Gfi1 Expression Is Controlled by Five Distinct Regulatory Regions Spread over 100 Kilobases, with Scl/Tal1, Gata2, PU.1, Erg, Meis1, and Runx1 Acting as Upstream Regulators in Early Hematopoietic Cells

ABSTRACT The growth factor independence 1 (Gfi1) gene was originally discovered in the hematopoietic system, where it functions as a key regulator of stem cell homeostasis, as well as neutrophil and T-cell development. Outside the blood system, Gfi1 is essential for inner-ear hair and intestinal secretory cell differentiation. To understand the regulatory hierarchies within which Gfi1 operates to control these diverse biological functions, we used a combination of comparative genomics, locus-wide chromatin immunoprecipitation assays, functional validation in cell lines, and extensive transgenic mouse assays to identify and characterize the complete ensemble of Gfi1 regulatory elements. This concerted effort identified five distinct regulatory elements spread over 100kb each driving expression in transgenic mice to a subdomain of endogenous Gfi1. Detailed characterization of an enhancer 35 kb upstream of Gfi1 demonstrated activity in the dorsal aorta region and fetal liver in transgenic mice, which was bound by key stem cell transcription factors Scl/Tal1, PU.1/Sfpi1, Runx1, Erg, Meis1, and Gata2. Taken together, our results reveal the regulatory regions responsible for Gfi1 expression and importantly establish that Gfi1 expression at the sites of hematopoietic stem cell (HSC) emergence is controlled by key HSC regulators, thus integrating Gfi1 into the wider HSC regulatory networks.

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The transcriptional program controlled by the stem cell leukemia gene Scl/Tal1 during early embryonic hematopoietic development

Blood ◽

10.1182/blood-2009-01-200048 ◽

2009 ◽

Vol 113 (22) ◽

pp. 5456-5465 ◽

Cited By ~ 81

Author(s):

Nicola K. Wilson ◽

Diego Miranda-Saavedra ◽

Sarah Kinston ◽

Nicolas Bonadies ◽

Samuel D. Foster ◽

...

Keyword(s):

Stem Cell ◽

Regulatory Networks ◽

Transcriptional Control ◽

Target Genes ◽

Fetal Liver ◽

Bioinformatic Analysis ◽

Regulatory Elements ◽

Hematopoietic Stem ◽

A Genome

The basic helix-loop-helix transcription factor Scl/Tal1 controls the development and subsequent differentiation of hematopoietic stem cells (HSCs). However, because few Scl target genes have been validated to date, the underlying mechanisms have remained largely unknown. In this study, we have used ChIP-Seq technology (coupling chromatin immunoprecipitation with deep sequencing) to generate a genome-wide catalog of Scl-binding events in a stem/progenitor cell line, followed by validation using primary fetal liver cells and comprehensive transgenic mouse assays. Transgenic analysis provided in vivo validation of multiple new direct Scl target genes and allowed us to reconstruct an in vivo validated network consisting of 17 factors and their respective regulatory elements. By coupling ChIP-Seq in model cell lines with in vivo transgenic validation and sophisticated bioinformatic analysis, we have identified a widely applicable strategy for the reconstruction of stem cell regulatory networks in which biologic material is otherwise limiting. Moreover, in addition to revealing multiple previously unrecognized links to known HSC regulators, as well as novel links to genes not previously implicated in HSC function, comprehensive transgenic analysis of regulatory elements provided substantial new insights into the transcriptional control of several important hematopoietic regulators, including Cbfa2t3h/Eto2, Cebpe, Nfe2, Zfpm1/Fog1, Erg, Mafk, Gfi1b, and Myb.

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microRNA Control of Hematopoietic Stem Cell Fitness and Myelopoiesis.

Blood ◽

10.1182/blood.v110.11.373.373 ◽

2007 ◽

Vol 110 (11) ◽

pp. 373-373 ◽

Cited By ~ 1

Author(s):

Chinavenmeni S. Velu ◽

Sarah Porteous ◽

Haiming Xu ◽

Avinash M. Baktula ◽

Philip Roehrs ◽

...

Keyword(s):

