Sandrina Kinet

Erythropoiesis is a highly coordinated process governed by lineage-defining transcription factors. While several master regulators have been characterized, many additional factors expressed in erythroid progenitors remain poorly understood. Here, we identify the epithelial-to-mesenchymal transition (EMT)–associated transcription factor ZEB1 as a previously unrecognized regulator of erythroid maturation. ZEB1 is expressed in murine and human erythroid progenitors, and its loss impairs proliferation and disrupts the erythroid transcriptional network. Mechanistically, ZEB1 interacts with KLF1 and governs both its expression dynamics and nuclear localization. In ZEB1-deficient cells, GATA2 silencing is delayed due to failure of KLF1 to properly accumulate in the nucleus, impairing KLF1-mediated repression of GATA2 and perturbing the GATA2–KLF1 regulatory switch that drives late-stage erythropoiesis. Consistent with these findings, ZEB1 downregulation hampers maturation of primary human CD34⁺-derived erythroid cells. Together, our findings establish KLF1 as a repressor of GATA2, and ZEB1 as a critical enforcer of erythroid transcriptional circuitry, and demonstrate that erythroid cells recruit EMT-associated factors to ensure lineage completion.

Hematopoietic stem cells (HSCs) possess the capacity for self-renewal and the sustained production of all mature blood cell lineages. It has been well established that a metabolic rewiring controls the switch of HSCs from a self-renewal state to a more differentiated state, but it is only recently that we have appreciated the importance of metabolic pathways in regulating the commitment of progenitors to distinct hematopoietic lineages. In the context of erythroid differentiation, an extensive network of metabolites, including amino acids, sugars, nucleotides, fatty acids, vitamins, and iron, is required for red blood cell (RBC) maturation. In this review, we highlight the multifaceted roles via which metabolites regulate physiological erythropoiesis as well as the effects of metabolic perturbations on erythroid lineage commitment and differentiation. Of note, the erythroid differentiation process is associated with an exceptional breadth of solute carrier (SLC) metabolite transporter upregulation. Finally, we discuss how recent research, revealing the critical impact of metabolic reprogramming in diseases of disordered and ineffective erythropoiesis, has created opportunities for the development of novel metabolic-centered therapeutic strategies.

Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. We discovered that SLC7A1/CAT1-dependent arginine uptake and its catabolism to spermidine control the erythroid specification of HSPCs via activation of eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on the metabolism of spermidine to hypusine and inhibiting hypusine synthesis abrogates erythropoiesis and diverts EPO-stimulated HSPCs to a myeloid fate. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A and induction of mitochondrial function partially rescues erythropoiesis in the absence of hypusine. Within the hypusine network, ribosomal proteins are highly enriched and we identify defective eIF5A hypusination in erythroid pathologies caused by abnormal ribosome biogenesis. Thus, eIF5A-dependent protein synthesis is critical in the branching of erythro-myeloid differentiation and attenuated eIF5A activity characterizes ribosomal protein-linked disorders of ineffective erythropoiesis.

DNA replication initiates from multiple genomic locations called replication origins. In metazoa, DNA sequence elements involved in origin specification remain elusive. Here, we examine pluripotent, primary, differentiating, and immortalized human cells, and demonstrate that a class of origins, termed core origins, is shared by different cell types and host ~80% of all DNA replication initiation events in any cell population. We detect a shared G-rich DNA sequence signature that coincides with most core origins in both human and mouse genomes. Transcription and G-rich elements can independently associate with replication origin activity. Computational algorithms show that core origins can be predicted, based solely on DNA sequence patterns but not on consensus motifs. Our results demonstrate that, despite an attributed stochasticity, core origins are chosen from a limited pool of genomic regions. Immortalization through oncogenic gene expression, but not normal cellular differentiation, results in increased stochastic firing from heterochromatin and decreased origin density at TAD borders.

The differentiation of CD4 T cells to a specific effector fate is metabolically regulated, integrating glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) with transcriptional and epigenetic changes. OXPHOS is tightly coordinated with the tricarboxylic acid (TCA) cycle but the precise role of TCA intermediates in CD4 T cell differentiation remain unclear. Here we demonstrate that α-ketoglutarate (αKG) inhibited regulatory T cell (Treg) generation while conversely, increasing Th1 polarization. In accord with these data, αKG promoted the effector profile of Treg-polarized chimeric antigen receptor-engineered T cells against the ErbB2 tumor antigen. Mechanistically, αKG significantly altered transcripts of genes involved in lipid-related processes, inducing a robust lipidome-wide remodelling and decreased membrane fluidity. A massive increase in storage and mitochondria lipids was associated with expression of mitochondrial genes and a significantly augmented OXPHOS. Notably, inhibition of succinate dehydrogenase activity, the bridge between the TCA cycle and the electron transport chain, enforced Treg generation. Thus, our study identifies novel connections between αKG, lipidome remodelling and OXPHOS in CD4 T cell fate decisions.

