Erythroid Differentiation Enhances RNA Mis-Splicing in SF3B1-Mutant Myelodysplastic Syndromes with Ring Sideroblasts.

Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly develop from hematopoietic stem cells (HSC) bearing mutations in the splicing factor SF3B1 (SF3B1mt). Direct studies into MDS-RS pathobiology have been limited by a lack of model systems that fully recapitulate erythroid biology and RS development and the inability to isolate viable human RS. Here, we combined successful direct RS isolation from patient samples, high-throughput multiomics analysis of cells encompassing the SF3B1mt stem-erythroid continuum, and functional assays to investigate the impact of SF3B1mt on erythropoiesis and RS accumulation. The isolated RS differentiated, egressed into the blood, escaped traditional nonsense-mediated decay (NMD) mechanisms, and leveraged stress-survival pathways that hinder wild-type hematopoiesis through pathogenic GDF15 overexpression. Importantly, RS constituted a contaminant of magnetically enriched CD34+ cells, skewing bulk transcriptomic data. Mis-splicing in SF3B1mt cells was intensified by erythroid differentiation through accelerated RNA splicing and decreased NMD activity, and SF3B1mt led to truncations in several MDS-implicated genes. Finally, RNA mis-splicing induced an uncoupling of RNA and protein expression, leading to critical abnormalities in proapoptotic p53 pathway genes. Overall, this characterization of erythropoiesis in SF3B1mt RS provides a resource for studying MDS-RS and uncovers insights into the unexpectedly active biology of the "dead-end" RS. SIGNIFICANCE: Ring sideroblast isolation combined with state-of-the-art multiomics identifies survival mechanisms underlying SF3B1-mutant erythropoiesis and establishes an active role for erythroid differentiation and ring sideroblasts themselves in SF3B1-mutant myelodysplastic syndrome pathogenesis.

Nuclear factor I-C overexpression promotes monocytic development and cell survival in acute myeloid leukemia.

Nuclear factor I-C (NFIC) belongs to a family of NFI transcription factors that binds to DNA through CAATT-boxes and are involved in cellular differentiation and stem cell maintenance. Here we show NFIC protein is significantly overexpressed in 69% of acute myeloid leukemia patients. Examination of the functional consequences of NFIC overexpression in HSPCs showed that this protein promoted monocytic differentiation. Single-cell RNA sequencing analysis further demonstrated that NFIC overexpressing monocytes had increased expression of growth and survival genes. In contrast, depletion of NFIC through shRNA decreased cell growth, increased cell cycle arrest and apoptosis in AML cell lines and AML patient blasts. Further, in AML cell lines (THP-1), bulk RNA sequencing of NFIC knockdown led to downregulation of genes involved in cell survival and oncogenic signaling pathways including mixed lineage leukemia-1 (MLL-1). Lastly, we show that NFIC knockdown in an ex vivo mouse MLL::AF9 pre-leukemic stem cell model, decreased their growth and colony formation and increased expression of myeloid differentiation markers Gr1 and Mac1. Collectively, our results suggest that NFIC is an important transcription factor in myeloid differentiation as well as AML cell survival and is a potential therapeutic target in AML.

An immunodominant NP105-113-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease.

NP105-113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105-113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105-113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105-113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105-113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.

The developing mouse coronal suture at single-cell resolution.

Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2+ osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1+/-; Tcf12+/- mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.

Multi-Modal Characterization of Monocytes in Idiopathic Pulmonary Fibrosis Reveals a Primed Type I Interferon Immune Phenotype.

