Delineating the heterogeneity of matrix-directed differentiation toward soft and stiff tissue lineages via single-cell profiling

Mesenchymal stromal/stem cells (MSCs) form a heterogeneous population of multipotent progenitors that contribute to tissue regeneration and homeostasis. MSCs assess extracellular elasticity by probing resistance to applied forces via adhesion, cytoskeletal, and nuclear mechanotransducers that direct differentiation toward soft or stiff tissue lineages. Even under controlled culture conditions, MSC differentiation exhibits substantial cell-to-cell variation that remains poorly characterized. By single-cell transcriptional profiling of nonconditioned, matrix-conditioned, and early differentiating cells, we identified distinct MSC subpopulations with distinct mechanosensitivities, differentiation capacities, and cell cycling. We show that soft matrices support adipogenesis of multipotent cells and early endochondral ossification of nonadipogenic cells, whereas intramembranous ossification and preosteoblast proliferation are directed by stiff matrices. Using diffusion pseudotime mapping, we outline hierarchical matrix-directed differentiation and perform whole-genome screening of mechanoresponsive genes. Specifically, top-ranked tropomyosin-1 is highly sensitive to stiffness cues both at RNA and protein levels, and changes in TPM1 expression determine the differentiation toward soft versus stiff tissue lineage. Consistent with actin stress fiber stabilization, tropomyosin-1 overexpression maintains YAP1 nuclear localization, activates YAP1 target genes, and directs osteogenic differentiation. Knockdown of tropomyosin-1 reversed YAP1 nuclear localization consistent with relaxation of cellular contractility, suppressed osteogenesis, activated early endochondral ossification genes after 3 d of culture in induction medium, and facilitated adipogenic differentiation after 1 wk. Our results delineate cell-to-cell variation of matrix-directed MSC differentiation and highlight tropomyosin-mediated matrix sensing.

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Delineating the heterogeneity of matrix-directed differentiation towards soft and stiff tissue lineages via single-cell profiling

10.1101/2020.11.23.394460 ◽

2020 ◽

Author(s):

Shlomi Brielle ◽

Danny Bavli ◽

Alex Motzik ◽

Yoav Kan-Tor ◽

Batia Avni ◽

...

Keyword(s):

Single Cell ◽

Transcriptional Profiling ◽

Directed Differentiation ◽

Protein Levels ◽

Multipotent Progenitors ◽

Multipotent Cells ◽

Single Cell Profiling ◽

Applied Forces ◽

Hierarchical Matrix ◽

Cell Variation

SummaryMesenchymal stromal/stem cells (MSCs) are a heterogeneous population of multipotent progenitors that contribute to tissue regeneration and homeostasis. MSCs assess extracellular elasticity by probing resistance to applied forces via adhesion, cytoskeletal, and nuclear mechanotransducers, that direct differentiation toward soft or stiff tissue lineages. Even under controlled conditions, MSC differentiation exhibits substantial cell-to-cell variation that remains poorly characterized. By single-cell transcriptional profiling of naïve, matrix-conditioned, and early differentiation state cells, we identified distinct MSC subpopulations with distinct mechanosensitivities, differentiation capacities, and cell cycling. We showed that soft matrices support adipogenesis of multipotent cells and endochondral ossification of non-adipogenic cells, whereas intramembranous ossification and pre-osteoblast proliferation are enhanced by stiff matrices. Using diffusion pseudotime mapping, we delineated hierarchical matrix-directed differentiation and identified mechanoresponsive genes. We found that tropomyosin-1 (TPM1) is highly sensitive to stiffness cues both at RNA and protein levels and that changes in expression of TPM1 determine adipogenic or osteogenic fates. Thus, cell-to-cell variation in tropomyosin-mediated matrix-sensing contributes to impaired differentiation with implications to the biomedical potential of MSCs.

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Deconvolution of single-cell multi-omics layers reveals regulatory heterogeneity

10.1101/316208 ◽

2018 ◽

Cited By ~ 5

Author(s):

Longqi Liu ◽

Chuanyu Liu ◽

Andrés Quintero ◽

Liang Wu ◽

Yue Yuan ◽

...

