P&F Projects 2022-2023
Termini Abstract Type A P&F 2023
While myeloablative treatments are commonly used to rid the body of cancerous cells, these regimens deplete normal hematopoietic cells. Further, myeloablation significantly remodels the physical and molecular features of the bone marrow microenvironment, putting patients at risk for developing life-threatening complications such as hemorrhage or infection. To accelerate hematopoietic recovery from these toxic therapies, it is imperative to identify the molecular regulators of hematopoietic regeneration. Prior approaches to visualize the regenerating bone marrow niche primarily used reporter mice or limited antibody panels for immunofluorescence imaging. In this proposal, we seek to use CODEX imaging to define the regenerating bone marrow niche with spatial and molecular precision.
Shahbazi Abstract Type A P&F 2023
Cells are in continuous communication and signaling with each other to prime the environment for their survival and growth. Hormones, growth factors, and cytokines are known signaling molecules that play a key role in this communication however, there is a fairly unknown communication mechanism that happens through the release of nanovesicles called exosomes. These tiny particles carry important protein or RNA signaling molecules that change the cellular behavior in the target cell, tissue, or organ. In this regard, bone marrow (BM) space is one of the complex and rich environments that homes various cell types with different roles in the human body. The focus of this study is to understand the exosomal communication with the hematopoietic stem and progenitor cells (HSPCs) in the bone marrow space that are responsible for the repopulation of the human blood including all myeloid and lymphoid progenies. Moreover, we will study the exosomal communication with a special population of HSPCs called long-term hematopoietic stem cells (LT-HSCs) that stand on top of the hematopoietic hierarchy and have this great capability of self-renewing and differentiation to different lineages. In this project, we will look at the interaction of exosomes released from bone marrow stromal cells with HSPCs and try to understand their effect on the self-renewal, expansion, and differentiation of HSPCs. We will approach this by delineating the role of exosomal communication using the ImageStream single-cell imaging platform, flow cytometry analysis, and colony-forming unit assay.
Thompson Abstract Type A P&F 2023
The purpose of this Pilot & Feasibility Grant is to elucidate the role of High Mobility Group A1 (HMGA1) chromatin regulators in clonal hematopoiesis (CH) and associated cardiovascular disease (CVD). CH results from expansion of hematopoietic stem cells (HSCs) harboring a mutation that provides a fitness advantage. In addition to myeloid malignancies, CH mutations are associated with increased risk of diverse cardiovascular diseases (CVD), including venous thromboembolism (VTE), atherosclerosis (AS), coronary artery disease (CAD), myocardial infarction (MI), and stroke, all independent of typical CVD risk factors such as hyperlipidemia, obesity, and diabetes. Importantly, incidence of CH is rising as our population ages and there is an unmet need for treatments to prevent CVD associated with CH. Indeed, individuals harboring CH mutations in TET2, DNMT3A, ASXL1, or JAK2 have an almost 2-fold higher risk for CVD; however, the underlying mechanisms are only beginning to emerge and further studies to identify therapeutic targets are needed. Here, we take a novel approach by focusing on the HMGA1 chromatin regulators in CVD associated with CH. Our
premise that HMGA1 plays a critical role in CVD associated with CH is based on the following preliminary data: 1) HMGA1 overexpression induces clonal expansion in adult stem cells by amplifying genes involved in inflammation and proliferation1. 2) In JAK2V617F transgenic mouse models of CH and myeloproliferative neoplasms (MPN), loss of just a single Hmga1 allele in hematopoietic stem cells (HSCs) dampens thrombocytosis, erythrocytosis, and neutrophilia, all of which are linked to CVD. 3) Moreover, HMGA1 deficiency prevents splenomegaly, megakaryocyte hyperplasia, and progression to myelofibrosis (MF). 4) Importantly, MF is characterized by excessive inflammatory signals from megakaryocytes and other progenitors, all of which could fuel CVD1. 5) HMGA1 also expands HSC with a monocyte-bias and increases IL6 expression, which are also linked to atherosclerosis (AS) and CVD. Together, these exciting results led us to the following hypotheses: 1) HMGA1 is required for the development of AS and other manifestations of CVD in CH through specific transcriptional networks, and, 2) HMGA1 networks expand specific stem and progenitor populations that increase pro-inflammatory signals. To begin to test this, we propose a strategic collaboration with Dr. Krause at YCCEH to elucidate aberrant inflammatory cytokine/chemokine networks induced by HMGA1
in well-established Tet2 mutant mouse models of atherosclerosis (AS) with the following Aims:
A) To identify HMGA1 transcriptional networks and the cell(s) of origin for Hmga1-driven, aberrant inflammatory signaling in Tet2 mutant CH using single cell RNA sequencing (scRNAseq) and,
B) To define HMGA1-dependent cytokines and chemokine pathways in Tet2 mutant mice with AS. Our focus will be on cytokines and networks that could be targeted in therapy. Thus, our collaborative studies with Dr. Diane Krause (Director of the Yale CCEH and expert in hematopoiesis), will not only reveal mechanistic insight into disease-related cytokine pathways in CH, but we also expect to discover new actionable mechanisms to prevent progression.
