scholarly journals Coordinated changes in gene expression kinetics underlie both mouse and human erythroid maturation

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Melania Barile ◽  
Ivan Imaz-Rosshandler ◽  
Isabella Inzani ◽  
Shila Ghazanfar ◽  
Jennifer Nichols ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Current Velocity ◽  
Fetal Liver ◽  
Step Change ◽  
Chimeric Mouse ◽  
Starting Point ◽  
Erythroid Maturation ◽  
Mutant Cells ◽  
Expression Kinetics

Abstract Background Single-cell technologies are transforming biomedical research, including the recent demonstration that unspliced pre-mRNA present in single-cell RNA-Seq permits prediction of future expression states. Here we apply this RNA velocity concept to an extended timecourse dataset covering mouse gastrulation and early organogenesis. Results Intriguingly, RNA velocity correctly identifies epiblast cells as the starting point, but several trajectory predictions at later stages are inconsistent with both real-time ordering and existing knowledge. The most striking discrepancy concerns red blood cell maturation, with velocity-inferred trajectories opposing the true differentiation path. Investigating the underlying causes reveals a group of genes with a coordinated step-change in transcription, thus violating the assumptions behind current velocity analysis suites, which do not accommodate time-dependent changes in expression dynamics. Using scRNA-Seq analysis of chimeric mouse embryos lacking the major erythroid regulator Gata1, we show that genes with the step-changes in expression dynamics during erythroid differentiation fail to be upregulated in the mutant cells, thus underscoring the coordination of modulating transcription rate along a differentiation trajectory. In addition to the expected block in erythroid maturation, the Gata1-chimera dataset reveals induction of PU.1 and expansion of megakaryocyte progenitors. Finally, we show that erythropoiesis in human fetal liver is similarly characterized by a coordinated step-change in gene expression. Conclusions By identifying a limitation of the current velocity framework coupled with in vivo analysis of mutant cells, we reveal a coordinated step-change in gene expression kinetics during erythropoiesis, with likely implications for many other differentiation processes.

scholarly journals Coordinated Changes in Gene Expression Kinetics Underlie both Mouse and Human Erythroid Maturation

2020 ◽  
Author(s):  
Melania Barile ◽  
Ivan Imaz-Rosshandler ◽  
Isabella Inzani ◽  
Shila Ghazanfar ◽  
Jennifer Nichols ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Current Velocity ◽  
Fetal Liver ◽  
Step Change ◽  
Chimeric Mouse ◽  
Starting Point ◽  
Erythroid Maturation ◽  
Mutant Cells ◽  
Expression Kinetics

AbstractSingle cell technologies are transforming biomedical research, including the recent demonstration that unspliced pre-mRNA present in single cell RNA-Seq permits prediction of future expression states. Here we applied this ‘RNA velocity concept’ to an extended timecourse dataset covering mouse gastrulation and early organogenesis. Intriguingly, RNA velocity correctly identified epiblast cells as the starting point, but several trajectory predictions at later stages were inconsistent with both real time ordering and existing knowledge. The most striking discrepancy concerned red blood cell maturation, with velocity-inferred trajectories opposing the true differentiation path. Investigating the underlying causes revealed a group of genes with a coordinated step-change in transcription, thus violating the assumptions behind current velocity analysis suites, which do not accommodate time-dependent changes in expression dynamics. Using scRNA-Seq analysis of chimeric mouse embryos lacking the major erythroid regulator Gata1, we show that genes with the step-changes in expression dynamics during erythroid differentiation fail to be up-regulated in the mutant cells, thus underscoring the coordination of modulating transcription rate along a differentiation trajectory. In addition to the expected block in erythroid maturation, the Gata1- chimera dataset revealed induction of PU.1 and expansion of megakaryocyte progenitors. Finally, we show that erythropoiesis in human fetal liver is similarly characterized by a coordinated step-change in gene expression. By identifying a limitation of the current velocity framework coupled with in vivo analysis of mutant cells, we reveal a coordinated step-change in gene expression kinetics during erythropoiesis, with likely implications for many other differentiation processes.

scholarly journals Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1092-1092
Author(s):  
Masahiro Marshall Nakagawa ◽  
Ryosaku Inagaki ◽  
Yutaka Kuroda ◽  
Yasuhito Nannya ◽  
Lanying Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Single Cell ◽  
Research Funding ◽  
Inflammatory Responses ◽  
Inflammatory Microenvironment ◽  
Single Cell Sequencing ◽  
Clonal Hematopoiesis ◽  
Otsuka Pharmaceutical ◽  
Mutant Cells

