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Tarik Möröy

Publications and source records attributed to Tarik Möröy.

At least 19 recordsLinked to original sources
PubMed2006

Gfi1b:green fluorescent protein knock-in mice reveal a dynamic expression pattern of Gfi1b during hematopoiesis that is largely complementary to Gfi1.

Gfi1b and Gfi1 are 37- and 55-kDa transcriptional repressors that share common features such as a 20-amino acid (aa) N-terminal SNAG domain, a nonconserved intermediary domain, and 6 highly conserved C-terminal zinc fingers. Both gene loci are under autoregulatory and cross-regulatory feedback control. We have generated a reporter mouse strain by inserting the cDNA for green fluorescent protein (GFP) into the Gfi1b gene locus which allowed us to follow Gfi1b expression during hematopoiesis and lymphopoiesis by measuring green fluorescence. We found highly dynamic expression patterns of Gfi1b in erythroid cells, megakaryocytes, and their progenitor cells (MEPS) where Gfi1 is not detected. Vice versa, Gfi1b could not be found in granulocytes, activated macrophages, or their granulomonocytic precursors (GMPs) or in mature naive or activated lymphocytes where Gfi1 is expressed, suggesting a complementary regulation of both loci during hematopoiesis. However, Gfi1b was found to be up-regulated in early stages of B-cell and in a subset of early T-cell development, where Gfi1 is also present, suggesting that cross-regulation of both loci exists but is cell-type specific.

Aging↗
PubMed2006

Auxiliary splice factor U2AF26 and transcription factor Gfi1 cooperate directly in regulating CD45 alternative splicing.

By alternative splicing, different isoforms of the transmembrane tyrosine phosphatase CD45 are generated that either enhance or limit T cell receptor signaling. We report here that CD45 alternative splicing is regulated by cooperative action of the splice factor U2AF26 and the transcription factor Gfi1. U2AF26 promoted formation of the less-active CD45RO by facilitating exon exclusion. Gfi1 antagonized that process by directly interacting with U2AF26, identifying a previously unknown link between a transcription factor and alternative splicing. The presence of Gfi1 led to formation of the more-active CD45RB, whereas loss of Gfi1 favored CD45RO production. We propose that the relative abundance of U2AF26 and Gfi1 determines the ratio of CD45 isoforms, thereby regulating T cell activation.

3T3 Cells↗
PubMed2006

Gfi1b alters histone methylation at target gene promoters and sites of gamma-satellite containing heterochromatin.

Gfi1b is a 37 kDa nuclear protein with six C2H2 zinc-finger domains that can silence transcription upon binding to specific target gene promoters. Here we show by using a chromatin immunoprecipitation and cloning protocol that Gfi1b also binds to gamma-satellite sequences that mainly occur in pericentric heterochromatin. Immuno-FISH experiments demonstrated that Gfi1b is localized at foci of pericentric heterochromatin identified by DAPI staining. Elevated levels of Gfi1b correlated with increased histone H3 lysine 9 dimethylation at sites neighboring gamma-satellite sequences but also at Gfi1b target gene promoters. In Gfi1b-deficient cells, however, a decrease of histone H3 lysine 9 trimethylation and a loss of heterochromatic structures was observed. Strikingly, we found that Gfi1b binds to both SUV39H1 and G9A histone methyl transferases, which provides a direct link between histone methylation and Gfi1b at heterochromatic and euchromatic sites. We propose that Gfi1b functions in heterochromatin formation and silencing of euchromatic transcription by recruiting histone methyl transferases to either gamma-satellite sequences or specific target gene promoters.

Animals↗
PubMed2006

Gfi1 and Gfi1b act equivalently in haematopoiesis, but have distinct, non-overlapping functions in inner ear development.

