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

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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Systematic integration of GATA transcription factors and epigenomes via IDEAS paints the regulatory landscape of mouse hematopoietic cells

10.1101/730358 ◽

2019 ◽

Author(s):

Ross C. Hardison ◽

Yu Zhang ◽

Cheryl A. Keller ◽

Guanjue Xiang ◽

Elisabeth Heuston ◽

...

Keyword(s):

Transcription Factors ◽

Hematopoietic Cells ◽

Cell Types ◽

Regulatory Elements ◽

Two Dimensions ◽

Specific Cell ◽

Hematopoietic Stem ◽

Cell Lineages ◽

Gata Factors ◽

Regulatory Landscape

SummaryMembers of the GATA family of transcription factors play key roles in the differentiation of specific cell lineages by regulating the expression of target genes. Three GATA factors play distinct roles in hematopoietic differentiation. In order to better understand how these GATA factors function to regulate genes throughout the genome, we are studying the epigenomic and transcriptional landscapes of hematopoietic cells in a model-driven, integrative fashion. We have formed the collaborative multi-lab VISION project to conduct ValIdated Systematic IntegratiON of epigenomic data in mouse and human hematopoiesis. The epigenomic data included nuclease accessibility in chromatin, CTCF occupancy, and histone H3 modifications for twenty cell types covering hematopoietic stem cells, multilineage progenitor cells, and mature cells across the blood cell lineages of mouse. The analysis used the Integrative and Discriminative Epigenome Annotation System (IDEAS), which learns all common combinations of features (epigenetic states) simultaneously in two dimensions - along chromosomes and across cell types. The result is a segmentation that effectively paints the regulatory landscape in readily interpretable views, revealing constitutively active or silent loci as well as the loci specifically induced or repressed in each stage and lineage. Nuclease accessible DNA segments in active chromatin states were designated candidate cis-regulatory elements in each cell type, providing one of the most comprehensive registries of candidate hematopoietic regulatory elements to date. Applications of VISION resources are illustrated for regulation of genes encoding GATA1, GATA2, GATA3, and Ikaros. VISION resources are freely available from our website http://usevision.org.

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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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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

Molecular and Cellular Biology ◽

10.1128/mcb.00032-10 ◽

2010 ◽

Vol 30 (15) ◽

pp. 3853-3863 ◽

Cited By ~ 44

Author(s):

Nicola K. Wilson ◽

Richard T. Timms ◽

Sarah J. Kinston ◽

Yi-Han Cheng ◽

S. Helen Oram ◽

...

Keyword(s):

Stem Cell ◽

Transgenic Mice ◽

Chromatin Immunoprecipitation ◽

Regulatory Networks ◽

Fetal Liver ◽

Regulatory Elements ◽

Regulatory Regions ◽

Hematopoietic Stem ◽

Upstream Regulators

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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Kit regulatory elements required for expression in developing hematopoietic and germ cell lineages

Blood ◽

10.1182/blood-2003-04-1296 ◽

2003 ◽

Vol 102 (12) ◽

pp. 3954-3962 ◽

Cited By ~ 57

Author(s):

Linda A. Cairns ◽

Emanuela Moroni ◽

Elena Levantini ◽

Alessandra Giorgetti ◽

Francesca G. Klinger ◽

...

Keyword(s):