Abstract Background Clonal hematopoiesis (CH) in apparently healthy individuals has been drawing an increasing attention of recent years due to its implication in the risk of hematopoietic malignancies and also in cardiovascular complications. However, our knowledge on CH has been largely based on genetic studies, while few functional analyses have been performed using human materials with most studies being confined to artificial models using mice. The major challenge here is the difficulty of isolating CH clones from wild-type (WT) cells in human bone marrow (BM) to elucidate the effect of CH-mutations. Methods To investigate cellular phenotypes of mutated and WT cells separately in CH, we developed a Fluidigm C1-based single cell-sequencing platform for simultaneous genotyping and gene expression analysis. We analyzed a total of 10,178 hematopoietic stem/progenitors (HSPCs) derived from BM of patients with (n=11) and without (n=17) CH. Results In the analysis of HSPCs from CH(−) elderly individuals, we found a significant positive correlation between age and expression of gene sets implicated in inflammatory responses including TGFβ signalling and IL-6. We next analysed CH(+) samples, including those with mutations in TET2, DNMT3A, SF3B1 and IDH1 in which we investigated the pathways enriched in differentially expressed genes between mutated and unmutated cells. Regardless of mutation type, mutant cells showed an upregulation of genes implicated in an enhanced cell proliferation, while genes related to inflammatory responses were significantly downregulated. These results suggest that mutant HSPCs show an enhanced cell proliferation and an attenuated response to an inflammatory microenvironment in aged BM, compared with endogenous WT counterparts. Based on these observations, we further investigated the role of the BM microenvironment in CH, in which we compared the phenotype of WT cells in age-matched CH(+) and CH(−) cases. Compared with those from CH(−) cases, WT cells from these CH(+) cases showed an enhanced response to proinflammatory cytokines, including IL-6, interferons and TNF, suggesting a possibility that CH(+) BM might be characterized by a more enhanced inflammatory microenvironment, compared with CH(−) BM. To understand the different phenotype of WT cells between CH(+) and CH(−) cases, we next investigated a possible effect of mutant cells on endogenous WT cells. Such an effect was first documented in an IDH1-mutated CH case. IDH1-mutated cells showed a gene expression profile suggestive of an enhanced cell proliferation compared with endogenous IDH1-WT cells (WT IDH1). However, on the basis of comparison of gene expression between WT cells from CH(−) cases, it was better explained by suppressed proliferation of endogenous WT cells, rather than enhanced proliferation of mutant cells. In agreement with this, mouse BM cells treated with 2-hydroxyglutalate (2HG), an oncometabolite produced by IDH1-mutant cells, mimicked the endogenous WT cells in IDH1-mutated cases, including downregulated E2F target genes and upregulated inflammation-related genes, compared with control BM cells. A non-cell autonomous effect of mutations was also seen in cases with TET2-mutated CH, in which not only TET2-mutated cells but also endogenous WT cells exhibited a significantly different gene expression profile, compared with those from CH(−) cases. Of interest, when BM cells from WT Ly5.1/5.2 mice were co-transplanted with those from heterozygous conditional Tet2 knock-out (Ly5.2) or WT (Ly5.2) mice into lethally irradiated mice (Ly5.1) and flow-sorted Lin − WT donor competitor cells were analysed using single cell sequencing, the WT HSC-like cells co-transplanted with Tet2-mutant competitors exhibited enhanced cell proliferation and IFNα and IFNγ pathway genes, compared with those co-transplanted with WT competitors. Conclusions Taken together, these results suggest that mutant cells in CH(+) BM have non-cell autonomous effects on endogenous WT cells, which might be responsible for an accelerated inflammatory microenvironment of aged BM, favor positive selection of CH-clones, and also affect the phenotype of endogenous WT cells, contributing to the pathogenesis of CH. Disclosures Nakagawa: Sumitomo Dainippon Pharma Oncology, Inc.: Research Funding. Inagaki: Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Yoda: Chordia Therapeutics Inc.: Research Funding. Ogawa: Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding.

scholarly journals Erythroid Maturation and Globin Gene Expression in Mice With Combined Deficiency of NF-E2 and Nrf-2

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3459-3466 ◽  
Author(s):  
Florence Martin ◽  
Jan M. van Deursen ◽  
Ramesh A. Shivdasani ◽  
Carl W. Jackson ◽  
Amber G. Troutman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Globin Gene ◽  
Liver Cells ◽  
Regulatory Sequences ◽  
Fetal Liver Cells ◽  
Erythroid Maturation ◽  
Deficient Mice ◽  
Globin Gene Expression ◽  
Combined Deficiency

NF-E2 binding sites, located in distant regulatory sequences, may be important for high level α- and β-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2–related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2–deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of α- and β-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2–deficient mice is not the result of compensation by Nrf-2.

