RESUMO
Vascular smooth muscle cells (SMC) possess a unique cytoplasticity, regulated by transcriptional, translational and phenotypic transformation in response to a diverse range of extrinsic and intrinsic pathogenic factors. The mature, differentiated SMC phenotype is physiologically typified transcriptionally by expression of genes encoding "contractile" proteins, such as SMα-actin (ACTA2), SM-MHC (myosin-11) and SM22α (transgelin). When exposed to various pathological conditions (e.g., pro-atherogenic risk factors, hypertension), SMC undergo phenotypic modulation, a bioprocess enabling SMC to de-differentiate in immature stages or trans-differentiate into other cell phenotypes. As recent studies suggest, the process of SMC phenotypic transformation involves five distinct states characterized by different patterns of cell growth, differentiation, migration, matrix protein expression and declined contractility. These changes are mediated via the action of several transcriptional regulators, including myocardin and serum response factor. Conversely, other factors, including Kruppel-like factor 4 and nuclear factor-κB, can inhibit SMC differentiation and growth arrest, while factors such as yin yang-1, can promote SMC differentiation whilst inhibiting proliferation. This article reviews recent advances in our understanding of regulatory mechanisms governing SMC phenotypic modulation. We propose the concept that transcription factors mediating this switching are important biomarkers and potential pharmacological targets for therapeutic intervention in cardiovascular disease.
Assuntos
Músculo Liso Vascular , Fator de Resposta Sérica , Actinas/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , NF-kappa B/metabolismo , Fenótipo , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismoRESUMO
AIMS: In-stent restenosis and late stent thrombosis are complications associated with the use of metallic and drug-coated stents. Strategies that inhibit vascular smooth muscle cell (SMC) proliferation without affecting endothelial cell (EC) growth would be helpful in reducing complications arising from percutaneous interventions. SMC hyperplasia is also a pathologic feature of graft stenosis and fistula failure. Our group previously showed that forced expression of the injury-inducible zinc finger (ZNF) transcription factor, yin yang-1 (YY1), comprising 414 residues inhibits neointima formation in carotid arteries of rabbits and rats. YY1 inhibits SMC proliferation without affecting EC growth in vitro. Identifying a shorter version of YY1 retaining cell-selective inhibition would make it more amenable for potential use as a gene therapeutic agent. METHODS AND RESULTS: We dissected YY1 into a range of shorter fragments (YY1A-D, YY1Δ) and found that the first two ZNFs in YY1 (construct YY1B, spanning 52 residues) repressed SMC proliferation. Receptor binding domain analysis predicts a three-residue (339KLK341) interaction domain. Mutation of 339KLK341 to 339AAA341 in YY1B (called YY1Bm) abrogated YY1B's ability to inhibit SMC but not EC proliferation and migration. Incubation of recombinant GST-YY1B and GST-YY1Bm with SMC lysates followed by precipitation with glutathione-agarose beads and mass spectrometric analysis identified a novel interaction between YY1B and BASP1. Overexpression of BASP1, like YY1, inhibited SMC but not EC proliferation and migration. BASP1 siRNA partially rescued SMC from growth inhibition by YY1B. In the rat carotid balloon injury model, adenoviral overexpression of YY1B, like full-length YY1, reduced neointima formation, whereas YY1Bm had no such effect. CD31+ immunostaining suggested YY1B could increase re-endothelialization in a 339KLK341-dependent manner. CONCLUSION: These studies identify a truncated form of YY1 (YY1B) that can interact with BASP1 and inhibit SMC proliferation, migration, and intimal hyperplasia after balloon injury of rat carotid arteries as effectively as full length YY1. We demonstrate the therapeutic potential of YY1B in vascular proliferative disease.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Lesões das Artérias Carótidas/terapia , Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Terapia Genética , Proteínas de Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Fator de Transcrição YY1/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Ligação a Calmodulina/genética , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Bovinos , Células Cultivadas , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Hiperplasia , Proteínas de Membrana/genética , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Coelhos , Ratos , Proteínas Repressoras/genética , Transdução de Sinais , Fator de Transcrição YY1/genéticaRESUMO