Stem Cell ◽

Cell Lines ◽

Chromatin Immunoprecipitation ◽

Target Genes ◽

Human Cancer ◽

Fetal Liver ◽

Embryonic Stem ◽

Wild Type ◽

Hematopoietic Stem ◽

Human Cancer Cell Lines

Abstract The Growth factor independent -1 (Gfi1) transcriptional repressor regulates both hematopoietic stem cell (HSC) self renewal and myeloid differentiation. Gfi1 null mice eventually die of HSC exhaustion, and Gfi1−/− HSC are not competitive in transplantation assays. Here we show that Gfi1 is a master regulator of microRNAs and that transcriptional control of a microRNA gene is critical for Gfi1-directed stem cell competitiveness and myelopoiesis. First, we show that the gene encoding miR21 is a direct transcriptional target of Gfi1. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal Gfi1 binding to specific DNA sequences upstream of the miR21 stem loop. Moreover, the expression of Gfi1 and miR21 is reciprocal in 1) wild type and Gfi1−/− marrow cells, 2) during normal differentiation from common myeloid progenitors (CMP) to granulocyte monocyte progenitors (GMP), and 3) during treatment-induced differentiation of human myeloid leukemia cell lines. Forced expression of Gfi1 lowers miR21 levels in wild type Lin− bone marrow cells and human cancer cell lines. Knockdown of Gfi1 expression with shRNA in human cancer cell lines increases miR21 expression. Moreover, conditional deletion of Gfi1 induces miR21 expression in primary murine hematopoietic cells, including sorted CMP and GMP. Thus, Gfi1 transcriptionally regulates miR21 in both human and murine hematopoietic cells. Interestingly, we find that the Ski oncoprotein/transcriptional corepressor is a direct target of miR-21. Subsequently, Ski is dramatically reduced in Gfi1−/− HSC and in wild type bone marrow Lin− cells forced to express miR21. Gfi1 may repress miR21 to maintain functional competence. Specifically, we find that Ski is a previously undescribed Gfi1 corepressor. Both endogenous Ski and Gfi1 physically interact. Synthetic Ski and Gfi1 proteins reveal that the interaction is both strong and specific. Chromatin immunoprecipitation reveals Ski and Gfi1 occupy several Gfi1 target genes. However, Ski function is critical as a corepressor on only a subset of Gfi1 target genes. To determine the importance of Ski corepression to Gfi1 induced biology, we examined two well established phenotypes of Gfi1 loss of function; HSC competitiveness and myelopoiesis. When Gfi1−/− embryonic stem cells are injected into a wild type blastocyst, they do not participate in hematopoiesis. Similarly, we find that when Ski−/− embryonic stem cells are injected into a blastocyst, they infrequently participate in hematopoeisis. Next, because Ski−/− animals die at or before birth, we examined the fitness of Ski−/− fetal liver HSC. In competitive transplantation assays, Ski−/− fetal liver HSC were significantly impaired in reconstitution compared to congenic wild-type competitor fetal-liver HSC. Moreover, Ski null HSC generated significantly less myeloid progeny. Thus, Ski−/− HSC display a partial phenocopy of Gfi1−/− hematopoiesis. We conclude that Gfi1 directly targets miR21 to control the expression of Ski, a corepressor for Gfi1, and that the Gfi1/Ski complex is critical to regulate a subset of Gfi1 target genes important for HSC fitness and myeloid cell production.

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Distinct Regulatory Networks Govern Human Hematopoietic Stem Cell Across Development

Blood ◽

10.1182/blood.v126.23.2375.2375 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2375-2375

Author(s):

Sasan Zandi ◽

John E. Dick ◽

Faiyaz Notta ◽

Naoya Takayama

Keyword(s):

Stem Cell ◽

Cell Biology ◽

Regulatory Networks ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Single Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

M Phase

Abstract Introduction: Much of our fundamental understanding of stem cell biology comes from studies of hematopoiesis where single cells produce differentiated progeny while still retaining the ability to produce daughter stem cells (self-renewal). The cardinal property of a stem cell, whether normal or malignant, is self-renewal; the key biological process that ensures the ability of the stem cell to maintain long-term clonal growth. However, our understanding of the molecular basis of self-renewal in human hematopoiesis is limited. At the embryonic stage fetal liver is the main source of hematopoiesis; from week 6 of gestation until before birth. At this stage HSCs are in a different microenvironment but capable of self-renewing and differentiation to the full spectrum of blood lineages. While murine studies uncovered several intrinsic differences between fetal and adult HSCs, a comprehensive analysis of human HSC compartment across development is lacking. In this study we have combined HSC purification methods and xenograft quantitative assay in conjunction with low input RNA sequencing and Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) to provide a comprehensive functional and molecular outlook of human stem cell compartment across development. Results: We followed the dynamics of four sub-fractions of CD34+CD38- divided by CD90 and CD49f expression across human blood development: fetal liver (hFL) and adult bone marrow (hBM). Using xenograft model, we identified human long, intermediate and short term HSCs in hFL and hBM. 5 single CD90+CD49f+ hFL cells were capable of sustaining the multilineage graft for over 52 weeks up to tertiary recipient, while BM cells only last for 20 weeks in the primary recipient. The frequency of LT-HSC in the CD90+CD49f+ compartment goes from 1/8 in hFL to 1/50 in hBM. hFL CD90-CD49f+ cells showed an intermediate repopulation capacity up to 44 weeks in secondary recipient. On average 10% of hFL long term HSC (LT-HSC) were in S/G2/M phase, in contrast only 0.4% of BM LT-HSC were in S/G2/M phase indicating that hFL HSCs are 20 times more in cycle compare to BM. We found that 320 genes were expressed differentially between LT-HSC and multipotent progenitors (MPP) in hBM as oppose to only 32 genes found to be differentially expressed in hFL (FDR<0.1). Interestingly, we found only 2 genes in common between these two groups. ERRBS showed an overall increase in methylation of HSC compartment in hBM compare to hFL and gradual demethylation of lineage associated genes in MPP. Conclusion: Our data indicate that there are distinct regulatory networks that govern hFL and hBM HSC self-renewal. We found very little differences in gene expression between all hFL HCS compartments (average 20 genes) compare to hBM (average 224), indicating that by adulthood self-renewal is becoming more restricted to the LT-HSC compartment. Disclosures No relevant conflicts of interest to declare.