Despite the effective clinical use of steroids for the treatment of Diamond Blackfan anemia (DBA), the mechanisms through which glucocorticoids regulate human erythropoiesis remain poorly understood. We report that the sensitivity of erythroid differentiation to dexamethasone is dependent on the developmental origin of human CD34⁺ progenitor cells, specifically increasing the expansion of CD34⁺ progenitors from peripheral blood (PB) but not cord blood (CB). Dexamethasone treatment of erythroid-differentiated PB, but not CB, CD34⁺ progenitors resulted in the expansion of a newly defined CD34⁺CD36⁺CD71^hiCD105^med immature colony-forming unit–erythroid (CFU-E) population. Furthermore, proteomics analyses revealed the induction of distinct proteins in dexamethasone-treated PB and CB erythroid progenitors. Dexamethasone treatment of PB progenitors resulted in the specific upregulation of p57^Kip2, a Cip/Kip cyclin–dependent kinase inhibitor, and we identified this induction as critical; shRNA-mediated downregulation of p57^Kip2, but not the related p27^Kip1, significantly attenuated the impact of dexamethasone on erythroid differentiation and inhibited the expansion of the immature CFU-E subset. Notably, in the context of DBA, we found that steroid resistance was associated with dysregulated p57^Kip2 expression. Altogether, these data identify a unique glucocorticoid-responsive human erythroid progenitor and provide new insights into glucocorticoid-based therapeutic strategies for the treatment of patients with DBA.

Studies of human erythropoiesis have relied, for the most part, on the in vitro differentiation of hematopoietic stem and progenitor cells (HSPC) from different sources. Here, we report that despite the common core erythroid program that exists between cord blood (CB)‐ and peripheral blood (PB)‐HSPC induced toward erythroid differentiation in vitro, significant functional differences exist. We undertook a comparative analysis of human erythropoiesis using these two different sources of HSPC. Upon in vitro erythroid differentiation, CB‐derived cells proliferated 4‐fold more than PB‐derived cells. However, CB‐derived cells exhibited a delayed kinetics of differentiation, resulting in an increased number of progenitors, notably colony‐forming unit (CFU‐E). The phenotypes of early erythroid differentiation stages also differed between the two sources with a significantly higher percentage of IL3R^‐GPA^‐CD34⁺CD36⁺ cells generated from PB‐ than CB‐HSPCs. This subset was found to generate both burst‐forming unit (BFU‐E) and CFU‐E colonies in colony‐forming assays. To further understand the differences between CB‐ and PB‐HSPC, cells at eight stages of erythroid differentiation were sorted from each of the two sources and their transcriptional profiles were compared. We document differences at the CD34, BFU‐E, poly‐ and orthochromatic stages. Genes exhibiting the most significant differences in expression between HSPC sources clustered into cell cycle‐ and autophagy‐related pathways. Altogether, our studies provide a qualitative and quantitative comparative analysis of human erythropoiesis, highlighting the impact of the developmental origin of HSPCs on erythroid differentiation.

T cells are stimulated by the engagement of antigen, cytokine, pathogen, and hormone receptors. While research performed over many years has focused on deciphering the molecular components of these pathways, recent data underscore the importance of the metabolic environment in conditioning responses to receptor engagement. The ability of T cells to undergo a massive proliferation and cytokine secretion in response to receptor signals requires alterations to their bioenergetic homeostasis, allowing them to meet new energetic and biosynthetic demands. The metabolic reprogramming of activated T cells is regulated not only by changes in intracellular nutrient uptake and utilization but also by nutrient and oxygen concentrations in the extracellular environment. Notably, the extracellular environment can be profoundly altered by pathological conditions such as infections and tumors, thereby perturbing the metabolism and function of antigen-specific T lymphocytes. This review highlights the interplay between diverse metabolic networks and the transcriptional/epigenetic states that condition T-cell differentiation, comparing the metabolic features of T lymphocytes with other immune cells. We further address recent discoveries in the metabolic pathways that govern T-cell function in physiological and pathological conditions.