Keyword(s):

Single Cell ◽

Target Genes ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Cell Populations ◽

Single Cell Level ◽

Complex Cell ◽

Cell Level ◽

Distinct Cell ◽

Cell Variation

AbstractIntegrative analysis of multi-omics layers at single cell level is critical for accurate dissection of cell-to-cell variation within certain cell populations. Here we report scCAT-seq, a technique for simultaneously assaying chromatin accessibility and the transcriptome within the same single cell. We show that the combined single cell signatures enable accurate construction of regulatory relationships between cis-regulatory elements and the target genes at single-cell resolution, providing a new dimension of features that helps direct discovery of regulatory patterns specific to distinct cell identities. Moreover, we generated the first single cell integrated maps of chromatin accessibility and transcriptome in human pre-implantation embryos and demonstrated the robustness of scCAT-seq in the precise dissection of master transcription factors in cells of distinct states during embryo development. The ability to obtain these two layers of omics data will help provide more accurate definitions of “single cell state” and enable the deconvolution of regulatory heterogeneity from complex cell populations.

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EPEN-21. IMPAIRED NEURONAL-GLIAL FATE SPECIFICATION IN PEDIATRIC EPENDYMOMA REVEALED BY SINGLE-CELL RNA-SEQ

Neuro-Oncology ◽

10.1093/neuonc/noaa222.158 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii311-iii312

Author(s):

Bernhard Englinger ◽

Johannes Gojo ◽

Li Jiang ◽

Jens M Hübner ◽

McKenzie L Shaw ◽

...

Keyword(s):

Dna Methylation ◽

Single Cell ◽

Regulatory Networks ◽

Target Genes ◽

Target Identification ◽

Rna Seq ◽

Cell Models ◽

Pediatric Ependymoma ◽

Glial Fate ◽

Anatomic Locations

Abstract Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets including IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

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Upregulation of COX4-2 via HIF-1α in Mitochondrial COX4-1 Deficiency

Cells ◽

10.3390/cells10020452 ◽

2021 ◽

Vol 10 (2) ◽

pp. 452

Author(s):

Liza Douiev ◽

Chaya Miller ◽

Shmuel Ruppo ◽

Hadar Benyamini ◽

Bassam Abu-Libdeh ◽

...

Keyword(s):

Oxygen Consumption ◽

Oxidative Phosphorylation ◽

Nuclear Localization ◽

Target Genes ◽

Compensatory Mechanism ◽

Terminal Electron Acceptor ◽

Human Foreskin Fibroblasts ◽

Mitochondrial Respiratory Complex ◽

Mrna Transcripts ◽

Oxphos System

Cytochrome-c-oxidase (COX) subunit 4 (COX4) plays important roles in the function, assembly and regulation of COX (mitochondrial respiratory complex 4), the terminal electron acceptor of the oxidative phosphorylation (OXPHOS) system. The principal COX4 isoform, COX4-1, is expressed in all tissues, whereas COX4-2 is mainly expressed in the lungs, or under hypoxia and other stress conditions. We have previously described a patient with a COX4-1 defect with a relatively mild presentation compared to other primary COX deficiencies, and hypothesized that this could be the result of a compensatory upregulation of COX4-2. To this end, COX4-1 was downregulated by shRNAs in human foreskin fibroblasts (HFF) and compared to the patient’s cells. COX4-1, COX4-2 and HIF-1α were detected by immunocytochemistry. The mRNA transcripts of both COX4 isoforms and HIF-1 target genes were quantified by RT-qPCR. COX activity and OXPHOS function were measured by enzymatic and oxygen consumption assays, respectively. Pathways were analyzed by CEL-Seq2 and by RT-qPCR. We demonstrated elevated COX4-2 levels in the COX4-1-deficient cells, with a concomitant HIF-1α stabilization, nuclear localization and upregulation of the hypoxia and glycolysis pathways. We suggest that COX4-2 and HIF-1α are upregulated also in normoxia as a compensatory mechanism in COX4-1 deficiency.

