Multiple ETS Family Proteins Regulate PF4 Gene Expression by Binding to the Same ETS Binding Site

Okada, Yoshiaki; Nobori, Haruaki; Shimizu, Mikiko; Watanabe, Miho; Yonekura, Motoki; Nakai, Takaaki; Kamikawa, Yuichiro; Wakimura, Atsuko; Funahashi, Nobuaki; Naruse, Hiroki; Watanabe, Ayako; Yamasaki, Daisuke; Fukada, So‐ichiro; Yasui, Kazuta; Matsumoto, Kayoko; Sato, Takahiro; Kitajima, Kenji; Nakano, Tsutomu; Aird, William C.; Doi, Takefumi

doi:10.1371/journal.pone.0024837

Cited by 15 publications

(19 citation statements)

References 34 publications

(46 reference statements)

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“…We studied two genes that are silenced in erythroid cells, Meis1 and Eng, which have erythroid-specific methylation peaks that contain consensus sites for ELF1, and both of these sites are bound by ELF1 in erythroblasts. Although ELF1 has not yet been reported to function as a transcriptional repressor, the redundant binding of ETS factors (Hollenhorst et al 2007;Okada et al 2011) combined with the recent descriptions of repressive functions of related ETS factors leads us to hypothesize that ETS factors may silence critical genes for proper erythroid development.…”

Section: Discussionmentioning

confidence: 99%

Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites

Hogart¹,

Lichtenberg²,

Ajay³

et al. 2012

Genome Res.

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DNA methylation is an essential epigenetic mark that is required for normal development. Knockout of the DNA methyltransferase enzymes in the mouse hematopoietic compartment reveals that methylation is critical for hematopoietic differentiation. To better understand the role of DNA methylation in hematopoiesis, we characterized genome-wide DNA methylation in primary mouse hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), and erythroblasts (ERYs). Methyl binding domain protein 2 (MBD) enrichment of DNA followed by massively parallel sequencing (MBD-seq) was used to map genome-wide DNA methylation. Globally, DNA methylation was most abundant in HSCs, with a 40% reduction in CMPs, and a 67% reduction in ERYs. Only 3% of peaks arise during differentiation, demonstrating a genomewide decline in DNA methylation during erythroid development. Analysis of genomic features revealed that 98% of promoter CpG islands are hypomethylated, while 20%-25% of non-promoter CpG islands are methylated. Proximal promoter sequences of expressed genes are hypomethylated in all cell types, while gene body methylation positively correlates with gene expression in HSCs and CMPs. Elevated genome-wide DNA methylation in HSCs and the positive association between methylation and gene expression demonstrates that DNA methylation is a mark of cellular plasticity in HSCs. Using de novo motif discovery, we identified overrepresented transcription factor consensus binding motifs in methylated sequences. Motifs for several ETS transcription factors, including GABPA and ELF1, are overrepresented in methylated regions. Our genomewide survey demonstrates that DNA methylation is markedly altered during myeloid differentiation and identifies critical regions of the genome and transcription factor programs that contribute to hematopoiesis.

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Section: Discussionmentioning

confidence: 99%

Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites

Hogart¹,

Lichtenberg²,

Ajay³

et al. 2012

Genome Res.

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“…) and because FLI‐1 does not synergistically activate the promoter with CBFβ. Previously, we demonstrated that GATA‐1 strongly and synergistically activates the PF4 promoter with FLI‐1 but not with the other ETS family proteins, including ETS‐1 . This suggests the possibility that the RUNX1/FLI‐1/GATA‐1 complex is formed in megakaryocytes and contributes to the strong promoter activation, but this requires further study.…”

Section: Discussionmentioning

confidence: 87%

“…In previous investigations of the mechanisms underlying megakaryocyte-specific gene expression, we identified several transcription factors, including ETS family proteins, ETS-1, FLI-1, and GABP, which regulate expression of platelet factor 4 (PF4), which is expressed specifically in megakaryocytes and platelets [12][13][14][15]. Furthermore, we demonstrated that RUNX1 suppresses megakaryocyte-specific genes, including PF4, in UT-7/ GM cells treated with thrombopoietin (TPO) [16].…”