Gfi1 is a transcriptional repressor essential for haematopoiesis and inner ear development. It shares with its paralogue Gfi1b an amino-terminal SNAG repressor domain and six carboxy-terminal zinc-finger motifs, but differs from Gfi1b in sequences separating these domains. Here, we describe two knock-in mouse models, in which the N-terminal SNAG repressor domain was mutated or in which the Gfi1 coding region was replaced by Gfi1b. Mouse mutants without an intact SNAG domain show the full phenotype of Gfi1 null mice. However, Gfi1:Gfi1b knock-in mice show almost normal pre-T-cell and neutrophil development, but lack properly formed inner ear hair cells. Hence, our findings show that an intact SNAG domain is essential for all functions of Gfi1 and that Gfi1b can replace Gfi1 functionally in haematopoiesis but, surprisingly, not in inner ear hair cell development, demonstrating that Gfi1 and Gfi1b have equivalent and domain-dependent, cell type-specific functions.

Animals↗
PubMed2006

The zinc finger protein Gfi1 acts upstream of TNF to attenuate endotoxin-mediated inflammatory responses in the lung.

Gfi1 is a 55-kD nuclear zinc finger protein that is differentially expressed in lymphoid and myeloid cells. Gfi1(-/-) mice show a very strong systemic response to the endotoxin LPS and die rapidly within 36 h with symptoms of septic shock. Here we report that the pathohysiological processes for this exaggerated inflammatory response take place in the lung. After LPS treatment, lungs of Gfi1(-/-) mice showed a rapid accumulation of mononuclear cells and a significant overproduction of inflammatory cytokines such as TNF, IL-1beta and IL-6. Increased cytokine production was also observed in blood-free perfused lungs from Gfi1(-/-) mice exposed to either LPS or overventilation. Alveolar macrophages but not airway epithelial cells from Gfi1(-/-) mice were found to be responsible for the enhanced cytokine production. Strikingly, when the TNF gene was deleted, Gfi1(-/-) animals were completely rescued from LPS hypersensitivity and had significantly lower IL-1beta and IL-6 levels. We conclude that the unrestrained endotoxin response of Gfi1(-/-) mice occurs mainly in the lung and that Gfi1 represents a novel factor limiting the inflammatory immune response of this organ, and propose that Gfi1 exerts its regulatory function in alveolar macrophages downstream of the LPS receptor (TLR4) and upstream of TNF.

Animals↗
PubMed2006

Regulation of pulmonary Pseudomonas aeruginosa infection by the transcriptional repressor Gfi1.

Pulmonary Pseudomonas aeruginosa infections are characterized by the release of proinflammatory mediators, focal induction of apoptosis in respiratory epithelial cells and internalization of the bacteria. Here, we demonstrate that the transcriptional regulator Gfi1 is critically involved in the regulation of proinflammatory cytokine release and the induction of apoptosis in respiratory epithelial cells and macrophages upon P. aeruginosa infection. Gfi1-deficient mice responded to a pulmonary P. aeruginosa infection with uncontrolled pulmonary release of interleukin (IL)-1 and tumour necrosis factor (TNF)-alpha, sepsis and death, which were delayed by injection of IL-1- and TNF-alpha-neutralizing antibodies. The uncontrolled release of cytokines seems to be caused by a failure of Gfi1-deficient respiratory epithelial cells in large to small bronchi and macrophages to respond to P. aeruginosa infection with an induction of apoptosis. Pharmacological inhibition of apoptosis in wild-type mice by intravenous injection of the broad-spectrum caspase inhibitor zVAD-cmk mimicked the phenotype of Gfi1-deficient mice and resulted in a profound sensitization of mice to P. aeruginosa, an increased release of cytokines, sepsis and death of the animals. Thus, Gfi1 controls apoptosis of respiratory epithelial cells and macrophages upon infection with P. aeruginosa. Inhibition of apoptosis by Gfi1 deficiency or caspase blockers sensitizes mice to P. aeruginosa infections, suggesting that apoptosis functions as a novel defence mechanisms in the regulation of the local innate immune response.

Animals↗
PubMed2005

Systemic lupus-erythematosus: deoxyribonuclease 1 in necrotic chromatin disposal.

