Combinatorial
            <i>Gata2</i>
            and Sca1 expression defines hematopoietic stem cells in the bone marrow niche

Suzuki, Norio; Onodera, Osamu; Minegishi, Naoko; Nishikawa, Mitsuo; Ohta, Takayuki; Takahashi, Satoru; Engel, James Douglas; Yamamoto, Masayuki

doi:10.1073/pnas.0508928103

Cited by 99 publications

(129 citation statements)

References 35 publications

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“…Rescue of the Gata2 Null Mutation with a Linked BAC Transgene-A Gata2 null mutant (EII-KI) mouse in which the green fluorescent protein (GFP) was integrated at the translational start site in Gata2 exon II has been previously characterized (23). GFP expression was detected in all of the anticipated organs and tissues (23).…”

Section: Resultsmentioning

confidence: 99%

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Defining the Functional Boundaries of the Gata2 Locus by Rescue with a Linked Bacterial Artificial Chromosome Transgene

Brandt

Khandekar

Suzuki

et al. 2008

Journal of Biological Chemistry

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Transcription factor GATA-2 is vital for both hematopoietic progenitor cell function and urogenital patterning. Transgenic mapping studies have shown that the hematopoietic and urogenital enhancers are located hundreds of kbp 5 and 3 to the Gata2 structural gene, and both are vital for embryonic development. Because the size of mammalian genes, including all of their associated regulatory elements, can exceed a megabase, transgenic complementation in mice has, in specific instances, proven to be a formidable hurdle. After incorporating the Gata2 structural gene as well as the distant hematopoietic and urogenital enhancers into a single, contiguous piece of DNA by fusing two bacterial artificial chromosomes (BACs) into one, we formally tested the hypothesis that the functional boundaries of this locus are contained within this contiguous genomic span. We show that two independent lines of transgenic mice bearing a multicopy 413-kbp-linked Gata2 BAC transgene (bearing sequences from ؊187 to ؉226 kbp of the locus) are able to fully rescue Gata2 null mutant embryonic lethality and that the rescued animals behave and reproduce normally. Surprisingly, the linked BAC confers expression in the ureteric epithelium, whereas sequences within any of the overlapping parental BACs and a yeast artificial chromosome that were originally tested do not, and thus these experiments also define a novel synthetic enhancer activity that has not been previously described. These genetic complementation studies define the required outer limits of the Gata2 locus and formally demonstrate that enhancers lying beyond those boundaries are not necessary for Gata2-regulated viability or fecundity.Transcription factor GATA-2 is exemplary of a vital developmental factor whose regulation reveals many of the intricacies of the controlling apparatus that is required for proper temporal and tissue-specific expression of a gene in many different tissues and organs that are critical for mammalian development. The evolutionarily conserved C 4 zinc finger GATA transcription factors play demonstrably crucial roles in embryogenesis. GATA-2, originally cloned from a chicken cDNA library (1), was shown to be essential for the proliferation and/or differentiation of early hematopoietic progenitors, as Gata2 null mutant mice die around mid-gestation from a block in primitive hematopoiesis (2). Further examination of Gata2 gain-of-function mice and in vitro differentiation of Gata2 null mutant ES cells underscored the fundamental conclusions from the initial loss of function experiments, and showed that GATA-2 plays a pivotal role in the proliferation of very early hematopoietic progenitors (3-5).We showed several years ago that Gata2 null mutant embryonic lethality could be rescued by complementation with a 247-kbp transgenic yeast artificial chromosome (YAC) 3 bearing sequences from Ϫ174 to ϩ73 kbp (revised endpoints relative to the Gata2 translational initiation site); the YAC contains all of the regulatory information required to rescue Gata2 function in p...

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Section: Resultsmentioning

confidence: 99%

“…GFP expression was detected in all of the anticipated organs and tissues (23). We mated the two independent linked BAC transgenic (Tg G2BAC ) lines (2.2 and 2.12) to Gata2 germ line mutant heterozygotes to generate Gata2 ϩ/Ϫ :Tg G2BAC compound mutant animals; none of the compound mutant offspring exhibited abnormal behavior.…”

Section: Resultsmentioning

confidence: 99%

Defining the Functional Boundaries of the Gata2 Locus by Rescue with a Linked Bacterial Artificial Chromosome Transgene

Brandt

Khandekar

Suzuki

et al. 2008

Journal of Biological Chemistry

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“…on May 10, 2018. by guest www.bloodjournal.org From when expressed in hematopoietic progenitor cells derived from embryonic stem cells. 27 GATA2 is expressed both in quiescent and proliferating hematopoietic stem/progenitor cells [28][29][30] and is rapidly degraded through ubiquitination-proteasome-dependent systems. 31 We found marked variation in GATA2 staining among cultured cells, 31 suggesting cell-to-cell fluctuations in GATA2 expression.…”