Idiopathic pulmonary fibrosis (IPF) is the most severe form of chronic lung fibrosis. Circulating monocytes have been implicated in immune pathology in IPF but their phenotype is unknown. In this work, we determined the immune phenotype of monocytes in IPF using multi-colour flow cytometry, RNA sequencing and corresponding serum factors, and mapped the main findings to amount of lung fibrosis and single cell transcriptomic landscape of myeloid cells in IPF lungs. We show that monocytes from IPF patients displayed increased expression of CD64 (FcγR1) which correlated with amount of lung fibrosis, and an amplified type I IFN response ex vivo. These were accompanied by markedly raised CSF-1 levels, IL-6, and CCL-2 in serum of IPF patients. Interrogation of single cell transcriptomic data from human IPF lungs revealed increased proportion of CD64hi monocytes and "transitional macrophages" with higher expression of CCL-2 and type I IFN genes. Our study shows that monocytes in IPF patients are phenotypically distinct from age-matched controls, with a primed type I IFN pathway that may contribute to driving chronic inflammation and fibrosis. These findings strengthen the potential role of monocytes in the pathogenesis of IPF.

Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a poor-prognosis childhood leukemia usually caused by RAS-pathway mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin-CD34+CD38-CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment, with RAS-pathway mutations as a "first hit," (2) mutations are acquired with both linear and branching patterns of clonal evolution, and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal interpatient heterogeneity of JMML LSCs, which are present in, but not confined to, the phenotypic HSC compartment. RNA sequencing of JMML LSC reveals up-regulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, and HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, and CD96), paving the way for LSC-directed disease monitoring and therapy in this disease.

Spatiotemporal analysis of human intestinal development at single-cell resolution.

Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.

Paracrine signalling by cardiac calcitonin controls atrial fibrogenesis and arrhythmia.

Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.

Transcriptomic profiling of the myeloma bone-lining niche reveals BMP signalling inhibition to improve bone disease.

Multiple myeloma is an incurable, bone marrow-dwelling malignancy that disrupts bone homeostasis causing skeletal damage and pain. Mechanisms underlying myeloma-induced bone destruction are poorly understood and current therapies do not restore lost bone mass. Using transcriptomic profiling of isolated bone lining cell subtypes from a murine myeloma model, we find that bone morphogenetic protein (BMP) signalling is upregulated in stromal progenitor cells. BMP signalling has not previously been reported to be dysregulated in myeloma bone disease. Inhibition of BMP signalling in vivo using either a small molecule BMP receptor antagonist or a solubilized BMPR1a-FC receptor ligand trap prevents trabecular and cortical bone volume loss caused by myeloma, without increasing tumour burden. BMP inhibition directly reduces osteoclastogenesis, increases osteoblasts and bone formation, and suppresses bone marrow sclerostin levels. In summary we describe a novel role for the BMP pathway in myeloma-induced bone disease that can be therapeutically targeted.

Single-cell assessment of transcriptome alterations induced by Scriptaid in early differentiated human haematopoietic progenitors during ex vivo expansion.

Priming haematopoietic stem/progenitor cells (HSPCs) in vitro with specific chromatin modifying agents and cytokines under serum-free-conditions significantly enhances engraftable HSC numbers. We extend these studies by culturing human CD133+ HSPCs on nanofibre scaffolds to mimic the niche for 5-days with the HDAC inhibitor Scriptaid and cytokines. Scriptaid increases absolute Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSPC numbers, while concomitantly decreasing the Lin-CD38-CD34+CD45RA-CD90- subset. Hypothesising that Scriptaid plus cytokines expands the CD90+ subset without differentiation and upregulates CD90 on CD90- cells, we sorted, then cultured Lin-CD34+CD38-CD45RA-CD90- cells with Scriptaid and cytokines. Within 2-days and for at least 5-days, most CD90- cells became CD90+. There was no significant difference in the transcriptomic profile, by RNAsequencing, between cytokine-expanded and purified Lin-CD34+CD38-CD45RA-CD49f+CD90+ cells in the presence or absence of Scriptaid, suggesting that Scriptaid maintains stem cell gene expression programs despite expansion in HSC numbers. Supporting this, 50 genes were significantly differentially expressed between CD90+ and CD90- Lin-CD34+CD38-CD45RA-CD49f+ subsets in Scriptaid-cytokine- and cytokine only-expansion conditions. Thus, Scriptaid treatment of CD133+ cells may be a useful approach to expanding the absolute number of CD90+ HSC, without losing their stem cell characteristics, both through direct effects on HSC and potentially also conversion of their immediate CD90- progeny into CD90+ HSC.

