Hematopoiesis has long served as a paradigm of stem cell biology and tissue homeostasis. In the past decade, the genomics revolution has ushered in powerful new methods for investigating the ...hematopoietic system that have provided transformative insights into its biology. As part of the advances in genomics, increasingly accurate deep sequencing and novel methods of cell tracking have revealed hematopoiesis to be more of a continuous and less of a discrete and punctuated process than originally envisioned. In part, this continuous nature of hematopoiesis is made possible by the emergent outcomes of vast, interconnected regulatory networks that influence cell fates and lineage commitment. It is also becoming clear how these mechanisms are modulated by genetic variation present throughout the population. This review describes how these recently uncovered complexities are reshaping our concept of tissue development and homeostasis while opening up a more comprehensive future understanding of hematopoiesis.
Liggett and Sankaran review our evolving understanding of the mechanisms of hematopoiesis, emphasizing how insights from genomics technologies are driving a deeper understanding of lineage commitment, gene regulatory mechanisms, and the effects of human genetic variation.
Anemia is a major source of morbidity and mortality worldwide. Here we review recent insights into how red blood cells (RBCs) are produced, the pathogenic mechanisms underlying various forms of ...anemia, and novel therapies derived from these findings. It is likely that these new insights, mainly arising from basic scientific studies, will contribute immensely to both the understanding of frequently debilitating forms of anemia and the ability to treat affected patients. Major worldwide diseases that are likely to benefit from new advances include the hemoglobinopathies (β-thalassemia and sickle cell disease); rare genetic disorders of RBC production; and anemias associated with chronic kidney disease, inflammation, and cancer. Promising new approaches to treatment include drugs that target recently defined pathways in RBC production, iron metabolism, and fetal globin-family gene expression, as well as gene therapies that use improved viral vectors and newly developed genome editing technologies.
Summary
Red blood cells (RBCs) are generated from haematopoietic stem and progenitor cells (HSPCs) through the step‐wise process of differentiation known as erythropoiesis. In this review, we discuss ...our current understanding of erythropoiesis and highlight recent advances in this field. During embryonic development, erythropoiesis occurs in three distinct waves comprising first, the yolk sac‐derived primitive RBCs, followed sequentially by the erythro‐myeloid progenitor (EMP) and HSPC‐derived definitive RBCs. Recent work has highlighted the complexity and variability that may exist in the hierarchical arrangement of progenitors responsible for erythropoiesis. Using recently defined cell surface markers, it is now possible to enrich for erythroid progenitors and precursors to a much greater extent than has been possible before. While a great deal of knowledge has been gained on erythropoiesis from model organisms, our understanding of this process is currently being refined through human genetic studies. Genes mutated in erythroid disorders can now be identified more rapidly by the use of next‐generation sequencing techniques. Genome‐wide association studies on erythroid traits in healthy populations have also revealed new modulators of erythropoiesis. All of these recent developments have significant promise not only for increasing our understanding of erythropoiesis, but also for improving our ability to intervene when RBC production is perturbed in disease.
Recent technological advances have enabled massively parallel chromatin profiling with scATAC-seq (single-cell assay for transposase accessible chromatin by sequencing). Here we present ATAC with ...select antigen profiling by sequencing (ASAP-seq), a tool to simultaneously profile accessible chromatin and protein levels. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA for clonal tracking, capturing three distinct modalities in single cells. ASAP-seq uses a bridging approach that repurposes antibody:oligonucleotide conjugates designed for existing technologies that pair protein measurements with single-cell RNA sequencing. Together with DOGMA-seq, an adaptation of CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) for measuring gene activity across the central dogma of gene regulation, we demonstrate the utility of systematic multi-omic profiling by revealing coordinated and distinct changes in chromatin, RNA and surface proteins during native hematopoietic differentiation and peripheral blood mononuclear cell stimulation and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.
Hematopoiesis requires balance between self-renewal of stem cells and differentiation into mature blood cells, orchestrated by pathways such as thrombopoietin signaling. In this issue of Cell, ...Tsutsumi et al. report the structure of the thrombopoietin ligand-receptor complex and demonstrate the potential to decouple its roles in self-renewal and hematopoietic differentiation.
Hematopoiesis requires balance between self-renewal of stem cells and differentiation into mature blood cells, orchestrated by pathways such as thrombopoietin signaling. In this issue of Cell, Tsutsumi et al. report the structure of the thrombopoietin ligand-receptor complex and demonstrate the potential to decouple its roles in self-renewal and hematopoietic differentiation.
Lineage tracing provides key insights into the fate of individual cells in complex organisms. Although effective genetic labeling approaches are available in model systems, in humans, most approaches ...require detection of nuclear somatic mutations, which have high error rates, limited scale, and do not capture cell state information. Here, we show that somatic mutations in mtDNA can be tracked by single-cell RNA or assay for transposase accessible chromatin (ATAC) sequencing. We leverage somatic mtDNA mutations as natural genetic barcodes and demonstrate their utility as highly accurate clonal markers to infer cellular relationships. We track native human cells both in vitro and in vivo and relate clonal dynamics to gene expression and chromatin accessibility. Our approach should allow clonal tracking at a 1,000-fold greater scale than with nuclear genome sequencing, with simultaneous information on cell state, opening the way to chart cellular dynamics in human health and disease.