Zheng Abstract Type B P&F 2023
ANKRD26 in Megakaryopoiesis (Ankyrin repeat domain-containing protein-26) is a highly conserved protein that is involved in platelet production. Mutations in the 5’ untranslated region (5’UTR) of ANKRD26 gene lead to ANKRD26 overexpression during megakaryocyte differentiation, resulting in impaired proplatelet formation. Point mutations and small size deletions in the 5’UTR of ANKRD26 have been identified in patients with inherited thrombocytopenia 2 (THC2), a life-long thrombocytopenia with a predisposition to developing hematological malignancies. However, little is known about the underlying mechanism of ANKRD26 mutation-associated thrombocytopenia. To explore the function of ANKRD26 and its role in megakaryopoiesis, we established and characterized the first animal model of THC2. Our preliminary results demonstrated that zebrafish ankrd26 mutants with mutations in the 5’UTR of ankrd26 resulted in overexpression of Ankrd26 and reduction of thrombocyte count, similar to the clinical presentation of THC2 patients. Moreover, we found that the level of ANKRD26 was positively corelated with the level of PRMT1 in the megakaryoblastic leukemia cells and in the platelets of immune thrombotic thrombocytopenic purpura patients. PRMT1 is an arginine methyltransferase for RUNX1 (runt-related transcription factor 1), which is a negative regulator for ANKRD26 expression. We hypothesize that dysregulation of the PRMT1-RUNX1-ANKRD26 pathway may affect both hereditary and acquired thrombocytopenia. Our study will further evaluate the role of this novel pathway in platelet development and production, providing a potential avenue for developing novel therapeutic strategies for patients with thrombocytopenia.
Omelianczyk Abstract Type B P&F 2023
Red blood cell (RBC)-mediated transport of oxygen throughout the body is a tightly controlled process. During hypoxia, the body increases blood adenosine levels to signal RBCs to release more oxygen. Adenosine is sensed in the RBC by the ADORA2B receptor, which initiates a signaling cascade that ultimately elevates levels of 2,3-bisphosphoglyceric acid (2,3-BPG) that binds hemoglobin to reduce its oxygen affinity. Acute hypoxia also reduces RBC fatty acid levels short-term, although these levels increase during prolonged hypoxia beyond steady-state levels. The mechanisms and biological implications of these changes are unclear. Sickle cell disease, which is caused by a single point mutation in the b-subunit of hemoglobin, phenocopies hypoxia, including elevated blood adenosine levels, active ADORA2B signaling, and increased levels of 2,3-BPG within the RBC. The release of oxygen from mutant hemoglobin, however, causes a conformational change, resulting in the characteristic sickling of the RBC. Sickle cell disease is widespread in sub-Saharan Africa, as it confers near complete protection from malaria, which is also endemic to this region. Recent work, however, indicates that this protection dwindles during infection by Plasmodium falciparum malaria parasites that harbor specific mutations that counter-act the protective effects of sickle trait and enable parasites to thrive in these patients. Intriguingly, two of the nonsynonymous parasite mutations that counteract sickle protection are in genes coding for proteins exported to the infected RBC. We posit that parasites actively interfere with RBC lipid signaling pathways to overcome the protective properties of sickle cell disease. We propose i) to unravel the lipid changes that underpin altered signaling in normoxic versus hypoxic conditions in RBCs from healthy and sickle-cell patients and ii) to understand how malaria parasites interfere with lipid signaling in RBCs and counteract sickle protection via specific adaptations. We expect to gain insights into red blood cell lipid metabolism beyond the current knowledge. This project will characterize the impact of ADORA2B signaling on lipid metabolism in healthy and sickling RBCs during hypoxia.