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Early embryonic expression ofFGF4/6/9gene and its role in the induction of mesenchyme and notochord inCiona savignyiembryos

Development ◽

10.1242/dev.129.7.1729 ◽

2002 ◽

Vol 129 (7) ◽

pp. 1729-1738 ◽

Cited By ~ 16

Author(s):

Kaoru S. Imai ◽

Nori Satoh ◽

Yutaka Satou

Keyword(s):

Cell Differentiation ◽

Nuclear Localization ◽

Target Genes ◽

Muscle Cells ◽

Cell Stage ◽

Ciona Savignyi ◽

Mesenchyme Cell ◽

Notochord Cells ◽

Mesenchyme Cells ◽

Endodermal Cells

In early Ciona savignyi embryos, nuclear localization of β-catenin is the first step of endodermal cell specification, and triggers the activation of various target genes. A cDNA for Cs-FGF4/6/9, a gene activated downstream of β-catenin signaling, was isolated and shown to encode an FGF protein with features of both FGF4/6 and FGF9/20. The early embryonic expression of Cs-FGF4/6/9 was transient and the transcript was seen in endodermal cells at the 16- and 32-cell stages, in notochord and muscle cells at the 64-cell stage, and in nerve cord and muscle cells at the 110-cell stage; the gene was then expressed again in cells of the nervous system after neurulation. When the gene function was suppressed with a specific antisense morpholino oligo, the differentiation of mesenchyme cells was completely blocked, and the fate of presumptive mesenchyme cells appeared to change into that of muscle cells. The inhibition of mesenchyme differentiation was abrogated by coinjection of the morpholino oligo and synthetic Cs-FGF4/6/9 mRNA. Downregulation of β-catenin nuclear localization resulted in the absence of mesenchyme cell differentiation due to failure of the formation of signal-producing endodermal cells. Injection of synthetic Cs-FGF4/6/9 mRNA in β-catenin-downregulated embryos evoked mesenchyme cell differentiation. These results strongly suggest that Cs-FGF4/6/9 produced by endodermal cells acts an inductive signal for the differentiation of mesenchyme cells. On the other hand, the role of Cs-FGF4/6/9 in the induction of notochord cells is partial; the initial process of the induction was inhibited by Cs-FGF4/6/9 morpholino oligo, but notochord-specific genes were expressed later to form a partial notochord.

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Exploring the Changing Landscape of Cell-to-Cell Variation After CTCF Knockdown via Single Cell RNA-seq

10.21203/rs.2.15870/v2 ◽

2019 ◽

Author(s):

Wei Wang ◽

Gang Ren ◽

Ni Hong ◽

Wenfei Jin

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Single Cell ◽

Zinc Finger ◽

Ctcf Binding ◽

Rna Seq ◽

Expression Noise ◽

Genome Wide ◽

Cell Variation ◽

Variable Genes

Abstract Background: CCCTC-Binding Factor (CTCF), also known as 11-zinc finger protein, participates in many cellular processes, including insulator activity, transcriptional regulation and organization of chromatin architecture. Based on single cell flow cytometry and single cell RNA-FISH analyses, our previous study showed that deletion of CTCF binding site led to a significantly increase of cellular variation of its target gene. However, the effect of CTCF on genome-wide landscape of cell-to-cell variation is unclear. Results: We knocked down CTCF in EL4 cells using shRNA, and conducted single cell RNA-seq on both wild type (WT) cells and CTCF-Knockdown (CTCF-KD) cells using Fluidigm C1 system. Principal component analysis of single cell RNA-seq data showed that WT and CTCF-KD cells concentrated in two different clusters on PC1, indicating gene expression profiles of WT and CTCF-KD cells were systematically different. Interestingly, GO terms including regulation of transcription, DNA binding, Zinc finger and transcription factor binding were significantly enriched in CTCF-KD-specific highly variable genes, indicating tissue-specific genes such as transcription factors were highly sensitive to CTCF level. The dysregulation of transcription factors potentially explain why knockdown of CTCF lead to systematic change of gene expression. In contrast, housekeeping genes such as rRNA processing, DNA repair and tRNA processing were significantly enriched in WT-specific highly variable genes, potentially due to a higher cellular variation of cell activity in WT cells compared to CTCF-KD cells. We further found cellular variation-increased genes were significantly enriched in down-regulated genes, indicating CTCF knockdown simultaneously reduced the expression levels and increased the expression noise of its regulated genes. Conclusions: To our knowledge, this is the first attempt to explore genome-wide landscape of cellular variation after CTCF knockdown. Our study not only advances our understanding of CTCF function in maintaining gene expression and reducing expression noise, but also provides a framework for examining gene function.