Yin Yang-1 (YY1) is a zinc finger protein and member of the GLI-Kruppel family that can activate or inactivate gene expression depending on interacting partners, promoter context and chromatin structure, and may be involved in the transcriptional control of â¼10% of the total mammalian gene set. A growing body of literature indicates that YY1 is overexpressed in multiple cancer types and that increased YY1 levels correlate with poor clinical outcomes in many cancers. However, the role of YY1 in the promotion or suppression of tumor growth remains controversial and its regulatory effects may be tumor cell type dependent at least in experimental systems. The molecular mechanisms responsible for the apparently conflicting roles of YY1 are not yet fully elucidated. This review highlights recent advances in our understanding of regulatory insights involving YY1 function in a range of cancer types. For example, YY1's roles in tumor growth involve stabilization of hypoxia-inducible factor HIF-1α in a p53 independent manner, negative regulation of miR-9 transcription, control of MYCT1 transcription, a novel miR-193a-5p-YY1-APC axis, intracellular ROS and mitochondrial superoxide generation, p53 reduction and EGFR activation, control of genes associated with mitochondrial energy metabolism and miRNA regulatory networks involving miR-7, miR-9, miR-34a, miR-186, miR-381, miR-584-3p and miR-635. On the other hand, tumor suppressor roles of YY1 appear to involve YY1 stimulation of tumor suppressor BRCA1, increased Bax transcription and apoptosis involving cytochrome c release and caspase-3/-7 cleavage, induction of heme oxygenase-1, inhibition of pRb phosphorylation and p21 binding to cyclin D1 and cdk4, reduced expression of long noncoding RNA of SOX2 overlapping transcript, and MUC4/ErbB2/p38/MEF2C-dependent downregulation of MMP-10. YY1 expression is associated with that of cancer stem cell markers SOX2, BMI1 and OCT4 across many cancers suggesting multidynamic regulatory control and groups of cancers with distinct molecular signatures. Greater understanding of the mechanistic roles of YY1 will in turn lead to the development of more specific approaches to modulate YY1 expression and activity with therapeutic potential.
Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Fator de Transcrição YY1/metabolismo , Animais , Biomarcadores , Proliferação de Células , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Ligação Proteica , Carga Tumoral , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Fator de Transcrição YY1/genéticaRESUMO
The GLI-Krüppel zinc finger factor yin yang-1 (YY1) is a complex protein that regulates a variety of processes including transcription, proliferation, development and differentiation. YY1 inhibits cell growth in a cell type-specific manner. The role played by YY1 in its control of tumor cell growth is unclear and controversial. We show here that YY1 can suppress the growth of different tumor cell types in vitro, including human breast carcinoma cells and glioblastoma cells. YY1 also blocked the growth of 13762 MAT mammary adenocarcinoma isografts in rats. YY1 inhibited 13762 MAT tumor growth by approximately 80% compared with the GFP alone group 21 days after injection. YY1 inhibited proliferating cell nuclear antigen (PCNA) expression and pRbSer249/Thr252 phosphorylation without influencing tumor microvascular density. Moreover, YY1 inhibited p21WAF1/Cip1 complex formation with cdk4 and cyclin D1. These findings demonstrate that YY1 can negatively regulate the growth of multiple malignant cell types.
Assuntos
Adenocarcinoma/enzimologia , Neoplasias da Mama/enzimologia , Proliferação de Células , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fator de Transcrição YY1/metabolismo , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microvasos/patologia , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Endogâmicos F344 , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Transfecção , Carga Tumoral , Fator de Transcrição YY1/genéticaRESUMO
Wound healing is a complex dynamic process involving a variety of cell types, including fibroblasts that express and respond to cytokines and growth factors in the local microenvironment. The mechanisms controlling gene expression after injury at a transcriptional level are poorly understood. Here we show that decreased expression of a key receptor, PDGF-receptor (R)-α, after fibroblast injury is due to the release and paracrine activity of TNF-α. TNF-α inhibits PDGF-R-α expression and this involves formation of a c-Fos-Yin Yang 1 (YY1) complex and histone deacetylase (HDAC) activity. c-Fos, induced by TNF-α, negatively regulates PDGF-R-α transcription. Small interfering RNA (siRNA) targeting c-Fos or the zinc finger transcription factor YY1 inhibits TNF-α suppression of PDGF-R-α expression. Coimmunoprecipitation studies show that TNF-α stimulates the formation of a complex between c-Fos with YY1. Furthermore, chromatin immunoprecipitation (ChIP) analysis reveals the enrichment of c-Fos, YY1, and HDAC-1 at the PDGF-R-α promoter in cells exposed to TNF-α. With suberoylanilide hydroxamic acid (SAHA) and HDAC-1 siRNA, we demonstrate that HDAC mediates TNF-α repression of PDGF-R-α. These findings demonstrate that transcriptional repression of PDGF-R-α after fibroblast injury involves paracrine activity of endogenous TNF-α, the formation of a c-Fos-YY1 complex, and negative regulatory activity by HDAC.