Stem Cell ◽

Fluorescent Protein ◽

Primordial Germ Cells ◽

Hematopoietic Cells ◽

Cell Types ◽

Regulatory Elements ◽

Mouse Development ◽

Hematopoietic Stem ◽

Hypersensitive Sites ◽

Green Fluorescent

Abstract The Kit (White) gene encodes the transmembrane receptor of stem cell factor/Kit ligand (KL) and is essential for the normal development/maintenance of pluripotent primordial germ cells (PGCs), hematopoietic stem cells (HSCs), melanoblasts, and some of their descendants. The molecular basis for the transcriptional regulation of Kit during development of these important cell types is unknown. We investigated Kit regulation in hematopoietic cells and PGCs. We identified 6 DNase I hypersensitive sites (HS1-HS6) within the promoter and first intron of the mouse Kit gene and developed mouse lines expressing transgenic green fluorescent protein (GFP) under the control of these regulatory elements. A construct driven by the Kit promoter and including all 6 HS sites is highly expressed during mouse development in Kit+ cells including PGCs and hematopoietic progenitors (erythroid blast-forming units and mixed colony-forming units). In contrast, the Kit promoter alone (comprising HS1) is sufficient to drive low-level GFP expression in PGCs, but unable to function in hematopoietic cells. Hematopoietic expression further requires the addition of the intronproximal HS2 fragment; HS2 also greatly potentiates the activity in PGCs. Thus, HS2 acts as an enhancer integrating transcriptional signals common to 2 developmentally unrelated stem cell/progenitor lineages. Optimal hematopoietic expression further requires HS3-HS6.

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First blood: the endothelial origins of hematopoietic progenitors

Angiogenesis ◽

10.1007/s10456-021-09783-9 ◽

2021 ◽

Author(s):

Giovanni Canu ◽

Christiana Ruhrberg

Keyword(s):

Stem Cells ◽

Molecular Mechanisms ◽

Vascular Endothelial Cells ◽

Fetal Liver ◽

Cell Types ◽

Yolk Sac ◽

Vascular Endothelial ◽

Hematopoietic Stem ◽

Close Proximity ◽

Clinical Interest

AbstractHematopoiesis in vertebrate embryos occurs in temporally and spatially overlapping waves in close proximity to blood vascular endothelial cells. Initially, yolk sac hematopoiesis produces primitive erythrocytes, megakaryocytes, and macrophages. Thereafter, sequential waves of definitive hematopoiesis arise from yolk sac and intraembryonic hemogenic endothelia through an endothelial-to-hematopoietic transition (EHT). During EHT, the endothelial and hematopoietic transcriptional programs are tightly co-regulated to orchestrate a shift in cell identity. In the yolk sac, EHT generates erythro-myeloid progenitors, which upon migration to the liver differentiate into fetal blood cells, including erythrocytes and tissue-resident macrophages. In the dorsal aorta, EHT produces hematopoietic stem cells, which engraft the fetal liver and then the bone marrow to sustain adult hematopoiesis. Recent studies have defined the relationship between the developing vascular and hematopoietic systems in animal models, including molecular mechanisms that drive the hemato-endothelial transcription program for EHT. Moreover, human pluripotent stem cells have enabled modeling of fetal human hematopoiesis and have begun to generate cell types of clinical interest for regenerative medicine.

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Expansion of mouse hematopoietic stem/progenitor cells in three-dimensional cocultures on growth-suppressed stromal cell layer

The International Journal of Artificial Organs ◽

10.1177/0391398819827596 ◽

2019 ◽

Vol 42 (7) ◽

pp. 374-379 ◽

Cited By ~ 1

Author(s):

Hirotoshi Miyoshi ◽

Chiaki Sato ◽

Yuichiro Shimizu ◽

Misa Morita

Keyword(s):

Progenitor Cells ◽

Mitomycin C ◽

Stromal Cells ◽

Fetal Liver ◽

Three Dimensional ◽

Hematopoietic Cells ◽

Liver Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Fetal Liver Cells

With the aim of establishing an effective method to expand hematopoietic stem/progenitor cells for application in hematopoietic stem cell transplantation, we performed ex vivo expansion of hematopoietic stem/progenitor cells derived from mouse fetal liver cells in three-dimensional cocultures with stromal cells. In these cocultures, stromal cells were first cultured within three-dimensional scaffolds to form stromal layers and then fetal liver cells containing hematopoietic cells were seeded on these scaffolds to expand the hematopoietic cells over the 2 weeks of coculture in a serum-containing medium without the addition of cytokines. Prior to coculture, stromal cell growth was suppressed by treatment with the DNA synthesis inhibitor mitomycin C, and its effect on hematopoietic stem/progenitor cell expansion was compared with that in control cocultures in which fetal liver cells were cocultured with three-dimensional freeze-thawed stromal cells. After coculture with mitomycin C-treated stromal cells, we achieved a several-fold expansion of the primitive hematopoietic cells (c-kit+hematopoietic progenitor cells >7.8-fold, and CD34+hematopoietic stem/progenitor cells >3.5-fold). Compared with control cocultures, expansion of hematopoietic stem/progenitor cells tended to be lower, although that of hematopoietic progenitor cells was comparable. Thus, our results suggest that three-dimensional freeze-thawed stromal cells have higher potential to expand hematopoietic stem/progenitor cells compared with mitomycin C-treated stromal cells.