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Expression of the leukemia oncogene Lmo2 is controlled by an array of tissue-specific elements dispersed over 100 kb and bound by Tal1/Lmo2, Ets, and Gata factors

Blood ◽

10.1182/blood-2008-11-187757 ◽

2009 ◽

Vol 113 (23) ◽

pp. 5783-5792 ◽

Cited By ~ 57

Author(s):

Josette-Renée Landry ◽

Nicolas Bonadies ◽

Sarah Kinston ◽

Kathy Knezevic ◽

Nicola K. Wilson ◽

...

Keyword(s):

Regulatory Networks ◽

Fetal Liver ◽

Lymphoblastic Leukemia ◽

Severe Combined Immunodeficiency ◽

Hematopoietic Cells ◽

Cell Types ◽

Regulatory Elements ◽

Proximal Promoter ◽

Hematopoietic Stem ◽

Conserved Noncoding Elements

Abstract The Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.

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Characterization of the new HLA‐B *35:482 , detected in a potential hematopoietic stem cell donor

HLA ◽

10.1111/tan.14150 ◽

2020 ◽

Author(s):

Anastasiia Ananeva ◽

Iuliia Sergeeva ◽

Raushania Gaifullina ◽

Elena Shagimardanova

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Hematopoietic Stem ◽

Cell Donor

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Characterization of hematopoietic stem cell subsets from patients with multiple myeloma after mobilization with plerixafor

Cytotherapy ◽

10.3109/14653249.2010.530652 ◽

2011 ◽

Vol 13 (4) ◽

pp. 459-466 ◽

Cited By ~ 27

Author(s):

Isabel Taubert ◽

Rainer Saffrich ◽

Abraham Zepeda-Moreno ◽

Isabelle Hellwig ◽

Volker Eckstein ◽

...

Keyword(s):

Multiple Myeloma ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Hematopoietic Stem

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HLF Expression Defines the Human Hematopoietic Stem Cell State.

Blood ◽

10.1182/blood.2021010745 ◽

2021 ◽

Author(s):

Bernhard Lehnertz ◽

Jalila Chagraoui ◽

Tara MacRae ◽

Elisa Tomellini ◽

Sophie Corneau ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Fetal Liver ◽

Cell Activity ◽

Mouse Strains ◽

Marker Genes ◽

Hematopoietic Stem ◽

Cell State ◽

Human Hscs ◽

Stem Cell State

Hematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and can regenerate all blood lineages following transplantation. Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. While several transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported. To identify genes with the most selective expression in human HSCs, we profiled population- and single-cell transcriptomes of un-expanded and ex vivo cultured cord blood-derived HSPCs as well as peripheral blood, adult bone marrow, and fetal liver. Based on these analyses, we propose the master transcription factor HLF (Hepatic Leukemia Factor) as one of the most specific HSC marker genes. To directly track its expression in human hematopoietic cells, we developed a genomic HLF reporter strategy, capable of selectively labeling the most immature blood cells based on a single engineered parameter. Most importantly, HLF-expressing cells comprise all of the stem cell activity in culture and in vivo during serial transplantation. Taken together, these results experimentally establish HLF as a defining gene of the human hematopoietic stem cell state and outline a new approach to continuously mark these cells with high fidelity.

