Cell fate decision in erythropoiesis: Insights from multiomics studies.

Every second, the body produces 2 million red blood cells through a process called erythropoiesis. Erythropoiesis is hierarchical in that it results from a series of cell fate decisions whereby hematopoietic stem cells progress toward the erythroid lineage. Single-cell transcriptomic and proteomic approaches have revolutionized the way we understand erythropoiesis, revealing it to be a gradual process that underlies a progressive restriction of fate potential driven by quantitative changes in lineage-specifying transcription factors. Despite these major advances, we still know very little about what cell fate decision entails at the molecular level. Novel approaches that simultaneously measure additional properties in single cells, including chromatin accessibility, transcription factor binding, and/or cell surface proteins are being developed at a fast pace, providing the means to exciting new advances in the near future. In this review, we briefly summarize the main findings obtained from single-cell studies of erythropoiesis, highlight outstanding questions, and suggest recent technological advances to address them.

Chromatin and transcription factor profiling in rare stem cell populations using CUT&Tag.

Muscle stem cells (MuSCs) are a rare stem cell population that provides myofibers with a remarkable capacity to regenerate after tissue injury. Here, we have adapted the Cleavage Under Target and Tagmentation technology to the mapping of the chromatin landscape and transcription factor binding in 50,000 activated MuSCs isolated from injured mouse hindlimb muscles. We have applied this same approach to human CD34+ hematopoietic stem and progenitor cells. This protocol could be adapted to any rare stem cell population. For complete details on the use and execution of this protocol, please refer to Robinson et al. (2021).

Single-cell profiling of human bone marrow progenitors reveals mechanisms of failing erythropoiesis in Diamond-Blackfan anemia.

Ribosome dysfunction underlies the pathogenesis of many cancers and heritable ribosomopathies. Here, we investigate how mutations in either ribosomal protein large (RPL) or ribosomal protein small (RPS) subunit genes selectively affect erythroid progenitor development and clinical phenotypes in Diamond-Blackfan anemia (DBA), a rare ribosomopathy with limited therapeutic options. Using single-cell assays of patient-derived bone marrow, we delineated two distinct cellular trajectories segregating with ribosomal protein genotypes. Almost complete loss of erythroid specification was observed in RPS-DBA. In contrast, we observed relative preservation of qualitatively abnormal erythroid progenitors and precursors in RPL-DBA. Although both DBA genotypes exhibited a proinflammatory bone marrow milieu, RPS-DBA was characterized by erythroid differentiation arrest, whereas RPL-DBA was characterized by preserved GATA1 expression and activity. Compensatory stress erythropoiesis in RPL-DBA exhibited disordered differentiation underpinned by an altered glucocorticoid molecular signature, including reduced ZFP36L2 expression, leading to milder anemia and improved corticosteroid response. This integrative analysis approach identified distinct pathways of erythroid failure and defined genotype-phenotype correlations in DBA. These findings may help facilitate therapeutic target discovery.

Absolute quantification of transcription factors in human erythropoiesis using selected reaction monitoring mass spectrometry.

Quantitative changes in transcription factor (TF) abundance regulate dynamic cellular processes, including cell fate decisions. Protein copy number provides information about the relative stoichiometry of TFs that can be used to determine how quantitative changes in TF abundance influence gene regulatory networks. In this protocol, we describe a targeted selected reaction monitoring (SRM)-based mass-spectrometry method to systematically measure the absolute protein concentration of nuclear TFs as human hematopoietic stem and progenitor cells differentiate along the erythropoietic lineage. For complete details on the use and execution of this protocol, please refer to Gillespie et al. (2020).

A Nuclear Stress Pathway that Parallels Cytoplasmic Stress Granule Formation.

Stress adaptation is exploited by cancer cells to survive and proliferate under adverse conditions. Survival pathways induced by stress are thus highly promising therapeutic targets. One key pathway involves formation of cytoplasmic stress granules, which regulate the location, stability, and translation of specific mRNAs. Here, we describe a transcriptional stress response that is triggered by similar stressors and characterized by accumulation of RepoMan (cell division cycle associated 2) at nuclear stress foci (nucSF). Formation of these structures is reversible, and they are distinct from known nuclear organelles and stress bodies. Immunofluorescence analysis revealed accumulation of heterochromatic markers, and increased association of RepoMan with the adenylate cyclase 2 (ADCY2) gene locus in stressed cells accompanied reduced levels of ADCY2 mRNA and protein. Quantitative comparison of the RepoMan interactome in stressed vs. unstressed cells identified condensin II as a nucSF factor, suggesting their functional association in the establishment and/or maintenance of these facultative heterochromatic domains.

Absolute Quantification of Transcription Factors Reveals Principles of Gene Regulation in Erythropoiesis.

Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy number of TFs in the nucleus is limited. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first gene regulatory network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs' cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that, in the nucleus, co-repressors are dramatically more abundant than co-activators at the protein level, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.

Single-Cell Proteomics Reveal that Quantitative Changes in Co-expressed Lineage-Specific Transcription Factors Determine Cell Fate.

Hematopoiesis provides an accessible system for studying the principles underlying cell-fate decisions in stem cells. Proposed models of hematopoiesis suggest that quantitative changes in lineage-specific transcription factors (LS-TFs) underlie cell-fate decisions. However, evidence for such models is lacking as TF levels are typically measured via RNA expression rather than by analyzing temporal changes in protein abundance. Here, we used single-cell mass cytometry and absolute quantification by mass spectrometry to capture the temporal dynamics of TF protein expression in individual cells during human erythropoiesis. We found that LS-TFs from alternate lineages are co-expressed, as proteins, in individual early progenitor cells and quantitative changes of LS-TFs occur gradually rather than abruptly to direct cell-fate decisions. Importantly, upregulation of a megakaryocytic TF in early progenitors is sufficient to deviate cells from an erythroid to a megakaryocyte trajectory, showing that quantitative changes in protein abundance of LS-TFs in progenitors can determine alternate cell fates.

Epigenetic Activation of Pro-angiogenic Signaling Pathways in Human Endothelial Progenitors Increases Vasculogenesis.

Human endothelial colony-forming cells (ECFCs) represent a promising source of adult stem cells for vascular repair, yet their regenerative capacity is limited. Here, we set out to understand the molecular mechanism restricting the repair function of ECFCs. We found that key pro-angiogenic pathways are repressed in ECFCs due to the presence of bivalent (H3K27me3/H3K4me3) epigenetic marks, which decreases the cells' regenerative potential. Importantly, ex vivo treatment with a combination of epigenetic drugs that resolves bivalent marks toward the transcriptionally active H3K4me3 state leads to the simultaneous activation of multiple pro-angiogenic signaling pathways (VEGFR, CXCR4, WNT, NOTCH, SHH). This in turn results in improved capacity of ECFCs to form capillary-like networks in vitro and in vivo. Furthermore, restoration of perfusion is accelerated upon transplantation of drug-treated ECFCs in a model of hindlimb ischemia. Thus, ex vivo treatment with epigenetic drugs increases the vascular repair properties of ECFCs through transient activation of pro-angiogenic signaling pathways.

UTX demethylase activity is required for satellite cell-mediated muscle regeneration.

The X chromosome-encoded histone demethylase UTX (also known as KDM6A) mediates removal of repressive trimethylation of histone H3 lysine 27 (H3K27me3) to establish transcriptionally permissive chromatin. Loss of UTX in female mice is embryonic lethal. Unexpectedly, male UTX-null mice escape embryonic lethality due to expression of UTY, a paralog that lacks H3K27 demethylase activity, suggesting an enzyme-independent role for UTX in development and thereby challenging the need for active H3K27 demethylation in vivo. However, the requirement for active H3K27 demethylation in stem cell-mediated tissue regeneration remains untested. Here, we employed an inducible mouse KO that specifically ablates Utx in satellite cells (SCs) and demonstrated that active H3K27 demethylation is necessary for muscle regeneration. Loss of UTX in SCs blocked myofiber regeneration in both male and female mice. Furthermore, we demonstrated that UTX mediates muscle regeneration through its H3K27 demethylase activity, as loss of demethylase activity either by chemical inhibition or knock-in of demethylase-dead UTX resulted in defective muscle repair. Mechanistically, dissection of the muscle regenerative process revealed that the demethylase activity of UTX is required for expression of the transcription factor myogenin, which in turn drives differentiation of muscle progenitors. Thus, we have identified a critical role for the enzymatic activity of UTX in activating muscle-specific gene expression during myofiber regeneration and have revealed a physiological role for active H3K27 demethylation in vivo.

UTX inhibition as selective epigenetic therapy against TAL1-driven T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous group of hematological tumors composed of distinct subtypes that vary in their genetic abnormalities, gene expression signatures, and prognoses. However, it remains unclear whether T-ALL subtypes differ at the functional level, and, as such, T-ALL treatments are uniformly applied across subtypes, leading to variable responses between patients. Here we reveal the existence of a subtype-specific epigenetic vulnerability in T-ALL by which a particular subgroup of T-ALL characterized by expression of the oncogenic transcription factor TAL1 is uniquely sensitive to variations in the dosage and activity of the histone 3 Lys27 (H3K27) demethylase UTX/KDM6A. Specifically, we identify UTX as a coactivator of TAL1 and show that it acts as a major regulator of the TAL1 leukemic gene expression program. Furthermore, we demonstrate that UTX, previously described as a tumor suppressor in T-ALL, is in fact a pro-oncogenic cofactor essential for leukemia maintenance in TAL1-positive (but not TAL1-negative) T-ALL. Exploiting this subtype-specific epigenetic vulnerability, we propose a novel therapeutic approach based on UTX inhibition through in vivo administration of an H3K27 demethylase inhibitor that efficiently kills TAL1-positive primary human leukemia. These findings provide the first opportunity to develop personalized epigenetic therapy for T-ALL patients.

