Platelet glycoprotein IIb gene expression as a model of megakaryocyte‐specific expression

Block, Karen; Poncz, Mortimer

doi:10.1002/stem.5530130205

Cited by 51 publications

(16 citation statements)

References 62 publications

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“…We have since shown that these quantitative differences in promoter strength were not speciesspecific differences in the promoter region, but due to differences in the cell systems studied. When the same rat ␣ IIb promoter constructs were restudied in a megakaryocytic cell line (HEL), the results seen were indistinguishable from the human data (12).…”

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confidence: 79%

An Sp1-binding Silencer Element Is a Critical Negative Regulator of the Megakaryocyte-specific αIIb Gene

Shou¹,

Baron²,

Poncz³

1998

Journal of Biological Chemistry

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The Sp1 family of transcription factors are often involved in the regulated expression of TATA-less genes, frequently enhancing gene transcription. In this paper, we demonstrate that an Sp1-binding element inhibits the expression of the megakaryocyte-specific ␣ IIb gene in all cell lines tested and that this inhibition is actively overcome only in megakaryocyte-like cell lines. We had noted previously in primary megakaryocytes that a 50-base pair (bp) deletion from ؊150 to ؊101 bp in the rat ␣ IIb promoter region resulted in increased expression. We now show that deletion of this region markedly increased expression in both megakaryocytic and nonmegakaryocytic cell lines, eliminating the tissue specificity of the ␣ IIb promoter. Electrophoretic mobility shift assays (EMSA) defined a single complex, which bound to a ؊145 to ؊125 bp subregion. Point mutations within this region, localized the critical point of binding around bases ؊136/؊135, and expression studies showed that introduction of the ؊136/؊135 mutation into the rat ␣ IIb promoter had a comparable result to that seen with the 50-bp deletion. EMSA studies with the homologous human ␣ IIb promoter region gave an identical migrating band. Southwestern blots of HeLa nuclear proteins with both the rat ؊145 to ؊125 DNA and its human homologue bound to a single ϳ110-kDa protein, the known molecular weight of Sp1. Confirmation that this region of the ␣ IIb gene promoter bound Sp1 was accomplished using EMSA studies with an Sp1 consensus probe, antiSp1 and -Sp3 antibodies, and recombinant Sp1 protein.Further support for the role of Sp1 in the silencing of the ␣ IIb promoter was obtained using a Gal4 binding site substitution for the silencer region of ␣ IIb and co-expression of near full-length Sp1/Gal4 fusion protein expression vectors. Ectopic reinsertion of the ؊150 to ؊101 bp region, back into the ؊150 to ؊101 bp deleted promoter, enhanced rather than decreased expression, suggesting that Sp1's inhibitory role at ؊136/؊135 depends on its local interactions. In summary, we believe that we have identified a cross-species, non-consensus Sp1-binding site that binds Sp1 and that acts as a silencer of ␣ IIb expression in many cell lines. A model is presented as to how this Sp1-binding silencer element contributes to the megakaryocyte-specific expression of ␣ IIb gene.Platelets have a central role in thrombus formation. These anuclear cytoplasmic fragments are derived from bone marrow megakaryocytes and are highly differentiated (1). One of the specialized features found on the platelet membrane is the ␣ IIb /␤ 3 (also known as glycoprotein IIb/IIIa or CD41b) integrin receptor (2). This receptor is densely packed on the platelet surface. Following platelet activation, this receptor binds fibrinogen and plays an important role in platelet aggregation (2). Normally, ␣ IIb /␤ 3 is only found on developing megakaryocytes and circulating platelets. This is due to the tissue-specific nature of the ␣ IIb subunit. While ␤ 3 is expressed in several different cell types (3), ...