This might open avenues for future studies ultimately novel treatment possibilities of patients suffering from acute and chronic hypoxia. Furthermore, the adaptation of Plasmodium falciparum to sickle cell blood puts large populations in sub-Saharan Africa at risk of severe malaria. This will further complicate the already slowing efforts to eradicate malaria. This work will help understand the underlying mechanism behind this newly discovered condition.
Shinha Abstract Type A P&F 2023
Myelodysplastic syndromes (MDS) are a heterogeneous group of blood disorders characterized by the ineffective production of mature blood cells from the bone marrow. Somatic mutations in genes encoding RNA splicing factors (SF) such as SRSF2 and U2AF1 are found in ~20% of MDS patients. These patients are generally associated with poor prognoses and are at an increased risk of leukemic transformation. The precise cellular and molecular mechanisms by which spliceosome gene mutations endow HSPCs with a competitive advantage in disease initiation and progression are still poorly understood. Several murine models have been developed to model the effects of somatic mutations in SRSF2 and U2AF1; however, the degree of splicing overlap between mouse and human was low due to low intronic sequence conservation across species. Moreover, cells are unable to tolerate lentiviral-based expression of splicing factors, which severely limits our ability to perform functional assays. To overcome this challenge, we are proposing to develop an AAV-based gene editing strategy to introduce the SRSF2P95H somatic mutation directly into the endogenous SRSF2 locus in primary human hematopoietic progenitors. Successful development of this technique will allow us to perform a wide range of molecular and functional assays to better understand how aberrant RNA splicing drives clonal blood diseases such as MDS.
Collins Abstract Type B P&F 2023
Hematopoiesis is a highly regulated process during which hematopoietic stem cells (HSCs) expand and differentiate, dynamically supplying a diversity of blood cells for the entire life of an organism. In vivo mouse models are essential for fundamental studies of hematopoiesis, providing numerous tools and methods for flexible in vivo experimentation that is restricted in in vivo studies of human hematopoiesis. Here, I propose to provide these critical tools by developing a protocol C-FiSHH; i.e., CRISPR-based Functional in vivo Screen of Human Hematopoiesis. I will build on state-of-the art technologies developed or adopted by my lab, following two Specific Aims. First, I will optimize a protocol to deliver a protein-barcoded gRNA/Cas9 complex in the humanized hematopoietic system of ‘MISTRG’ mice that I helped develop. Second, I will assess the feasibility of an in vivo singlecell CRISPR screen of human hematopoiesis. As a proof-of-principle, I will design a gRNA library that targets genes essential for the development of specific hematopoietic lineages. Once optimized, my technology will be highly versatile and will enable functional screens to identify mechanisms underlying fundamental characteristics of human hematopoiesis, such as the long-term maintenance of self-renewing HSCs, lineage commitment, and the role of cytokines in these processes. I expect to provide a necessary complement to the high-dimensional mapping of human hematopoiesis transcriptomes, adding a functional perspective to descriptive studies.
Guo Abstract Type A P&F 2023
Proliferation followed by differentiation of the granulocyte and macrophage progenitors (GMPs) sustains the rapid cellular turnover of the myeloid lineages. Their essentiality is highlighted by the side effects of many chemotherapy drugs designed to target the proliferating cancer cells. This highly proliferative state, however, is transient for a given GMP cell, which exits from the proliferative state upon differentiation. My lab previously found that GMPs divide every 6-8 hours at their peak proliferative state, and this state lasts for ~1-2 days, at least in vitro, when cell cycle slows upon differentiation. These proliferative progenitors possess enormous regenerative potential; agents that can tap into this regenerative power without transformation would provide ample therapeutic opportunities. In our search for such agents, we discovered a small molecule to yield orders of magnitude more expansion without impairing subsequent differentiation. While experiments are being planned to test its effect in vivo, we propose to identify the molecular target(s) for this small molecule compound. We have teamed up with renowned Yale experts in medicinal chemistry and target identification, Dr. Craig Crews, and have obtained critical evidence to support that this compound may act by metabolic reprogramming. Therefore, we seek the CCEH Type A support from the University of Utah Metabolomics Core to test our hypothesis. Overall, the experiments in this proposal could unveil a novel class of chemical agents with pharmacologic potential for hematopoietic regeneration.