Assuntos
Histona Desacetilases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Sequência de Bases , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histona Desacetilases/genética , Ácidos Hidroxâmicos/metabolismo , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/genética , Interferência de RNA , RNA Interferente Pequeno , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Vorinostat , Fator de Transcrição YY1/genéticaRESUMO
Conditions resulting in retinal angiogenesis and edema (exudative age-related macular degeneration, diabetic retinopathy, retinal vein occlusion and retinopathy of prematurity) are major causes of visual impairment, with significant impact on quality of life. There has been increasing clinical usage of anti-vascular endothelial growth factor (anti-VEGF) agents to stop retinal angiogenesis and resolve intraretinal fluid arising from these conditions. However, anti-VEGFs have not been completely successful in curing these conditions, and a range of emerging treatments aimed at supplementing or competing with anti-VEGF agents are being developed. We will discuss the proposed merits these emerging agents bring to the treatment arsenal and how they compare with anti-VEGFs with regards to therapeutic activity, potency, specificity and safety. This review will also highlight recent pre-clinical research findings and suggest where future research might be directed.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Edema/complicações , Edema/tratamento farmacológico , Neovascularização Patológica/complicações , Neovascularização Patológica/tratamento farmacológico , Retina , Vasos Retinianos/patologia , Animais , Anticorpos Monoclonais/farmacologia , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , Retina/efeitos dos fármacos , Retina/patologia , Vasos Retinianos/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
RATIONALE: induction of heme oxygenase (HO)-1 protects against experimental atherosclerotic diseases, and certain pharmacological HO-1 inducers, like probucol, inhibit the proliferation of vascular smooth muscle cells and, at the same time, promote the growth of endothelial cells in vivo and in vitro. OBJECTIVE: because such cell-specific effects are reminiscent of the action of the transcription factor Yin Yang (YY)1, we tested the hypothesis that there is a functional relationship between HO-1 and YY1. METHODS AND RESULTS: we report that probucol increases the number of YY1(+) cells in rat carotid artery following balloon injury at a time coinciding with increased HO-1 expression. The drug also induces the expression of YY1 mRNA and protein in rat aortic smooth muscle cells (RASMCs) in vitro, as do other known HO-1 inducers (tert-butylhydroquinone and hemin) and overexpression of HO-1 using a human HMOX1 cDNA plasmid. Conversely, overexpression of YY1 induces expression of HO-1 in RASMCs. Induction of YY1 expression is dependent on HO-1 enzyme activity and its reaction product CO, because pharmacological inhibition of heme oxygenase activity or CO scavenging block, whereas exposure of RASMCs to a CO-releasing molecule increases, YY1 expression. Furthermore, RNA interference knockdown of YY1 prevents probucol or adeno-HO-1 from inhibiting RASMC proliferation in vitro and neointimal formation in vivo. CONCLUSIONS: our findings show, for the first time, that HO-1 functionally interplays with the multifunctional transcription factor YY1 and that this interplay explains some of the protective activities of HO-1.