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Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex

Development ◽

10.1242/dev.126.22.5137 ◽

1999 ◽

Vol 126 (22) ◽

pp. 5137-5148 ◽

Cited By ~ 8

Author(s):

H.D. Ryoo ◽

T. Marty ◽

F. Casares ◽

M. Affolter ◽

R.S. Mann

Keyword(s):

Nuclear Localization ◽

Target Genes ◽

Dominant Negative ◽

Homeodomain Protein ◽

Hox Proteins ◽

Trimeric Complex ◽

Binding Residue ◽

Dominant Negative Form

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.

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Evaluation of tools for highly variable gene discovery from single-cell RNA-seq data

Briefings in Bioinformatics ◽

10.1093/bib/bby011 ◽

2018 ◽

Vol 20 (4) ◽

pp. 1583-1589 ◽

Cited By ~ 32

Author(s):

Shun H Yip ◽

Pak Chung Sham ◽

Junwen Wang

Keyword(s):

Gene Expression ◽

Single Cell ◽

Differentially Expressed Gene ◽

Embryonic Stem ◽

Rna Seq ◽

Software Packages ◽

Homogeneous Cell ◽

Variable Gene ◽

Cell Variation ◽

Larger Sample

Abstract Traditional RNA sequencing (RNA-seq) allows the detection of gene expression variations between two or more cell populations through differentially expressed gene (DEG) analysis. However, genes that contribute to cell-to-cell differences are not discoverable with RNA-seq because RNA-seq samples are obtained from a mixture of cells. Single-cell RNA-seq (scRNA-seq) allows the detection of gene expression in each cell. With scRNA-seq, highly variable gene (HVG) discovery allows the detection of genes that contribute strongly to cell-to-cell variation within a homogeneous cell population, such as a population of embryonic stem cells. This analysis is implemented in many software packages. In this study, we compare seven HVG methods from six software packages, including BASiCS, Brennecke, scLVM, scran, scVEGs and Seurat. Our results demonstrate that reproducibility in HVG analysis requires a larger sample size than DEG analysis. Discrepancies between methods and potential issues in these tools are discussed and recommendations are made.

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Nuclear localization of β-catenin and expression of target genes are associated with increased Wnt secretion in oral dysplasia

Oral Oncology ◽

10.1016/j.oraloncology.2019.05.010 ◽

2019 ◽

Vol 94 ◽

pp. 58-67 ◽

Cited By ~ 6

Author(s):

Montserrat Reyes ◽

Daniel Peña-Oyarzun ◽

Andrea Maturana ◽

Vicente A. Torres

Keyword(s):

Nuclear Localization ◽

Target Genes ◽

Oral Dysplasia

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Estrogen-induced transcription at individual alleles is independent of receptor level and active conformation but can be modulated by coactivators activity

Nucleic Acids Research ◽

10.1093/nar/gkz1172 ◽

2020 ◽

Vol 48 (4) ◽

pp. 1800-1810

Author(s):

Fabio Stossi ◽

Radhika D Dandekar ◽

Maureen G Mancini ◽

Guowei Gu ◽

Suzanne A W Fuqua ◽

...

Keyword(s):

Single Cell ◽

Target Genes ◽

Hormonal Stimulation ◽

Receptor Level ◽

Molecule Inhibitor ◽

Before And After ◽

Allele Specific ◽

Activity State ◽

Estrogen Stimulation

Abstract Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.

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