Unravelling Heterogeneity of Stem and Progenitor Cells in Myeloid Neoplasms Through Single Cell Multi-omics

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-31-SCI-31
Author(s):  
Adam J. Mead
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Single Cell ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Clonal Evolution ◽  
Genomic Analysis ◽  
Stem And Progenitor Cells ◽  
Cancer Medicine ◽  
Mutant Cells

Intratumoural heterogeneity (ITH) underlies many of the challenges we face in cancer medicine, including therapy-resistance, disease progression/evolution and relapse after seemingly effective therapy. Distinct tumour cell subpopulations selectively evade therapy and drive disease-progression and technologies that reveal key aspects of ITH are therefore critical for the application of precision cancer medicine. Although bulk genomic analysis has without question provided many insights into genetic ITH, this approach faces a number of fundamental limitations: ITH in cancer occurs at many levels, not restricted to genetics (mutations) but also other factors, such as presence of cancer stem cells in some tumours. Furthermore, bulk genomic analysis reveals patterns of somatic mutations, but not their molecular consequences within distinct (and therapy-resistant) cancer subclones. Whilst many of the scientific questions relating to ITH have remained the same over many decades, our ability to address these questions has advanced dramatically not least because of advances in technology. Ultimately, as the unit of evolution and clonal selection by therapy in cancer is the cell, techniques that resolve heterogeneity at the single-cell level are ideally placed to unravel ITH and provide entirely new insights into cancer biology, with enormous potential to accelerate the development of new approaches to improve outcomes for patients. However, the lack of coverage across key mutation hotspots when studying cancers using single-cell RNA-sequencing techniques has precluded the correlation of genetic and transcriptional readouts from the same single cell, limiting their application to the study of tumors. To overcome such limitation, we developed TARGET-seq, a single cell multi-omic method for the high-sensitivity detection of mutations within single cells in parallel with whole transcriptome analysis. TARGET-seq achieved extremely low allelic dropout rates, allowing resolution of clonal hierarchies with over 98% accuracy, while obtaining unbiased high quality transcriptomes from the same single cell. We have applied TARGET-seq to the study of over ten thousand haematopoietic stem and progenitor cells (HSPCs) from JAK2-mutant myeloproliferative neoplasms. This analysis revealed a high degree of genetic heterogeneity, identifying both linear and branching patterns of clonal evolution. At the transcriptome level different genetic subclones showed distinct transcriptional signatures, indicating that each of them was molecularly distinct. Wild-type cells from MPN patients also showed disrupted gene expression as compared to cells from normal donors, upregulating molecular pathways associated with inflammation (TNFα, TGFβ and IFN signalling). This suggests cell-extrinsic effects disrupting gene expression in non-mutant cells, which has been shown to have prognostic significance and might underlie therapy response. Moreover, TARGET-seq analysis allowed us to identify putative biomarkers of JAK2V617F mutant cells, including novel therapeutic targets to selectively eradicate JAK2-mutant cells and importantly, potential candidates for antibody-based immunotherapy. Analysis of samples from MPN patients undergoing disease transformation to Acute Myeloid Leukemia (sAML) revealed striking patterns of clonal evolution in different immunophenotypically-defined cell types. We identified pre-leukemic and leukemic subclones emerging from hematopoietic stem cells rather than more mature progenitors, in contrast to evolution patterns in de novo AML, which might indicate different cancer stem cell reservoirs. In summary, TARGET-seq allowed us to identify distinct and biologically relevant molecular signatures of different genetic subclones of HSPCs in myeloproliferative neoplasms. TARGET-seq could also be broadly applied to the study of other types of tumours, providing a powerful tool for biomarker and therapeutic target discovery for precision medicine. Disclosures Mead: Bristol Myers-Squibb: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel/accommodation expenses, Research Funding, Speakers Bureau; CTI: Honoraria, Research Funding; Celgene: Consultancy, Research Funding.

scholarly journals Reconstructing Early Erythroid Development In Vivo Using Single-Cell Transcriptomics

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1195-1195
Author(s):  
Betsabeh Khoramian Tusi ◽  
Samuel Wolock ◽  
Caleb Weinreb ◽  
Yung Hwang ◽  
Daniel Hidalgo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Single Cell ◽  
Fetal Liver ◽  
Terminal Differentiation ◽  
Molecular Study ◽  
S Phase ◽  
Hematopoietic Tissue ◽  
Erythroid Progenitors ◽  
Erythroid Development