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confidence: 79%

An Sp1-binding Silencer Element Is a Critical Negative Regulator of the Megakaryocyte-specific αIIb Gene

Shou¹,

Baron²,

Poncz³

1998

Journal of Biological Chemistry

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“…During megakaryopoiesis, the expression of megakaryocytic genes, such as GPIIb, GPIX, and C-MPL, is strictly regulated. An early marker of the megakaryocytic lineage, gpIIb (CD41) (63,64), combines with the more broadly expressed ␤ 3 (gpIIIa, CD61) subunit to form the gpIIb/IIIa heterodimer receptor that regulates cell adhesion and functions in aggregation of activated platelets by binding fibrinogen, fibronectin, vitronectin, collagen, and von Willebrand factor (63,64). gpIX is required for assembly of the gpIb⅐V⅐IX complex (65) that provides the binding site for the von Willebrand receptor and is critical for initial adhesion of platelets to damaged blood vessels.…”

Section: Discussionmentioning

confidence: 99%

Ets-dependent Regulation of Target Gene Expression during Megakaryopoiesis

Jackers¹,

Szalai²,

Moussa

et al. 2004

Journal of Biological Chemistry

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Megakaryopoiesis is the process by which hematopoietic stem cells in the bone marrow differentiate into mature megakaryocytes. The expression of megakaryocytic genes during megakaryopoiesis is controlled by specific transcription factors. Fli-1 and GATA-1 transcription factors are required for development of megakaryocytes and promoter analysis has defined in vitro functional binding sites for these factors in several megakaryocytic genes, including GPIIb, GPIX, and C-MPL. Herein, we utilize chromatin immunoprecipitation to examine the presence of Ets-1, Fli-1, and GATA-1 on these promoters in vivo. Fli-1 and Ets-1 occupy the promoters of GPIIb, GPIX, and C-MPL genes in both Meg-01 and CMK11-5 cells. Whereas GPIIb is expressed in both Meg-01 and CMK11-5 cells, GPIX and C-MPL are only expressed in the more differentiated CMK11-5 cells. Thus, in vivo occupancy by an Ets factor is not sufficient to promote transcription of some megakaryocytic genes. GATA-1 and Fli-1 are both expressed in CMK11-5 cells and co-occupy the GPIX and C-MPL promoters. Transcription of all three megakaryocytic genes is correlated with the presence of acetylated histone H3 and phosphorylated RNA polymerase II on their promoters. We also show that exogenous expression of GATA-1 in Meg-01 cells leads to the expression of endogenous c-mpl and gpIX mRNA. Whereas GPIIb, GPIX, and C-MPL are direct target genes for Fli-1, both Fli-1 and GATA-1 are required for formation of an active transcriptional complex on the C-MPL and GPIX promoters in vivo. In contrast, GPIIb expression appears to be independent of GATA-1 in Meg-01 cells.Ets family members each contain a conserved winged helixloop-helix DNA binding (ETS) domain that allows recognition of purine-rich DNA sequences with a core GGA(A/T) consensus, designated EBS 1 (Ets binding sequence) (1-3). These transcription factors have critical roles in the transcriptional control of genes important in development, morphogenesis, proliferation, and angiogenesis. Ets factors can function as either positive or negative transcriptional regulators (4, 5) and additional binding of other transcription factors to cis-elements located near EBSs contributes to regulated transcription of specific target genes. Thus, in addition to binding to DNA, Ets transcription factors participate in protein interactions that affect their functions (6, 7).Fli-1, a member of the Ets gene family of transcription factors, performs functions critical for normal development and oncogenesis (for a review, see Ref. 8). Fli-1 is preferentially expressed in cells of hematopoietic lineages and vascular endothelial cells, and has been shown to transcriptionally activate genes, including the stem cell leukemia gene (9), tenascin (10), the stress response gene GADD153 (11), the anti-apoptotic gene Bcl-2 (12) and several megakaryocytic specific genes (see below). Fli-1 also forms ternary complexes through interaction with SRF to bind to SRE elements of fos and Egr-1 promoters (13, 14). Fli-1 protein interaction with other regulatory proteins...