Epigenetic regulation of endothelial-cell-mediated vascular repair.

Maintenance of vascular integrity is essential for the prevention of vascular disease and for recovery following cardiovascular, cerebrovascular and peripheral vascular events including limb ischemia, heart attack and stroke. Endothelial stem/progenitor cells have recently gained considerable interest due to their potential use in stem cell therapies to mediate revascularization after ischemic injury. Therefore, there is an urgent need to understand fundamental mechanisms regulating vascular repair in specific cell types to develop new beneficial therapeutic interventions. In this review, we highlight recent studies demonstrating that epigenetic mechanisms (including post-translational modifications of DNA and histones as well as non-coding RNA-mediated processes) play essential roles in the regulation of endothelial stem/progenitor cell functions through modifying chromatin structure. Furthermore, we discuss the potential of using small molecules that modulate the activities of epigenetic enzymes to enhance the vascular repair function of endothelial cells and offer insight on potential strategies that may accelerate clinical applications.

Trichostatin A enhances vascular repair by injected human endothelial progenitors through increasing the expression of TAL1-dependent genes.

A major goal of cell therapy for vascular diseases is to promote revascularization through the injection of endothelial stem/progenitor cells. The gene regulatory mechanisms that underlie endothelial progenitor-mediated vascular repair, however, remain elusive. Here, we identify the transcription factor TAL1/SCL as a key mediator of the vascular repair function of primary human endothelial colony-forming cells (ECFCs). Genome-wide analyses in ECFCs demonstrate that TAL1 activates a transcriptional program that promotes cell adhesion and migration. At the mechanistic level, we show that TAL1 upregulates the expression of migratory and adhesion genes through recruitment of the histone acetyltransferase p300. Based on these findings, we establish a strategy that enhances the revascularization efficiency of ECFCs after ischemia through ex vivo priming with the histone deacetylase inhibitor TSA. Thus, small molecule epigenetics drugs are effective tools for modifying the epigenome of stem/progenitor cells prior to transplantation as a means to enhance their therapeutic potential.

A method for cell type marker discovery by high-throughput gene expression analysis of mixed cell populations.

BACKGROUND: Gene transcripts specifically expressed in a particular cell type (cell-type specific gene markers) are useful for its detection and isolation from a tissue or other cell mixtures. However, finding informative marker genes can be problematic when working with a poorly characterized cell type, as markers can only be unequivocally determined once the cell type has been isolated. We propose a method that could identify marker genes of an uncharacterized cell type within a mixed cell population, provided that the proportion of the cell type of interest in the mixture can be estimated by some indirect method, such as a functional assay. RESULTS: We show that cell-type specific gene markers can be identified from the global gene expression of several cell mixtures that contain the cell type of interest in a known proportion by their high correlation to the concentration of the corresponding cell type across the mixtures. CONCLUSIONS: Genes detected using this high-throughput strategy would be candidate markers that may be useful in detecting or purifying a cell type from a particular biological context. We present an experimental proof-of-concept of this method using cell mixtures of various well-characterized hematopoietic cell types, and we evaluate the performance of the method in a benchmark that explores the requirements and range of validity of the approach.

Lentiviral-mediated knockdown during ex vivo erythropoiesis of human hematopoietic stem cells.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments. Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Differential genomic targeting of the transcription factor TAL1 in alternate haematopoietic lineages.

TAL1/SCL is a master regulator of haematopoiesis whose expression promotes opposite outcomes depending on the cell type: differentiation in the erythroid lineage or oncogenesis in the T-cell lineage. Here, we used a combination of ChIP sequencing and gene expression profiling to compare the function of TAL1 in normal erythroid and leukaemic T cells. Analysis of the genome-wide binding properties of TAL1 in these two haematopoietic lineages revealed new insight into the mechanism by which transcription factors select their binding sites in alternate lineages. Our study shows limited overlap in the TAL1-binding profile between the two cell types with an unexpected preference for ETS and RUNX motifs adjacent to E-boxes in the T-cell lineage. Furthermore, we show that TAL1 interacts with RUNX1 and ETS1, and that these transcription factors are critically required for TAL1 binding to genes that modulate T-cell differentiation. Thus, our findings highlight a critical role of the cellular environment in modulating transcription factor binding, and provide insight into the mechanism by which TAL1 inhibits differentiation leading to oncogenesis in the T-cell lineage.