Inhibition of glycogen synthase kinase-3 (Gsk3) supports mouse embryonic stem cells (ESCs) by modulating Tcf3, but the critical targets downstream of Tcf3 are unclear. We analyzed the intersection ...between genome localization and transcriptome data sets to identify genes repressed by Tcf3. Among these, manipulations of Esrrb gave distinctive phenotypes in functional assays. Knockdown and knockout eliminated response to Gsk3 inhibition, causing extinction of pluripotency markers and loss of colony forming capability. Conversely, forced expression phenocopied Gsk3 inhibition or Tcf3 deletion by suppressing differentiation and sustaining self-renewal. Thus the nuclear receptor Esrrb is necessary and sufficient to mediate self-renewal downstream of Gsk3 inhibition. Leukaemia inhibitory factor (LIF) regulates ESCs through Stat3, independently of Gsk3 inhibition. Consistent with parallel operation, ESCs in LIF accommodated Esrrb deletion and remained pluripotent. These findings highlight a key role for Esrrb in regulating the naive pluripotent state and illustrate compensation among the core pluripotency factors.
► Esrrb is the principal target of Tcf3 repression in the pluripotency network ► Esrrb is essential for self-renewal downstream of Gsk3 inhibition ► Esrrb potently suppresses differentiation and sustains ESC self-renewal ► Esrrb is a core pluripotency factor but can be compensated by LIF/Stat3
Inhibition of glycogen synthase kinase 3 keeps embryonic stem cells in a naïve ground state. Surprisingly this effect is mediated mainly by upregulating expression of a single transcription factor Esrrb. Esrrb function can be compensated by independent activation of Stat3, however, demonstrating the plasticity of the core network underpinning pluripotency.
Hematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for ...the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.
► Genome-wide TF binding analysis in primary human megakaryocyte ► Regulatory network reconstruction from TF, epigenetic, and expression data ► Simultaneous binding of five TFs marks hematopoietic regulators ► Five-factor binding reveals eight genes essential for zebrafish blood development
Hematopoietic stem and progenitor cells (HSPCs) maintain the adult blood system, and their dysregulation causes a multitude of diseases. However, the differentiation journeys toward specific ...hematopoietic lineages remain ill defined, and system-wide disease interpretation remains challenging. Here, we have profiled 44 802 mouse bone marrow HSPCs using single-cell RNA sequencing to provide a comprehensive transcriptional landscape with entry points to 8 different blood lineages (lymphoid, megakaryocyte, erythroid, neutrophil, monocyte, eosinophil, mast cell, and basophil progenitors). We identified a common basophil/mast cell bone marrow progenitor and characterized its molecular profile at the single-cell level. Transcriptional profiling of 13 815 HSPCs from the c-Kit mutant (W41/W41) mouse model revealed the absence of a distinct mast cell lineage entry point, together with global shifts in cell type abundance. Proliferative defects were accompanied by reduced Myc expression. Potential compensatory processes included upregulation of the integrated stress response pathway and downregulation of proapoptotic gene expression in erythroid progenitors, thus providing a template of how large-scale single-cell transcriptomic studies can bridge between molecular phenotypes and quantitative population changes.
•Single-cell transcriptional landscape of 44 802 hematopoietic stem/progenitor cells defines entry points to 8 different blood lineages.•Comparison with 13 815 c-Kit mutant cells identifies pleiotropic changes in cell type abundance and underlying molecular profiles.
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During mouse embryonic development, pluripotent cells rapidly divide and diversify, yet the regulatory programs that define the cell repertoire for each organ remain ill-defined. To delineate ...comprehensive chromatin landscapes during early organogenesis, we mapped chromatin accessibility in 19,453 single nuclei from mouse embryos at 8.25 days post-fertilization. Identification of cell-type-specific regions of open chromatin pinpointed two TAL1-bound endothelial enhancers, which we validated using transgenic mouse assays. Integrated gene expression and transcription factor motif enrichment analyses highlighted cell-type-specific transcriptional regulators. Subsequent in vivo experiments in zebrafish revealed a role for the ETS factor FEV in endothelial identity downstream of ETV2 (Etsrp in zebrafish). Concerted in vivo validation experiments in mouse and zebrafish thus illustrate how single-cell open chromatin maps, representative of a mammalian embryo, provide access to the regulatory blueprint for mammalian organogenesis.
Metazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. ...To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development.
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•Comprehensive genome-scale resource for studying embryonic blood cell specification•Genome-scale definition of cis elements driving differential gene expression•A gene regulatory network model for hematopoiesis aiding reprogramming experiments•Analysis suggests a role for TEAD factors in hematopoietic specification
Goode, Obier, Vijayabaskar et al. isolate cells at six different stages of hematopoietic differentiation, starting from embryonic stem cells, and perform a comprehensive multi-omics analysis of this developmental pathway. The data identify regulators of hematopoietic specification and highlight the minimum requirements for the reprogramming of non-blood cells to blood.