Assuntos
Células Endoteliais/patologia , Heme Oxigenase-1/fisiologia , Miócitos de Músculo Liso/patologia , Fator de Transcrição YY1/fisiologia , Animais , Artérias Carótidas/patologia , Proliferação de Células , Células Cultivadas , Endotélio Vascular/patologia , Heme Oxigenase-1/genética , Humanos , Hiperplasia , Músculo Liso Vascular , Miócitos de Músculo Liso/metabolismo , Probucol/farmacologia , RNA Mensageiro/análise , Ratos , Fatores de Transcrição/fisiologia , Ativação Transcricional , Túnica Íntima/patologia , Fator de Transcrição YY1/análise , Fator de Transcrição YY1/genéticaRESUMO
Coronary artery bypass graft failure represents an unsolved problem in interventional cardiology and heart surgery. Late occlusion of autologous saphenous vein bypass grafts is a consequence of neointima formation underpinned by smooth muscle cell (SMC) migration and proliferation. Poor long term patency and the lack of pharmacologic agents that prevent graft failure necessitate effective alternative therapies. Our objective here was to evaluate the effect of targeted inhibition of the bZIP transcription factor c-Jun on intimal hyperplasia in human saphenous veins and vein graft stenosis after autologous end-to-side transplantation. DNAzymes targeting c-Jun attenuated intimal hyperplasia in human saphenous vein explants. Adenovirus-forced c-Jun expression stimulated SMC proliferation, proliferating cell nuclear antigen, and MMP-2 expression. c-Jun DNAzymes abrogated Adeno-c-Jun-inducible SMC growth and wound repair and reduced intimal thickening in jugular veins of New Zealand white rabbits 4 weeks after autologous end-to-side transplantation to carotid arteries. Conversely, in a DNAzyme-free setting, Adeno-c-Jun potentiated neointima formation in the veins compared with Adeno-LacZ. Inducible c-Jun expression is ERK1/2- and JNK-dependent but p38-independent. Injury- and shear-inducible c-Jun controls early growth response-1. These data demonstrate that strategies targeting c-Jun may be useful for the prevention of vein graft stenosis. Control of one important shear-responsive transcription factor by another indicates the existence of transcriptional amplification mechanisms that magnify the vascular response to cell injury or stress through inducible transcriptional networks.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Veias Jugulares/metabolismo , Veia Safena/metabolismo , Túnica Íntima/metabolismo , Adenoviridae/genética , Animais , Western Blotting , Artérias Carótidas/cirurgia , Linhagem Celular , Células Cultivadas , DNA Catalítico/genética , DNA Catalítico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Flavonoides/farmacologia , Humanos , Hiperplasia , Técnicas In Vitro , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Veias Jugulares/patologia , Veias Jugulares/transplante , Masculino , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Coelhos , Ratos , Veia Safena/patologia , Veia Safena/cirurgia , Estresse Mecânico , Transfecção , Transplante Autólogo , Túnica Íntima/patologia , Túnica Íntima/cirurgiaRESUMO
Vascular injury initiates a cascade of phenotype-altering molecular events. Transcription factor function in this process, particularly that of negative regulators, is poorly understood. We demonstrate here that the forced expression of the injury-inducible GLI-Krüppel zinc finger protein Yin Yang-1 (YY1) inhibits neointima formation in human, rabbit and rat blood vessels. YY1 inhibits p21(WAF1/Cip1) transcription, prevents assembly of a p21(WAF1/Cip1)-cdk4-cyclin D1 complex, and blocks downstream pRb(Ser249/Thr252) phosphorylation and expression of PCNA and TK-1. Conversely, suppression of endogenous YY1 elevates levels of p21(WAF1/Cip1), PCNA, pRb(Ser249/Thr252) and TK-1, and increases intimal thickening. YY1 binds Sp1 and prevents its occupancy of a distinct element in the p21(WAF1/Cip1) promoter without YY1 itself binding the promoter. Additionally, YY1 induces ubiquitination and proteasome-dependent degradation of p53, decreasing p53 immunoreactivity in the artery wall. These findings define a new role for YY1 as both an inducer of p53 instability in smooth muscle cells, and an indirect repressor of p21(WAF1/Cip1) transcription, p21(WAF1/Cip1)-cdk4-cyclin D1 assembly and intimal thickening.
Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ciclinas/metabolismo , Complexos Multiproteicos/metabolismo , Miócitos de Músculo Liso/metabolismo , Túnica Íntima/crescimento & desenvolvimento , Fator de Transcrição YY1/metabolismo , Animais , Artérias/citologia , Artérias/crescimento & desenvolvimento , Linhagem Celular , Ciclina D , Quinase 4 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Ciclinas/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Complexos Multiproteicos/genética , Miócitos de Músculo Liso/citologia , Antígeno Nuclear de Célula em Proliferação/biossíntese , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica/fisiologia , Coelhos , Ratos , Elementos de Resposta/fisiologia , Proteína do Retinoblastoma/biossíntese , Proteína do Retinoblastoma/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Timidina Quinase/biossíntese , Timidina Quinase/genética , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Túnica Íntima/citologia , Fator de Transcrição YY1/genéticaRESUMO
Atherosclerosis and restenosis are common vascular disorders that involve excess proliferation of smooth muscle cells (SMCs) in the artery wall. In this study we demonstrate the anti-mitogenic, pro-apoptotic role of the zinc finger transcription factor Sp1 in vascular SMCs and define the underlying molecular mechanism via its capacity to repress the expression of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 at the level of transcription, mRNA, and protein. SMC proliferation inducible by a dominant-negative mutant form of Sp1 was abrogated by antisense strategies targeting p21WAF1/Cip1. Conversely, antisense p21WAF1/Cip1 induced apoptosis in SMCs overexpressing dominant-negative-Sp1. p21WAF1/Cip1 overexpression alone stimulated proliferation and inhibited apoptosis. Sp1 down-regulated p21WAF1/Cip1 expression in SMCs. Sp1 blocked assembly of cyclin D1-Cdk4-p21WAF1/Cip1 complex formation whose integrity is critical for G1->S transition. Moreover, Rb phosphorylation, which lies immediately downstream of the cyclin D1-Cdk4-p21WAF1/Cip1 complex, was blocked either by Sp1 overexpression or antisense p21WAF1/Cip1. These findings, using complementary approaches, demonstrate the inverse relationship between Sp1 and p21WAF1/Cip1 in SMCs and the capacity of Sp1 to regulate SMC proliferation and apoptosis via its repression of p21WAF1/Cip1.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclina D1/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Endotélio Vascular/citologia , Miócitos de Músculo Liso/patologia , Proteínas Proto-Oncogênicas , Fator de Transcrição Sp1/fisiologia , Transcrição Gênica , Animais , Apoptose , Artérias/metabolismo , Western Blotting , Bromodesoxiuridina/farmacologia , Ciclo Celular , Divisão Celular , Linhagem Celular , Células Cultivadas , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Regulação para Baixo , Etoposídeo/farmacologia , Genes Dominantes , Mutação , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos WKY , Proteína do Retinoblastoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fatores de Tempo , Dedos de ZincoRESUMO
Friend leukemia integration 1 (Fli-1) is a member of the Ets family of transcriptional activators that has been shown to be an important regulator during megakaryocytic differentiation. We undertook a two-hybrid screen of a K562 cDNA library to identify transcription factors that interacted with Fli-1 and were potential regulators of megakaryocyte development. Here we report the physical interaction of Fli-1 with GATA-1, a well-characterized, zinc finger transcription factor critical for both erythroid and megakaryocytic differentiation. We map the minimal domains required for the interaction and show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. GATA-1 has previously been shown to interact with the Ets domain of the Fli-1-related protein PU.1, and the two proteins appear to inhibit each other's activity. In contrast, we demonstrate that GATA-1 and Fli-1 synergistically activate the megakaryocyte-specific promoters GPIX and GPIbalpha in transient transfections. Quantitative electrophoretic mobility shift assays using oligonucleotides derived from the GPIX promoter containing Ets and GATA binding motifs reveal that Fli-1 and GATA-1 exhibit cooperative DNA binding in which the binding of GATA-1 to DNA is increased approximately 26-fold in the presence of Fli-1 (from 4.2 to 0.16 nM), providing a mechanism for the observed transcriptional synergy. To test the effect on endogenous genes, we stably overexpressed Fli-1 in K562 cells, a line rich in GATA-1. Overexpression of Fli-1 induced the expression of the endogenous GPIX and GPIbalpha genes as measured by Northern blot and fluorescence-activated cell sorter analysis. This work suggests that Fli-1 and GATA-1 work together to activate the expression of genes associated with the terminal differentiation of megakaryocytes.