Scalf Abstract Type B P&F 2022
Proliferation followed by differentiation of the granulocyte and macrophage progenitors (GMPs) sustains the rapid cellular turnover of the myeloid lineages. Their essentiality is highlighted by the side effects of many chemotherapy drugs designed to target the proliferating cancer cells. This highly proliferative state, however, is transient for a given GMP cell, which exits from the proliferative state upon differentiation. My lab previously found that GMPs divide every 6-8 hours at their peak proliferative state, and this state lasts for ~1-2 days, at least in vitro, when cell cycle slows upon differentiation. These proliferative progenitors possess enormous regenerative potential; agents that can tap into this regenerative power without transformation would provide ample therapeutic opportunities. In our search for such agents, I discovered a small molecule to display such activity, yielding orders of magnitude more expansion without impairing subsequent differentiation. My proposal is to identify the molecular target(s) for the small molecule and to test whether it can promote hematopoietic recovery following injury in vivo. We have teamed up with renowned Yale experts in medicinal chemistry and target identification, Dr. Craig Crews, and have obtained critical evidence to support feasibility using a targeted proteomic approach to unveil the molecular target(s)/mechanism for the small molecule. In a second aim as supported by encouraging preliminary results, I will administer the small molecule to mice post irradiation and 5’ fluorouracil to test whether it promotes hematopoietic/myeloid recovery post injury. Overall, the experiments in this proposal could unveil a novel class of chemical agents with pharmacologic potential for hematopoietic regeneration.
Klein Abstract Type B P&F 2022
This collaborative proposal targets fatty acid oxidation (FAO) to expand human hematopoietic stem cells (HSCs) in chemically defined culture conditions. Independent research paths in the Tong and Klein labs have converged on the unexpected finding that FAO activation enhances HSC function during ex vivo expansion. The Tong lab has found that genetic disruption of the adaptor protein LNK, which expands long-term HSCs >10-fold, activates a metabolic program that increases FAO and reduces oxidative stress. Independently, a high throughput screen in the Klein lab identified drugs that induce FAO through activation of PPARa as potent enhancers of ex vivo HSC expansion. Here we propose to test the hypothesis that FAO activation is required for HSC expansion and that PPARa agonists can be used for therapeutic HSC expansion ex vivo. Investigation of metabolic regulators of hematopoiesis is a new area of research for both the Tong and Klein labs that could have substantial therapeutic impact. The significance of this proposal includes: 1. The ability to expand human HSCs in umbilical cord blood units will substantially increase the number of available HLA matches for patients needing HSC transplant, notably patients with myelodysplastia, advanced myeloproliferative disorders, and bone marrow failure. 2. Gene editing techniques for the treatment of inherited blood disorders are limited by the loss of long term (LT)-HSCs during ex vivo manipulations; our low cytokine culture conditions maintain LT-HSCs ex vivo without loss of reconstituting activity, providing an ideal platform for therapeutic gene editing. At a basic science level, our preliminary data and published work from others identify a role for FAO in the maintenance of LT-HSCs. However, the mechanisms by which FAO enhances HSC function have not been explored. Furthermore, while PPARa agonists are widely used clinically to treat hyperlipidemia (e.g. gemfibrozil/lopid), these drugs have not previously been applied to therapeutic HSC expansion. Our data are too preliminary for an R01 submission; these high risk/high gain experiments will require transplantation studies in mice and additional metabolic analyses to support a collaborative R01 submission to the NIDDK in the future.
Karnik Abstract Type A P&F 2022
Bone marrow (BM) is the primary site of hematopoiesis in mammals and where hematopoietic stem cells (HSCs) reside. As the age advances, the BM architecture changes and the hematopoietic potential of HSCs declines. The pathways involved with this decline in HSC regenerative potential are not fully understood. However, we know that from previous studies by our group and others on dissociated bone marrow, the cells of the hematopoietic niche (HN) such as osteomacs (OMs), megakaryocytes (MKs), and osteoblasts (OBs) are involved in maintaining the HSC function. These studies use established techniques such as flow cytometry and immunofluorescence which use dissociated BM either to quantify the cells of the HN or the tissue to visualize up to three cell markers at a time. However, it is important to understand the spatial relationships of the cells of the HN with each other in an unperturbed BM to better comprehend the interactions of these cells in the BM niche and how they change as the age advances. A multiplex high-resolution imaging technology, CODEX, that can visualize up to 60 markers simultaneously can overcome the limitations of traditional techniques to visualize the entire niche. We propose to use the CODEX to image the HN to study the spatial relationships between the cells of HN in young and old mice to better understand the HN architectural landscape and to develop the methodology for CODEX use so it can become a research and investigative tool for other hematology investigators. Plotting the architectural landscape of the BM of both young and old mice will enhance our understanding of the relationship between these cells in the BM niche and to help us develop the methodology and protocols of the CODEX so it can become a research and investigative tool for non-malignant hematology investigators.