Colonic epithelial cell diversity in health and inflammatory bowel disease.

The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.

Cellular polarity modulates drug resistance in primary colorectal cancers via orientation of the multidrug resistance protein ABCB1.

Colonic epithelial cells are highly polarised with a lumen-facing apical membrane, termed the brush border, and a basal membrane in contact with the underlying extracellular matrix (ECM). This polarity is often maintained in cancer tissue in the form of neoplastic glands and has prognostic value. We compared the cellular polarity of several ex vivo spheroid colonic cancer cultures with their parental tumours and found that those grown as non-attached colonies exhibited apical brush border proteins on their outer cellular membranes. Transfer of these cultures to an ECM, such as collagen, re-established the centralised apical polarity observed in vivo. The multidrug resistance protein ABCB1 also became aberrantly polarised to outer colony membranes in suspension cultures, unlike cultures grown in collagen, where it was polarised to central lumens. This polarity switch was dependent on the presence of serum or selected serum components, including epidermal growth factor (EGF), transforming growth factor-ß1 (TGF-ß1) and insulin-like growth factor-1 (IGF-1). The apical/basal orientation of primary cancer colon cultures cultured in collagen/serum was modulated by α2ß1 integrin signalling. The polarisation of ABCB1 in colonies significantly altered drug uptake and sensitivity, as the outward polarisation of ABCB1 in suspension colonies effluxed substrates more effectively than ECM-grown colonies with ABCB1 polarised to central lumens. Thus, serum-free suspension colonies were more resistant to a variety of anti-cancer drugs than ECM-grown colonies. In conclusion, the local stroma, or absence thereof, can have profound effects on the sensitivity of colorectal cultures to drugs that are ABCB1 substrates. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Does osteogenic potential of clonal human bone marrow mesenchymal stem/stromal cells correlate with their vascular supportive ability?

BACKGROUND: Human bone marrow-derived mesenchymal stem/stromal cells (hBM MSCs) have multiple functions, critical for skeletal formation and function. Their functional heterogeneity, however, represents a major challenge for their isolation and in developing potency and release assays to predict their functionality prior to transplantation. Additionally, potency, biomarker profiles and defining mechanisms of action in a particular clinical setting are increasing requirements of Regulatory Agencies for release of hBM MSCs as Advanced Therapy Medicinal Products for cellular therapies. Since the healing of bone fractures depends on the coupling of new blood vessel formation with osteogenesis, we hypothesised that a correlation between the osteogenic and vascular supportive potential of individual hBM MSC-derived CFU-F (colony forming unit-fibroblastoid) clones might exist. METHODS: We tested this by assessing the lineage (i.e. adipogenic (A), osteogenic (O) and/or chondrogenic (C)) potential of individual hBM MSC-derived CFU-F clones and determining if their osteogenic (O) potential correlated with their vascular supportive profile in vitro using lineage differentiation assays, endothelial-hBM MSC vascular co-culture assays and transcriptomic (RNAseq) analyses. RESULTS: Our results demonstrate that the majority of CFU-F (95%) possessed tri-lineage, bi-lineage or uni-lineage osteogenic capacity, with 64% of the CFU-F exhibiting tri-lineage AOC potential. We found a correlation between the osteogenic and vascular tubule supportive activity of CFU-F clones, with the strength of this association being donor dependent. RNAseq of individual clones defined gene fingerprints relevant to this correlation. CONCLUSIONS: This study identified a donor-dependent correlation between osteogenic and vascular supportive potential of hBM MSCs and important gene signatures that support these functions that are relevant to their bone regenerative properties.

Invasive Salmonella exploits divergent immune evasion strategies in infected and bystander dendritic cell subsets.