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“…Collectively, loss of megakaryocyte-derived platelets is one possible cause of the hemorrhagic phenotype we observed. Ets factors have been shown to activate transcription driven by the megakaryocyte-specific membrane glycoprotein gpIIb promoter (7,21), the platelet glycoprotein Iba promoter (15), the glycoprotein IX promoter (4), the platelet factor 4 promoter (24), and the promoter of mpl (10), the receptor for thromobopoietin. These studies support the hypothesis that one or more ETS transcription factors play a role in the regulation of transcription of megakaryocytic and platelet-specific genes.…”

Section: (Iv) It Has Recently Been Demonstrated That Fli1 Protein Is mentioning

confidence: 99%

Hemorrhage, Impaired Hematopoiesis, and Lethality in Mouse Embryos Carrying a Targeted Disruption of the Fli1Transcription Factor

Spyropoulos

Pharr

Lavenburg

et al. 2000

Molecular and Cellular Biology

274

297

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The Ets family of transcription factors have been suggested to function as key regulators of hematopoeisis. Here we describe aberrant hematopoeisis and hemorrhaging in mouse embryos homozygous for a targeted disruption in the Ets family member, Fli1. Mutant embryos are found to hemorrhage from the dorsal aorta to the lumen of the neural tube and ventricles of the brain (hematorrhachis) on embryonic day 11.0 (E11.0) and are dead by E12.5. Histological examinations and in situ hybridization reveal disorganization of columnar epithelium and the presence of hematomas within the neuroepithelium and disruption of the basement membrane lying between this and mesenchymal tissues, both of which express Fli1 at the time of hemorrhaging. Livers from mutant embryos contain few pronormoblasts and basophilic normoblasts and have drastically reduced numbers of colony forming cells. These defects occur with complete penetrance of phenotype regardless of the genetic background (inbred B6, hybrid 129/B6, or outbred CD1) or the targeted embryonic stem cell line used for the generation of knockout lines. Taken together, these results provide in vivo evidence for the role of Fli1 in the regulation of hematopoiesis and hemostasis.The human FLI1 gene, which we originally cloned from the leukemia T-cell line, CEM, is a member of the Ets gene family of transcription factors (38). As observed with all other members of the Ets gene family, FLI1 encodes a protein that retains a region of conserved sequence, the Ets domain (37, 38). This minimal 85-amino-acid region has been shown to be the DNAbinding domain. Ets proteins bind to DNA sequences that contain a consensus GGA(A/T) core motif (Ets-binding site) and, in the majority of cases, function as transcriptional activators. Ets proteins control the expression of genes that are critical for the control of cellular proliferation, differentiation, and programmed cell death. The presence of multiple Ets family proteins in a variety of cell types and the overlapping DNA-binding specificity of the Ets proteins have made it difficult to identify target genes that are specific for individual Ets factors. The generation and analysis of targeted disruptions in individual family members, coupled with the identification of such target genes, however, is one approach to understanding the role of Ets transcription factors in normal and dysregulated development. A variety of studies including the analysis of expression of members of the Ets transcription factor family in hematopoietic tissues and cell lines and the generation and analysis of targeted mutations in Ets gene family members in mouse suggest that they play important roles in the regulation of normal hematopoietic development (13,14,29).FLI1 was found to be highly related to the human ERG gene, and we originally named it ERGB to reflect this homology (38). Sequence alignments of the predicted 452-amino-acid protein product of human FLI1 with those of the ERG and mouse Fli1 products (5) demonstrated 80 and 96% similarity, respectively. FLI1 ha...

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Platelet glycoprotein IIb gene expression as a model of megakaryocyte‐specific expression

Cited by 51 publications

References 62 publications

An Sp1-binding Silencer Element Is a Critical Negative Regulator of the Megakaryocyte-specific αIIb Gene

An Sp1-binding Silencer Element Is a Critical Negative Regulator of the Megakaryocyte-specific αIIb Gene

Ets-dependent Regulation of Target Gene Expression during Megakaryopoiesis

Hemorrhage, Impaired Hematopoiesis, and Lethality in Mouse Embryos Carrying a Targeted Disruption of the Fli1Transcription Factor

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