Section: Introductionmentioning

confidence: 94%

“…RUNX1 synergistically activates PF4 expression in combination with ETS family proteins RUNX1 is known to interact with ETS family proteins ETS-1 and FLI-1, which we have previously identified as transcriptional activators of PF4 [12,26]. To investigate whether RUNX1 synergistically activates the PF4 promoter in combination with ETS family proteins, cotransfection assays were performed using HepG2 cells.…”

Section: Up-regulated Exogenous Runx1 Expression Enhances Pf4 Expressmentioning

confidence: 99%

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RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4gene expression in combination with ETS family proteins

Okada

Watanabe

Nakai

et al. 2013

Journal of Thrombosis and Haemostasis

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WC, Doi T. RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins. J Thromb Haemost 2013; 11: 1742-50.Summary. Background: Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runtrelated transcription factor 1 (RUNX1) of FPD patients. Objectives: To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype. Methods: We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFb), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells. Results: We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFb, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFb, and FLI-1. Conclusions: RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.

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“…Although GABPα was expressed throughout neutrophil differentiation, protein levels declined in differentiating SCF ER-Hoxb8 and HL-60 cells despite the increase in LBR levels. This result is not unprecedented, since GABP expression also declines during megakaryocyte differentiation induced by thrombopoietin in hematopoietic progenitors (54), and therefore GABPα may not be required during final maturation of certain myeloid lineages. By comparison, both PU.1 and C/EBPε exhibit significantly increased expression during differentiation of each mouse myeloid model examined.…”

Section: Discussionmentioning

confidence: 99%

Cooperative Activity of GABP with PU.1 or C/EBPε Regulates Lamin B Receptor Gene Expression, Implicating Their Roles in Granulocyte Nuclear Maturation

Malu

Garhwal

Pelletier

et al. 2016

The Journal of Immunology

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Nuclear segmentation is a hallmark feature of mammalian neutrophil differentiation, but the mechanisms that control this process are poorly understood. Gene expression in maturing neutrophils requires combinatorial actions of lineage-restricted and more widely expressed transcriptional regulators. Examples include interactions of the widely expressed ETS transcription factor, GA-binding protein (GABP), with the relatively lineage-restricted ETS factor, PU.1, and with CCAAT enhancer binding proteins, C/EBPα and C/EBPε. Whether such cooperative interactions between these transcription factors also regulate the expression of genes encoding proteins that control nuclear segmentation is unclear. We investigated the roles of ETS and C/EBP family transcription factors in regulating the gene encoding the lamin B receptor (LBR), an inner nuclear membrane protein whose expression is required for neutrophil nuclear segmentation. Although C/EBPε was previously shown to bind the Lbr promoter, surprisingly, we found that neutrophils derived from Cebpe null mice exhibited normal Lbr gene and protein expression. Instead, GABP provided transcriptional activation through the Lbr promoter in the absence of C/EBPε, and activities supported by GABP were greatly enhanced by either C/EBPε or PU.1. Both GABP and PU.1 bound Ets sites in the Lbr promoter in vitro, and in vivo within both early myeloid progenitors and differentiating neutrophils. These findings demonstrate that GABP, PU.1, and C/EBPε cooperate to control transcription of the gene encoding LBR, a nuclear envelope protein that is required for the characteristic lobulated morphology of mature neutrophils.

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Multiple ETS Family Proteins Regulate PF4 Gene Expression by Binding to the Same ETS Binding Site

Cited by 15 publications

References 34 publications

Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites

Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites

RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4gene expression in combination with ETS family proteins

Cooperative Activity of GABP with PU.1 or C/EBPε Regulates Lamin B Receptor Gene Expression, Implicating Their Roles in Granulocyte Nuclear Maturation

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