Non-typhoidal Salmonella (NTS) are highly prevalent food-borne pathogens. Recently, a highly invasive, multi-drug resistant S. Typhimurium, ST313, emerged as a major cause of bacteraemia in children and immunosuppressed adults, however the pathogenic mechanisms remain unclear. Here, we utilize invasive and non-invasive Salmonella strains combined with single-cell RNA-sequencing to study the transcriptome of individual infected and bystander monocyte-derived dendritic cells (MoDCs) implicated in disseminating invasive ST313. Compared with non-invasive Salmonella, ST313 directs a highly heterogeneous innate immune response. Bystander MoDCs exhibit a hyper-activated profile potentially diverting adaptive immunity away from infected cells. MoDCs harbouring invasive Salmonella display higher expression of IL10 and MARCH1 concomitant with lower expression of CD83 to evade adaptive immune detection. Finally, we demonstrate how these mechanisms conjointly restrain MoDC-mediated activation of Salmonella-specific CD4+ T cell clones. Here, we show how invasive ST313 exploits discrete evasion strategies within infected and bystander MoDCs to mediate its dissemination in vivo.

Single-molecule DNA-mapping and whole-genome sequencing of individual cells.

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation-renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893-4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell's genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing.

Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device.

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.

Ezh2 and Runx1 Mutations Collaborate to Initiate Lympho-Myeloid Leukemia in Early Thymic Progenitors.

Lympho-myeloid restricted early thymic progenitors (ETPs) are postulated to be the cell of origin for ETP leukemias, a therapy-resistant leukemia associated with frequent co-occurrence of EZH2 and RUNX1 inactivating mutations, and constitutively activating signaling pathway mutations. In a mouse model, we demonstrate that Ezh2 and Runx1 inactivation targeted to early lymphoid progenitors causes a marked expansion of pre-leukemic ETPs, showing transcriptional signatures characteristic of ETP leukemia. Addition of a RAS-signaling pathway mutation (Flt3-ITD) results in an aggressive leukemia co-expressing myeloid and lymphoid genes, which can be established and propagated in vivo by the expanded ETPs. Both mouse and human ETP leukemias show sensitivity to BET inhibition in vitro and in vivo, which reverses aberrant gene expression induced by Ezh2 inactivation.

Hepcidin is regulated by promoter-associated histone acetylation and HDAC3.

Hepcidin regulates systemic iron homeostasis. Suppression of hepcidin expression occurs physiologically in iron deficiency and increased erythropoiesis but is pathologic in thalassemia and hemochromatosis. Here we show that epigenetic events govern hepcidin expression. Erythropoiesis and iron deficiency suppress hepcidin via erythroferrone-dependent and -independent mechanisms, respectively, in vivo, but both involve reversible loss of H3K9ac and H3K4me3 at the hepcidin locus. In vitro, pan-histone deacetylase inhibition elevates hepcidin expression, and in vivo maintains H3K9ac at hepcidin-associated chromatin and abrogates hepcidin suppression by erythropoietin, iron deficiency, thalassemia, and hemochromatosis. Histone deacetylase 3 and its cofactor NCOR1 regulate hepcidin; histone deacetylase 3 binds chromatin at the hepcidin locus, and histone deacetylase 3 knockdown counteracts hepcidin suppression induced either by erythroferrone or by inhibiting bone morphogenetic protein signaling. In iron deficient mice, the histone deacetylase 3 inhibitor RGFP966 increases hepcidin, and RNA sequencing confirms hepcidin is one of the genes most differentially regulated by this drug in vivo. We conclude that suppression of hepcidin expression involves epigenetic regulation by histone deacetylase 3.Hepcidin controls systemic iron levels by inhibiting intestinal iron absorption and iron recycling. Here, Pasricha et al. demonstrate that the hepcidin-chromatin locus displays HDAC3-mediated reversible epigenetic modifications during both erythropoiesis and iron deficiency.