Leibold Abstract Type A P&F 2022
Due to its presence in proteins involved in hemoglobin synthesis, DNA synthesis and mitochondrial respiration, eukaryotic cells require iron for growth and proliferation. Regulation of cellular iron content is crucial: excess cellular iron catalyzes the generation of reactive oxygen species that damage DNA and proteins, while cellular iron deficiency causes cell cycle arrest and cell death. Dysregulation of iron homeostasis caused by iron deficiency or iron excess leads to hematological, neurodegenerative and metabolic disorders. Vertebrate iron metabolism is controlled post-transcriptionally by iron-regulatory protein 2 (Irp2). Irp2 binds to iron-responsive elements (IREs) in the mRNAs of proteins involved in iron uptake (transferrin receptor 1), sequestration (ferritin) and export (ferroportin), and regulates the translation or stability of these mRNAs. Our previous work show that Irp2 is regulated by iron-dependent proteolysis by the FBXL5 SCF-ubiquitin ligase. We also discovered a novel iron-independent mechanism for regulating Irp2 RNA-binding activity during the cell cycle. Irp2 is phosphorylated at S157 by Cdk1/cyclin B during G2/M and dephosphorylated by Cdc14A during mitotic exit. Irp2-S157 phosphorylation blocks its interaction with RNA to increase ferritin synthesis and decrease TfR1 mRNA stability during mitosis. The significance of S157 phosphorylation was investigated in mice where Ser157 was mutated to Ala157 (Irp2A/A)). Irp2A/A mice display anemia, defective erythroid terminal differentiation and dysregulated systemic iron metabolism. Our overall objective is to determine the role of Irp2-S157 phosphorylation in hematopoiesis. Our goal here is characterize the anemia in WT and Irp2A/A mice. For these studies, we propose to utilize the University of Utah Center for Iron & Heme Disorders Core for quantification of iron and heme content in tissue samples from WT and Irp2A/A mice.
Mayday Abstract Type B P&F 2022
RNA binding motif protein 15 (RBM15) is a key regulator of N6-methyladenosine (m6A) epitranscriptome modification and is essential for recruitment of the m6A writer protein complex to target RNAs. It has been shown that RBM15 is important for hematopoietic stem cell (HSC) maintenance and quiescence, but its role in the fate specification of hematopoietic progenitor cells (HPCs) remains poorly understood. RBM15 is part of the recurrent t(1;22) translocation associated with infantile acute megakaryoblastic leukemia; therefore, investigation into its role in megakaryocyte differentiation and maturation is warranted. Understanding the mechanistic role of RBM15 in the process of megakaryocyte maturation will not only provide potential avenues of investigation for treatment of AMKL but will also shed light on the role of this protein and the m6A epitranscriptome in megakaryopoiesis. This proposal aims to understand the transcriptomic targets of RBM15 (Aim 1) and the m6A epitranscriptomic modifications regulated by RBM15 (Aim 2) in a model of megakaryopoiesis. Using enhanced crosslinking and immunoprecipitation sequencing techniques, we will characterize these targets at single nucleotide resolution. Comparison to RNA-seq will provide insight into the consequences of RBM15 binding and modification on target transcripts. By determining the exact nucleotide bound by RBM15 and the exact adenosine modified nearby, we will gain detailed insight into the epitranscriptome dynamics involved in hematopoietic differentiation. Completion of this project will illuminate the transcriptome and epitranscriptome interactions of RBM15 and will provide candidate genes that may be critical for downstream mechanistic effects driving megakaryopoiesis. By filling gaps in the field, this work will lay the foundation for further investigation of the epitranscriptome in hematopoietic fate decision.