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The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development

Blood ◽

10.1182/blood.v85.1.96.bloodjournal85196 ◽

1995 ◽

Vol 85 (1) ◽

pp. 96-105 ◽

Cited By ~ 149

Author(s):

PE Young ◽

S Baumhueter ◽

LA Lasky

Keyword(s):

Blood Vessels ◽

Matrix Protein ◽

Vascular System ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Embryoid Bodies ◽

Extracellular Matrix Protein ◽

Hematopoietic Stem ◽

Endothelial Growth Factor ◽

High Degree

The processes of angiogenesis and hematopoiesis require a high degree of coordination during embryogenesis. Whereas much is understood about the development of the vascular system in avian embryos, little information has been attained in mammals, predominantly because there are no specific markers for either blood vessels or hematopoietic cells in any developing mammalian system. We have recently shown that murine CD34 (mCD34) is expressed on the vascular endothelium in all organs and tissues of the adult mouse as well as on a small percentage of presumably hematopoietic stem cells in the bone marrow and fetal liver. Here we show that mCD34 is also expressed on the endothelium of blood vessels and on a subset of hematopoietic-like cells throughout murine development. mCD34 is first observed on the yolk sac endothelium of day 7.5 embryos and on a subset of hematopoietic cells within these yolk sacs. mCD34 expression is maintained on vessels and hematopoietic cells in all organs and tissues throughout embryogenesis. In addition, mCD34 is localized on growth conelike filopodial processes that appear at the budding edge of newly sprouted capillaries. Double staining of capillaries for mCD34 and laminin shows that these growth conelike processes seem to be free of laminin, whereas the formed capillaries seem to be coated with this extracellular matrix protein. Analysis of vessels in developing brain shows that these filopodial processes seem to be directed toward the ventricular epithelium, a previously described site of vascular endothelial growth factor synthesis. Finally, we show that the vascular structures of developing murine embryoid bodies also express mCD34. These data suggest that mCD34 is a useful marker for the analysis of the development of the blood vascular system in murine embryos.

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A Kinetic Model for the Homing and Migration of Prenatally Transplanted Marrow

Blood ◽

10.1182/blood.v94.9.3251 ◽

1999 ◽

Vol 94 (9) ◽

pp. 3251-3257 ◽

Cited By ~ 40

Author(s):

Aimen F. Shaaban ◽

Heung Bae Kim ◽

Ross Milner ◽

Alan W. Flake

Keyword(s):

Stem Cell ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Donor Cell ◽

Cell Phenotype ◽

Functional Assay ◽

Cell Frequency ◽

Color Flow ◽

Hematopoietic Stem ◽

Kinetic Profile

Abstract Currently little is known about the mechanisms regulating the homing and the early engraftment of prenatally transplanted hematopoietic cells due to the lack of a relevant functional assay. In this study, we have defined a reproducible kinetic profile of the homing and the early engraftment events in a murine model of prenatal stem cell transplantation. Light density mononuclear cells (LDMCs) from adult C57Pep3b and SJL/J marrow were transplanted by intraperitoneal (IP) injection into C57BL/6 fetuses (106 LDMCs/fetus) at 14 days of gestation. The fetuses were sacrificed at early time points (1.5 to 96 hours) after transplantation. Recipient fetal liver and cord blood were analyzed for donor cell frequency and donor cell phenotype by dual color flow cytometry. Pertinent findings included the following: (1) a triphasic kinetic profile exists after in utero hematopoietic stem cell (HSC) transplantation (homing of circulating donor cells, rapid reduction of donor cell frequency, and donor cell competitive equilibration); (2) homing to the fetal liver is nonselective and reflects the phenotypic profile of the donor population; and (3) the kinetics after the prenatal transplantation of congenic or fully allogeneic cells are identical. This model will facilitate a systematic analysis of the mechanisms that regulate the homing of prenatally transplanted hematopoietic cells.