Section: Introductionmentioning

confidence: 99%

Cell-cycle–dependent oscillation of GATA2 expression in hematopoietic cells

Koga

Yamada

Abe

et al. 2007

Blood

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In vitro manipulation of hematopoietic stem cells (HSCs) is a key issue in both transplantation therapy and regenerative medicine, and thus new methods are required to achieve HSC expansion with self-renewal. GATA2 is a transcription factor controlling pool size of HSCs. Of interest, continuous overexpression of GATA2 does not induce HSC proliferation. In this report, we demonstrate that GATA2 expression, in leukemic and normal hematopoietic cells, oscillates during the cell cycle, such that expression is high in S phase but low in G 1 /S and M phase.GATA2 binding to target Bcl-X gene also oscillates in accordance with GATA2 expression. Using a green fluorescent protein (GFP)-GATA2 fusion protein, we demonstrate cell-cycle-specific activity of proteasome-dependent degradation of GATA2. Immunoprecipitation/immunoblotting analysis demonstrated phosphorylation of GATA2 at cyclin-dependent kinase (Cdk)-consensus motifs, S/T 0 P ؉1 , and interaction of GATA2 with Cdk2/ cyclin A2-, Cdk2/cyclin A2-, and Cdk4/ cyclin D1-phosphorylated GATA2 in vitro. Mutants in phosphorylation motifs exhibited altered expression profiles of GFP-GATA2 domain fusion proteins. These results indicate that GATA2 phosphorylation by Cdk/cyclin systems is responsible for the cell-cycle-dependent regulation of GATA2 expression, and suggest the possibility that a cell-cycle-specific "onoff" response of GATA2 expression may control hematopoietic-cell proliferation and survival. IntroductionHematopoietic stem cells (HSCs) give rise to the immunohematopoietic system throughout the lifetime of host animals. 1 HSCs have distinctive systems regulating their proliferation and differentiation. These systems maintain steady-state hematopoiesis, and respond to stresses, including infection, hypoxia, and blood loss. 1,2 Two types of cell division occur in HSCs: one type produces differentiated daughter cells to supply mature blood cells, while the other type reproduces HSCs (self-renewal division) to maintain life-long hematopoiesis. 1,2 Of interest, in the bone marrow reservoir of HSCs, more than 70% of bone marrow HSCs are quiescent (G 0 phase), maintaining high proliferative capacity and pluripotency. 3 In contrast, their quiescent mature progeny generally lack the capacity for growth and division.Directing the expansion of HSCs in vitro is an urgent problem that must be solved to meet the needs of transplantation therapy and to fulfill the promise of regenerative medicine. 4 HSC self-renewal and maturation divisions appear to be controlled, at least in part, by transcription factors (MEL/ELF4, 5 Gfi1 6,7 ), and also by cellcycle regulatory factors (cyclin-dependent kinase (Cdk) inhibitor, p21 Cip1/Waf1 , 8 p18 INK4C 9 phosphatase and tensin homologue (PTEN), 10 c-Myc, 11 and Myc antagonist, Mad 1 12 ). Analysis of mice deficient in Gfi1, 6,7 p21 Cip1/Waf1 , 8 or PTEN 10 revealed increased marrow HSC cycling and subsequent exhaustion of HSCs, and suggest possible cooperation between transcription factors and cell-cycle regulators.GATA2 is a member of th...

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“…Of the six GATA factors, GATA1, GATA2, and GATA3 are abundantly expressed in hematopoietic tissues and are referred to as hematopoietic GATA factors. Each GATA factor is nonredundantly required in the specific cell lineages (1), such that GATA3 is expressed in Th2 lymphoid cells (2) and GATA2 is expressed in immature multipotent progenitors and hematopoietic stem cells (3). GATA1 is expressed in erythroid lineage cells, megakaryocytic lineage cells, eosinophils, and mast cells.…”

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confidence: 99%

“…GATA1 is mainly required for erythropoiesis and megakaryopoiesis (4 -6). The complete lack of Gata1 expression leads mice to embryonic lethality by embryonic 11.5 days (E11.5) 3 due to insufficient primitive erythropoiesis (7). Strict regulations of Gata1 and Gata2 genes by a transcription factor network, including GATA factors, are important for the maintenance of hematopoietic homeostasis (8).…”

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confidence: 99%

N- and C-terminal Transactivation Domains of GATA1 Protein Coordinate Hematopoietic Program

Kaneko

Kobayashi

Yamamoto

et al. 2012

Journal of Biological Chemistry

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Background:The N-terminal transactivation domain (N-TAD) is requisite for GATA1 function in vivo. Results: The C-terminal region of GATA1 works as TAD, and fetal hematopoiesis is perturbed in the GATA1-deficient mice rescued with GATA1 lacking C-terminal TAD activity. Conclusion: GATA1 has two TADs, which differentially work. Significance: The complex nature of molecular mechanisms is underlying how GATA1 works for the maintenance of hematopoietic homeostasis.

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Combinatorial Gata2 and Sca1 expression defines hematopoietic stem cells in the bone marrow niche

Cited by 99 publications

References 35 publications

Defining the Functional Boundaries of the Gata2 Locus by Rescue with a Linked Bacterial Artificial Chromosome Transgene

Defining the Functional Boundaries of the Gata2 Locus by Rescue with a Linked Bacterial Artificial Chromosome Transgene

Cell-cycle–dependent oscillation of GATA2 expression in hematopoietic cells

N- and C-terminal Transactivation Domains of GATA1 Protein Coordinate Hematopoietic Program

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