Prasada Rao Jarajapu Abstract Type B P&F 2022
Diabetes increases risk for cardiovascular diseases. Chronic inflammation and oxidative stress appear to be underlying mechanisms of diabetic dysfunction in cardiovascular tissues. Systemic inflammation is due to myelopoietic bias in the stem/progenitor cells and the resulting increased generation of pro-inflammatory monocyte-macrophages. TERT is a subunit of telomerase that is responsible for telomere maintenance and chromosomal stability. Independent of telomerase, TERT regulates hematopoiesis and mitochondrial functions. Our preliminary studies discovered that diabetic CD34+ hematopoietic stem/progenitor cells (HSPCs) express one or more deletion variants of TERT, α-, β- or αβ-variants, while nondiabetic cells do not. Deletion variants of TERT are known to oppose full-length TERT function. The presence of variants correlated with decreased telomerase activity and elevated mitochondrial reactive oxygen species (mitoROS) in diabetic CD34+ cells compared to the nondiabetic. Furthermore, we found that silencing of TGF1 blocked TERT-splicing that accompanied decreased mitoROS and reversed myelopoiesis. This pilot proposal tests the hypothesis that splice variants of TERT mediate increased myelopoiesis and mitochondrial oxidative stress in diabetic CD34+ HSPCs. Splice variants will be silenced by using morpholino-oligonucleotide sequences complementary to specific intron-exon or exon-intron boundaries of the TERT pre-mRNA sequence of TERT, which block the alternate splice sites α- (intron 5/exon 6) and β- (intron 6/exon 7). We will test the impact of this molecular modification on myelopoietic potential in ex vivo and in vivo. A novel mouse model NSGS mouse that support myelopoietic differentiation of human CD34+ cells will be used. Beneficial effects of the TERT variants’ knockdown on mitochondrial functions including oxidative stress, metabolism - glycolysis and oxidative phosphorylation, and mitoDNA damage will be evaluated. This pilot study will enhance our understanding of TERT physiology in diabetic HSPC
Xiao Abstract Type B P&F 2022
Iron is an essential micronutrient required for many biological processes, including erythropoiesis, but excess iron is toxic due to its ability to generate reactive oxygen species. Aberrant regulation of the master iron hormone hepcidin is a major underlying cause of most iron disorders including anemia of chronic disease, iron refractory iron deficiency anemia, beta-thalassemia, and hemochromatosis. Hepcidin functions by binding and inducing degradation of the sole iron exporter ferroportin to reduce iron influx into the circulation from dietary sources and body stores. Liver hepcidin expression is regulated by several different signals that indicate whether the body needs more or less iron, including serum and tissue iron levels, erythropoietic drive, and inflammation. Previous studies have discovered that the bone morphogenetic protein (BMP)-SMAD signaling pathway is a central regulator of hepcidin transcription in response to most of its known signals. However, there are many facets of hepcidin regulation that are still not understood, including how hepcidin is regulated by homeostatic iron regulator HFE. Mutations in HFE are the most common cause of hereditary hemochromatosis, an autosomal recessive iron overload disorder with a prevalence of 1 in 300 individuals in the United States. Although HFE mutations were linked to hereditary hemochromatosis over 20 years ago, the precise mechanisms of action of HFE in hepcidin regulation are still unclear. The current working model is that HFE deficiency leads to impaired SMAD signaling responses to BMP ligands. In the current proposal, preliminary data will be presented using a novel mouse model demonstrating that HFE also regulates hepcidin through a SMAD-independent pathway. Herein, I propose to perform bulk RNA sequencing (Seq) and single cell RNA-seq in this mouse model as a discovery approach to identify the unknown SMAD-independent signaling pathway(s) governing hepcidin regulation by HFE. This work holds the promise to shift the paradigm in understanding the mechanism of action of HFE and to discover a novel signaling pathway governing hepcidin and iron homeostasis regulation that may ultimately identify new therapeutic targets for treating iron disorders.
Belot Abstract Type B P&F 2022
Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biological functions and is the major source of bioavailable iron in the human diet. The current dogma states that mammalian heme levels within cells are controlled by balance between heme biosynthesis and heme catabolism. In the past few years, the Hamza lab has led the field by contributing to the discovery of eukaryotic heme transporters and heme trafficking pathways. Our overarching, paradigm-shifting hypothesis is that cellular heme levels are not only maintained by internal heme synthesis (cell-autonomous), but also by distally located proteins which signal systemic heme requirements to a heme trafficking network (cell-nonautonomous). Although emerging evidence supports the existence of a cell-nonautonomous heme communication system in mammals, this concept has remained unexplored. Our recent preliminary studies have revealed an unexpected and novel finding that adult mice under heme depletion and acute need for red blood cells production upregulate erythroid HRG1 expression. This strongly suggests that heme homeostasis during erythropoiesis involves previously unappreciated mechanism(s). Thus, the overall goal of the proposed studies is to use stress erythropoiesis condition (chronic phlebotomies) in different mouse model (WT, HRG1 KO, HRG1EpoR/EpoR mice) to elucidate whether HRG1 heme importer could participate in hemoglobinization of red blood cells. We plan to (a) elucidate the role of HRG1 in erythroid progenitors, and (b) identify the heme-responsive cell-nonautonomous pathway using RNA-seq in WT and HRG1-KO mice.