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A 2-kb c-mpl promoter fragment is sufficient to direct expression to the megakaryocytic lineage and sites of embryonic hematopoiesis in transgenic mice

Blood ◽

10.1182/blood-2002-01-0281 ◽

2002 ◽

Vol 100 (3) ◽

pp. 1072-1074 ◽

Cited By ~ 13

Author(s):

Sandra Ziegler ◽

Kurt Bürki ◽

Radek C. Skoda

Keyword(s):

Transgenic Mice ◽

Reporter Gene ◽

Fetal Liver ◽

Regulatory Elements ◽

Yolk Sac ◽

Placental Alkaline Phosphatase ◽

Hematopoietic Progenitors ◽

Thrombopoietin Receptor ◽

Human Placental Alkaline Phosphatase ◽

Dna Fragment

Abstract Thrombopoietin receptor c-mpl is expressed on hematopoietic progenitors and cells of the megakaryocytic lineage. The c-mpl promoter may, therefore, be useful for directing the expression of transgenes. We tested whether a 2-kb genomic DNA fragment comprising the putative c-mpl regulatory elements and most of the 5′-untranslated region of mouse c-mpl is able to direct the expression of a reporter gene to hematopoietic cells in transgenic mice. As a reporter gene we used the human placental alkaline phosphatase (PLAP). In adult transgenic mice, PLAP expression was specifically detected in megakaryocytes and platelets. Embryos showed PLAP reporter gene expression already in the yolk sac at embryonic day 6.5 (E6.5) and in blood islands at E7.5. At E9.5, expression was found in blood vessels of the yolk sac and the embryo proper, followed by high levels of expression in the fetal liver at E11.5. Expression in E6.5 yolk sac is compatible with a function of c-mpl and its ligand, thrombopoietin, in the earliest stages of embryonic hematopoiesis.

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Replication Stress Response and CDKN1A Engagement Constrain Fetal Hematopoietic Stem Cell Pool Size in Fanconi Anemia

Blood ◽

10.1182/blood-2019-125669 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 107-107

Author(s):

Makiko Mochizuki-Kashio ◽

Young Me Yoon ◽

Theresa N Menna ◽

Markus Grompe ◽

Peter Kurre

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Nuclear Localization ◽

Fanconi Anemia ◽

Fetal Liver ◽

Replication Stress ◽

Pool Size ◽

Hematopoietic Stem ◽

Independent Functions

Bone marrow (BM) failure is the principal source of morbidity and mortality in Fanconi Anemia (FA) patients. Recessively inherited germline mutations in one of 25 genes lead to deficits by in a pathway central to DNA crosslink repair. Functionally, FA proteins protect adult hematopoietic stem cells (HSC) from p53 mediated apoptosis elicited by alkylating agents, a range of experimental inflammatory cues or aldehyde exposure. However, these mechanisms do not seem to account for depleted hematopoietic stem and progenitor cell pools in very young FA patients, or the spontaneous, non-apoptotic and p53-independent fetal HSC deficits observed in murine models. Building on our previous observation of a quantitatively constrained fetal HSC pool in FA mice (Fancd2-/-), the current experiments reveal the specific developmental timeframe for the onset of stem cell deficits during HSC expansion in the fetal liver (FL). Cell cycle studies using an EdU/BrdU pulse chase protocol reveal delays in S-phase entry and progression at E13.5. Building on the role of FA proteins (FANCM, FANCI and FANCD2) in countering experimental replication stress (RS) in cell line models, we reasoned that rapid rates of proliferation required during expansion in the FL may similarly confer RS on the FA HSC pool. Experiments in E13.5 FL HSC confirmed the predicted increase in single stranded DNA and accumulation of nuclear replication associated protein (pRpa), along with activation of pChk1, a critical cell cycle checkpoint in cells under RS. For comparison, pChk1 in unperturbed adult cells was no different between Fancd2-/- and WT. The data are also consistent with gains in RAD51 and alkaline comet assays we previously published (Yoon et al., Stem Cell Reports 2016). The cell cycle regulator Cdkn1a (p21) is considered a canonical downstream component of the p53 response in adult FA HSC, but it also performs p53 independent functions in the RS response that coincide with its role in the nucleus. Here, we observed an increase in nuclear localization of p21 in Fancd2-/- FL HSC. TGF-β is a critical developmental morphogen that regulates p21 activity, and TGF-β inhibitors can partially reverse adult FA HSC function along with suppression of NHEJ mediated DNA repair. To test regulation of p21 in fetal HSC under RS, we first treated WT FL HSC with aphidicolin to experimentally simulate RS and found that SD208, a small molecule TGF-β-R1 inhibitor, completely rescued the p21 nuclear localization. We then went on to demonstrate that pharmacological inhibition of TGF-β signaling also reversed the nuclear p21 translocation in FA FL HSC (under physiological RS) and functionally rescued the primitive myeloid progenitor colony formation (CFU-GEMM) in vitro. Altogether, our data show that HSC deficits in FA first emerge in the fetal liver, where rapid fetal expansion causes RS that elicits pChk1 activation and nuclear p21 translocation, which restrain cell cycle progression and act as principal mechanisms limiting fetal HSC pool size in FA. Our experiments suggest a central and p53-independent role for p21 in fetal FA HSC regulation. Detailed knowledge of the physiological role of FA proteins in fetal phenotype HSC has the potential to lead to new therapies that delay or rescue hematopoietic failure in FA patients. Disclosures No relevant conflicts of interest to declare.

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