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The Ly-6E.1 (Sca-1) gene requires a 3' chromatin-dependent region for high-level gamma-interferon-induced hematopoietic cell expression

Blood ◽

10.1182/blood.v87.7.2750.bloodjournal8772750 ◽

1996 ◽

Vol 87 (7) ◽

pp. 2750-2761 ◽

Cited By ~ 17

Author(s):

A Sinclair ◽

B Daly ◽

E Dzierzak

Keyword(s):

Consensus Sequence ◽

Hematopoietic Cells ◽

Regulatory Elements ◽

Dnase I ◽

Gamma Interferon ◽

Hematopoietic Cell ◽

Hematopoietic Stem ◽

Dnase I Hypersensitive Sites ◽

Hypersensitive Sites ◽

High Level

The Ly-6E.1/A.2 gene product recognized by the Sca-1 antibody has been found on murine hematopoietic stem cells and some hematopoietic precursors, T lymphocytes, and nonhematopoietic cell lineages, suggesting a complex array of gene regulatory elements. The ability to use the Ly6E.1/A.2 transcriptional regulatory elements to direct expression of heterologous genes will allow for the manipulation of these cells during development and in hematopoietic cell transplantations. To identify the elements necessary for high-level expression, we have made deletion constructs of Ly-6E.1 gene flanking regions containing DNase I hypersensitive sites, tested them for expression in hematopoietic cells, and have performed kinetic analyses to correlate the appearance of hypersensitive sites with gene transcription and protein expression. We show that a 3′ region containing two DNase I hypersensitive sites at +8.7 and +8.9 kb is required for high-level, gamma-interferon (gamma-IFN)-induced expression of the Ly-6E.1 gene and that a consensus sequence for a gamma-IFN-responsive element localizes to the +8.7 site. We also provide a description of allele- and cell-specific DNase I hypersensitive site patterns of the Ly-6E.1 and Ly-6A.2 genes. Taken together, these data indicate that while both 5′ and 3′ hypersensitive sites are rapidly induced with gamma-IFN, the 3′ most distal hypersensitive sites are involved in directing high levels of expression of Sca-1 in hematopoietic cells.

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Hoxb4-deficient mice undergo normal hematopoietic development but exhibit a mild proliferation defect in hematopoietic stem cells

Blood ◽

10.1182/blood-2003-10-3557 ◽

2004 ◽

Vol 103 (11) ◽

pp. 4126-4133 ◽

Cited By ~ 86

Author(s):

Ann C. M. Brun ◽

Jon Mar Björnsson ◽

Mattias Magnusson ◽

Nina Larsson ◽

Per Leveén ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Hox Genes ◽

Fetal Liver ◽

Severe Combined Immunodeficiency ◽

Hematopoietic Stem ◽

Cell Counts ◽

Mild Reduction

Abstract Enforced expression of Hoxb4 dramatically increases the regeneration of murine hematopoietic stem cells (HSCs) after transplantation and enhances the repopulation ability of human severe combined immunodeficiency (SCID) repopulating cells. Therefore, we asked what physiologic role Hoxb4 has in hematopoiesis. A novel mouse model lacking the entire Hoxb4 gene exhibits significantly reduced cellularity in spleen and bone marrow (BM) and a subtle reduction in red blood cell counts and hemoglobin values. A mild reduction was observed in the numbers of primitive progenitors and stem cells in adult BM and fetal liver, whereas lineage distribution was normal. Although the cell cycle kinetics of primitive progenitors was normal during endogenous hematopoiesis, defects in proliferative responses of BM Lin- Sca1+ c-kit+ stem and progenitor cells were observed in culture and in vivo after the transplantation of BM and fetal liver HSCs. Quantitative analysis of mRNA from fetal liver revealed that a deficiency of Hoxb4 alone changed the expression levels of several other Hox genes and of genes involved in cell cycle regulation. In summary, the deficiency of Hoxb4 leads to hypocellularity in hematopoietic organs and impaired proliferative capacity. However, Hoxb4 is not required for the generation of HSCs or the maintenance of steady state hematopoiesis.

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