This paper reconsiders the contemporary moral reading of women’s oppression, and revises our understanding of the practical reasons for action a victim of mistreatment acquires through her unjust ...circumstances. The paper surveys various ways of theorising victims’ moral duties to resist their own oppression, and considers objections to prior academic work arguing for the existence of an imperfect Kantian duty of resistance to oppression grounded in self-respect. These objections suggest (1) that such a duty is victim blaming; (2) that it distorts the normative direction of self-regarding duties; and (3) that consequentialist reasons are inapt for justifying self-regarding ethical responsibilities. The paper then argues that the need for normative coherence in our very concept of a moral duty is of paramount importance, and especially so in the fight against patriarchal oppression. Accordingly, we should acknowledge the salient differences between pro tanto or defeasible moral reasons and fully fledged moral duties identifying agent-relative obligatory action. The paper concludes that we better respect and defend women’s rights when first we understand them as having, at best, defeasible moral reasons to oppose their oppression; and second, ensure that we make adequate allowance for a woman’s interpretative right to choose how to respond to her oppressive circumstances.
Gains and losses in DNA methylation are prominent features of mammalian cell types. To gain insight into the mechanisms that promote shifts in DNA methylation and contribute to changes in cell fate, ...including malignant transformation, we performed genome-wide mapping of 5-methylcytosine and 5-hydroxymethylcytosine in purified mouse hematopoietic stem cells. We discovered extended regions of low methylation (canyons) that span conserved domains frequently containing transcription factors and are distinct from CpG islands and shores. About half of the genes in these methylation canyons are coated with repressive histone marks, whereas the remainder are covered by activating histone marks and are highly expressed in hematopoietic stem cells (HSCs). Canyon borders are demarked by 5-hydroxymethylcytosine and become eroded in the absence of DNA methyltransferase 3a (Dnmt3a). Genes dysregulated in human leukemias are enriched for canyon-associated genes. The new epigenetic landscape we describe may provide a mechanism for the regulation of hematopoiesis and may contribute to leukemia development.
Cellular decision-making is mediated by a complex interplay of external stimuli with the intracellular environment, in particular transcription factor regulatory networks. Here we have determined the ...expression of a network of 18 key haematopoietic transcription factors in 597 single primary blood stem and progenitor cells isolated from mouse bone marrow. We demonstrate that different stem/progenitor populations are characterized by distinctive transcription factor expression states, and through comprehensive bioinformatic analysis reveal positively and negatively correlated transcription factor pairings, including previously unrecognized relationships between Gata2, Gfi1 and Gfi1b. Validation using transcriptional and transgenic assays confirmed direct regulatory interactions consistent with a regulatory triad in immature blood stem cells, where Gata2 may function to modulate cross-inhibition between Gfi1 and Gfi1b. Single-cell expression profiling therefore identifies network states and allows reconstruction of network hierarchies involved in controlling stem cell fate choices, and provides a blueprint for studying both normal development and human disease.
Recent advances in molecular profiling provide descriptive datasets of complex differentiation landscapes including the haematopoietic system, but the molecular mechanisms defining progenitor states ...and lineage choice remain ill‐defined. Here, we employed a cellular model of murine multipotent haematopoietic progenitors (Hoxb8‐FL) to knock out 39 transcription factors (TFs) followed by RNA‐Seq analysis, to functionally define a regulatory network of 16,992 regulator/target gene links. Focussed analysis of the subnetworks regulated by the B‐lymphoid TF Ebf1 and T‐lymphoid TF Gata3 revealed a surprising role in common activation of an early myeloid programme. Moreover, Gata3‐mediated repression of Pax5 emerges as a mechanism to prevent precocious B‐lymphoid differentiation, while Hox‐mediated activation of Meis1 suppresses myeloid differentiation. To aid interpretation of large transcriptomics datasets, we also report a new method that visualises likely transitions that a progenitor will undergo following regulatory network perturbations. Taken together, this study reveals how molecular network wiring helps to establish a multipotent progenitor state, with experimental approaches and analysis tools applicable to dissecting a broad range of both normal and perturbed cellular differentiation landscapes.
SYNOPSIS
Principles of gene regulation underlying cellular differentiation remain poorly understood. Combining sequencing and computational methods, this resource defines the complex transcription factor (TF) network of multipotent blood progenitors, and reveals how coregulation by lineage‐associated transcription factors maintains murine hematopoiesis.
Individual depletion of 39 TFs identifies the gene networks depending on them in a defined lympho‐myeloid progenitor cell line model.
Some 17,000 TF‐target interactions reveal co‐regulation, TF regulatory hierarchies, and target gene modules.
Lymphoid TFs Gata3 and Ebf1 contribute to a CEBPA‐dependent early myeloid program.
Scoring inferred directions of cellular transition using single‐cell RNA sequencing landscapes aids interpretation of the perturbation data.
Functional definition of a transcription factor network reveals regulatory interdependencies during early blood development.
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