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•Somatic mtDNA mutations can track cellular relationships and hierarchies in vitro•Single-cell genomic assays faithfully detect mtDNA mutations•Lineage inference can be combined with gene expression or chromatin state profiles•mtDNA mutations enable studies of clonal architecture in human health and disease
Using single-cell sequencing technologies, somatic mutations in mtDNA can be used as natural genetic barcodes to study cellular states and clonal dynamics.
Genome-wide association studies (GWAS) have successfully identified thousands of associations between common genetic variants and human disease phenotypes, but the majority of these variants are ...non-coding, often requiring genetic fine-mapping, epigenomic profiling, and individual reporter assays to delineate potential causal variants. We employ a massively parallel reporter assay (MPRA) to simultaneously screen 2,756 variants in strong linkage disequilibrium with 75 sentinel variants associated with red blood cell traits. We show that this assay identifies elements with endogenous erythroid regulatory activity. Across 23 sentinel variants, we conservatively identified 32 MPRA functional variants (MFVs). We used targeted genome editing to demonstrate endogenous enhancer activity across 3 MFVs that predominantly affect the transcription of SMIM1, RBM38, and CD164. Functional follow-up of RBM38 delineates a key role for this gene in the alternative splicing program occurring during terminal erythropoiesis. Finally, we provide evidence for how common GWAS-nominated variants can disrupt cell-type-specific transcriptional regulatory pathways.
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•A massively parallel reporter assay was developed to screen for functional variation•Variants identified by this assay are enriched for orthogonal measures of function•Functional GWAS variants alter activity of master transcription factors•The target gene RBM38 was linked to its GWAS phenotype and regulates mRNA splicing
A cost-effective, scalable, and allele-specific assay is used to systematically screen for functional non-coding genetic variation affecting red blood cell traits.
Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and β-thalassemia. BCL11A has been identified as a key regulator of HbF silencing, although its ...precise mechanisms of action remain incompletely understood. Recent studies have identified pathogenic mutations that cause heterozygous loss-of-function of BCL11A and result in a distinct neurodevelopmental disorder that is characterized by persistent HbF expression. While the majority of cases have deletions or null mutations causing haploinsufficiency of BCL11A, several missense variants have also been identified. Here, we perform functional studies on these variants to uncover specific liabilities for BCL11A's function in HbF silencing. We find several mutations in an N-terminal C2HC zinc finger that increase proteasomal degradation of BCL11A. We also identify a distinct C-terminal missense variant in the fifth zinc finger domain that we demonstrate causes loss-of-function through disruption of DNA binding. Our analysis of missense variants causing loss-of-function in vivo illuminates mechanisms by which BCL11A silences HbF and also suggests potential therapeutic avenues for HbF induction to treat sickle cell disease and β-thalassemia.
A recent study1 demonstrates how hematopoietic stem cells (HSCs) contribute minimally to blood and immune cell production during development and only become active postnatally. The work also reveals ...how Mecom expression can be used to distinguish rare HSCs from the more abundant progenitors that arise to maintain embryonic hematopoiesis.
A recent study1 demonstrates how hematopoietic stem cells (HSCs) contribute minimally to blood and immune cell production during development and only become active postnatally. The work also reveals how Mecom expression can be used to distinguish rare HSCs from the more abundant progenitors that arise to maintain embryonic hematopoiesis.
MECOM deficiency is a recently identified inborn error of immunity and inherited bone marrow failure syndrome caused by haploinsufficiency of the hematopoietic transcription factor MECOM. It is ...unique among inherited bone marrow failure syndromes, many of which present during later childhood or adolescence, because of the early age of onset and severity of the pancytopenia, emphasizing the importance and gene dose dependency of
MECOM
during hematopoiesis. B-cell lymphopenia and hypogammaglobulinemia have been described in a subset of patients with MECOM deficiency. While the mechanisms underlying the B-cell deficiency are currently unknown, recent work has provided mechanistic insights into the function of MECOM in hematopoietic stem cell (HSC) maintenance. MECOM binds to regulatory enhancers that control the expression of a network of genes essential for HSC maintenance and self-renewal. Heterozygous mutations, as seen in MECOM-deficient bone marrow failure, lead to dysregulated MECOM network expression. Extra-hematopoietic manifestations of MECOM deficiency, including renal and cardiac anomalies, radioulnar synostosis, clinodactyly, and hearing loss, have been reported. Individuals with specific genotypes have some of the systemic manifestations with isolated mild thrombocytopenia or without hematologic abnormalities, highlighting the tissue specificity of mutations in some MECOM domains. Those infants with MECOM-associated bone marrow failure require HSC transplantation for survival. Here, we review the expanding cohort of patient phenotypes and accompanying genotypes resulting in MECOM deficiency, and the proposed mechanisms underlying MECOM regulation of human HSC maintenance and B-cell development.
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