Abstract Erythroid differentiation may be divided into two broad stages: early development, and terminal differentiation. Early development was first explored using the colony-formation potential of hematopoietic tissue. This approach identified multi-potential progenitors (MPP) and unipotential erythroid progenitors that form 'bursts' (BFUe) and smaller colonies (CFUe). Early erythroid development is followed by erythroid terminal differentiation (ETD), which profoundly remodels erythroblasts into enucleated red cells. The molecular study of ETD was fundamentally transformed with the development of cell-surface marker strategies that identify sequential stage-specific erythroblasts in hematopoietic tissue. By contrast, there have been no strategies that systematically identify the entire cellular and molecular trajectory of the early erythroid lineage as it first arises from the MPP and progresses to the point where the ETD program is activated. To address this gap, we undertook single-cell transcriptomics using the InDrop-seq platform (Klein et al. Cell 161:1187 2015). We analyzed Kit+ cells in the bone marrow of mice in the basal state, mice stimulated with Erythropoietin (Epo) for 48 hours, or fetal liver cells. We used Next-Generation Sequencing data to construct k-nearest neighbor (kNN) graphs of cell states for each condition, and extracted the erythroid trajectory using Population Balance Analysis (PBA), a novel computational approach that predicts differentiation fates from single cell RNA profiles. We identified early erythroid developmental stages based on the modeled probability of erythroid commitment, gene expression dynamics, and the architecture of the kNN graph. By screening for appropriate cell surface markers, we developed a flow-cytometric strategy that isolates sub-populations corresponding to regions of the kNN graph, including sub-regions of the erythroid trajectory. This allowed validation of gene expression patterns and of cell fate predictions using in vitro colony formation assays. The earliest stage in the erythroid trajectory, the Erythroid/Basophil MPP stage (E/B-MPP) contains progenitors that emerge from MPPs, predicted to remain mutli-potential but be biased towards bipotential erythroid/basophil and eryrthroid/megakaryocytic fates. This stage is characterized by rapidly changing gene expression profiles. Genes whose expression correlates with the probability of erythroid commitment include both known transcriptional regulators of erythropoiesis such as GATA1, GATA2, Ldb1 and Klf1, as well as novel candidates. Downstream from the E/B-MPP, the two-dimensional projection of the kNN graph becomes a narrow bottleneck, indicating a transient stage. Here the erythroid trajectory contains cells with rapidly increasing probability of erythroid commitment (Emerging Erythroid Progenitors, EEP). The bottleneck connects to a bulge-like region in which progenitors have an extremely high probability of attaining the erythroid fate (committed erythroid progenitors, CEP). This region contains the majority of the marrow's committed erythroid progenitors, and functions as an amplification module, increasing in size in Epo-stimulated marrow and in the fetal liver. Functionally, cells in the bottleneck region give rise to multifocal erythroid colonies (early and late BFUe), whereas cells in the CEP amplification module give rise to unifocal erythroid colonies, including the CFUe. Therefore, the ability of a progenitor to give rise to either multifocal or unifocal colonies correlates closely with molecular stage. Cells in the CEP module express a unique set of genes, induced at the module entry, and repressed at its exit. These include growth-factor receptors mst1r, ryk and il17ra. Their ligands, MSP, Wnt5a and IL17a, are novel regulators of erythropoiesis, either stimulating or inhibiting the formation of erythroid colonies. Exit from the CEP module is marked by a rapid switch, in which the repression of CEP genes coincides with induction of the ETD program. Remarkably, this switch is synchronized with expression of G1/S and S phase genes, underlying a role for S phase progression in ETD activation (Pop et al., PLoS Biology 2010). Our work charts the erythroid trajectory of murine hematopoietic tissue, identifying developmental milestones, setting the stage for their molecular study and for discovery of novel erythroid regulators. Disclosures Klein: OneCell Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Erythroid Maturation and Globin Gene Expression in Mice With Combined Deficiency of NF-E2 and Nrf-2

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3459-3466 ◽  
Author(s):  
Florence Martin ◽  
Jan M. van Deursen ◽  
Ramesh A. Shivdasani ◽  
Carl W. Jackson ◽  
Amber G. Troutman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Globin Gene ◽  
Liver Cells ◽  
Regulatory Sequences ◽  
Fetal Liver Cells ◽  
Erythroid Maturation ◽  
Deficient Mice ◽  
Globin Gene Expression ◽  
Combined Deficiency

Abstract NF-E2 binding sites, located in distant regulatory sequences, may be important for high level α- and β-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2–related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2–deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of α- and β-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2–deficient mice is not the result of compensation by Nrf-2.

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