽  
Author(s):  
Johan Reimegård ◽  
Marcus Danielsson ◽  
Marcel Tarbier ◽  
Jens Schuster ◽  
Sathishkumar Baskaran ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Embryonic Stem ◽  
Single Cell Protein ◽  
Cell Protein ◽  
Depth Analysis ◽  
Mrna And Protein Expression

ABSTRACTCombined measurements of mRNA and protein expression in single cells enables in-depth analysis of cellular states. We present single-cell protein and RNA co-profiling (SPARC), an approach to simultaneously measure global mRNA and large sets of intracellular protein in individual cells. Using SPARC, we show that mRNA expression fails to accurately reflect protein abundance at the time of measurement in human embryonic stem cells, although the direction of changes of mRNA and protein expression are in agreement during cellular differentiation. Moreover, protein levels of transcription factors better predict their downstream effects than do the corresponding transcripts. We further show that changes of the balance between protein and mRNA expression levels can be applied to infer expression kinetic trajectories, revealing future states of individual cells. Finally, we highlight that mRNA expression may be more varied among cells than levels of the corresponding proteins. Overall, our results demonstrate that mRNA and protein measurements in single cells provide different and complementary information regarding cell states. Accordingly, SPARC can offer valuable insights in gene expression programs of single cells.

scholarly journals Single-cell analysis of transcriptome and DNA methylome in human oocyte maturation

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241698
Author(s):  
Bo Yu ◽  
Naresh Doni Jayavelu ◽  
Stephanie L. Battle ◽  
Jessica C. Mar ◽  
Timothy Schimmel ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Oocyte Maturation ◽  
Gene Expression Regulation ◽  
Single Cell Analysis ◽  
Reproductive Potential ◽  
Cpg Methylation ◽  
Fertility Treatment ◽  
Human Oocyte

Oocyte maturation is a coordinated process that is tightly linked to reproductive potential. A better understanding of gene regulation during human oocyte maturation will not only answer an important question in biology, but also facilitate the development of in vitro maturation technology as a fertility treatment. We generated single-cell transcriptome and used our previously published single-cell methylome data from human oocytes at different maturation stages to investigate how genes are regulated during oocyte maturation, focusing on the potential regulatory role of non-CpG methylation. DNMT3B, a gene encoding a key non-CpG methylation enzyme, is one of the 1,077 genes upregulated in mature oocytes, which may be at least partially responsible for the increased non-CpG methylation as oocytes mature. Non-CpG differentially methylated regions (DMRs) between mature and immature oocytes have multiple binding motifs for transcription factors, some of which bind with DNMT3B and may be important regulators of oocyte maturation through non-CpG methylation. Over 98% of non-CpG DMRs locate in transposable elements, and these DMRs are correlated with expression changes of the nearby genes. Taken together, this data indicates that global non-CpG hypermethylation during oocyte maturation may play an active role in gene expression regulation, potentially through the interaction with transcription factors.

Abstract LB-076: Single-cell analysis of transcription factors provides deeper characterization of cancer cells

2017 ◽  
Author(s):  
Camila Egidio ◽  
Robert Durruthy-Durruthy ◽  
Michael Gonzales ◽  
Manisha Ray ◽  
Jason McKinney
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Cancer Cells ◽  
Single Cell Analysis ◽  
Cell Analysis

scholarly journals Multiplexed single-cell proteomics using SCoPE2

2021 ◽  
Author(s):  
Aleksandra A Petelski ◽  
Edward Emmott ◽  
Andrew Leduc ◽  
R. Gray Huffman ◽  
Harrison Specht ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cost Effective ◽  
Protein Analysis ◽  
Single Cell Protein ◽  
Protein Quantification ◽  
Cell Protein ◽  
Label Free ◽  
Cost Effective Method

Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying over 1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Isobaric carrier based multiplexed single-cell proteomics is a scalable, reliable, and cost-effective method that can be fully automated and implemented on widely available equipment. It uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. Here we describe an automated Single Cell ProtEomics (SCoPE2) workflow that allows analyzing about 200 single cells per 24 hours using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.

Sign in / Sign up

Export Citation Format

Share Document