Systemic lupus-erythematosus is an auto-immune-disease characterized by pathogenic anti-nuclear auto-antibodies. These form immune-complexes that after deposition at basal membranes at various locations initiate inflammatory reactions. There is a clear genetic and gender predisposition (females are affected 10 times more frequently), but also infectious agents and further environmental factors have been shown to be causative for the initiation of the disease. It has been suggested that the auto-antibodies arise after release and/or inefficient removal of nuclear components during cell death (defective cellular "waste disposal" theory). So far, increased apoptotic cell death has been made responsible, but recent data suggest that defective cellular waste disposal during/after necrosis may also lead to the release and prolonged exposure of nuclear components. Here, we concentrate on chromatin disposal during necrosis and the involvement of Deoxyribonuclease 1 in this process with respect to its possible role in the prevention of anti-nuclear auto-immunity.

Animals↗
PubMed2005

The oncogenic serine/threonine kinase Pim-1 directly phosphorylates and activates the G2/M specific phosphatase Cdc25C.

The proto-oncogene Pim-1 encodes a serine-threonine kinase which is a downstream effector of cytokine signaling and can enhance cell cycle progression by altering the activity of several cell cycle regulators among them the G1 specific inhibitor p21(Waf), the phosphatase Cdc25A and the kinase C-TAK1. Here, we demonstrate by using biochemical assays that Pim-1 can interact with the phosphatase Cdc25C and is able to directly phosphorylate the N-terminal region of the protein. Cdc25C is functionally related to Cdc25A but acts specifically at the G2/M cell cycle transition point and can be inactivated by C-TAK1-mediated phosphorylation. Immuno-fluorescence experiments showed that Pim-1 and Cdc25C co-localize in the cytoplasm of both epithelial and myeloid cells. We find that phosphorylation by Pim-1 enhances the phosphatase activity of Cdc25C and in transfected cells that are arrested in G2/M by bleomycin, Pim-1 can enhance progression into G1. Therefore, we propose that Pim-1 activates Cdc25C by a direct phosphorylation and can thereby assume the function of a positive cell cycle regulator at the G2/M transition.

Animals↗
PubMed2005

Runx3 regulates integrin alpha E/CD103 and CD4 expression during development of CD4-/CD8+ T cells.

During thymic T cell development, immature CD4+CD8+ double-positive (DP) thymocytes develop either into CD4+CD8- Th cells or CD4-CD8+ CTLs. Differentially expressed primary factors inducing the fate of these cell types are still poorly described. The transcription factor Runx3/AML-2 Runx, runt [corrected] dominant factor; AML, acute myeloid leukemia is expressed specifically during the development of CD8 single-positive (SP) thymocytes, where it silences CD4 expression. Deletion of murine Runx3 results in a reduction of CD8 SP T cells and concomitant accumulation of CD4+CD8+ T cells, which cannot down-regulate CD4 expression in the thymus and periphery. In this study we have investigated the role of Runx3 during thymocyte development and CD4 silencing and have identified integrin alpha(E)/CD103 on CD8 SP T cells as a new potential target gene of Runx3. We demonstrate that Runx3 is necessary not only to repress CD4, but also to induce CD103 expression during development of CD8 SP T cells. In addition, transgenic overexpression of Runx3 reduced CD4 expression during development of DP thymocytes, leading to a reduced number of CD4 SP thymocytes and an increased number of CD8 SP thymocytes. This reversal is not caused by redirection of specific MHC class II-restricted cells to the CD8 lineage. Overexpression of Runx3 also up-regulated CD103 expression on a subpopulation of CD4 SP T cells with characteristics of regulatory T cells. Thus, Runx3 is a main regulator of CD4 silencing and CD103 induction and thus contributes to the phenotype of CD8 SP T cells during thymocyte development.

Amino Acid Sequence↗
PubMed2005

Direct transcriptional repression of the genes encoding the zinc-finger proteins Gfi1b and Gfi1 by Gfi1b.

Gfi1b is a 37 kDa transcriptional repressor with six zinc-finger domains that is differentially expressed during hemato- and lymphopoiesis. We show here that transcription from the Gfi1b gene locus is silenced in the spleen but not in the bone marrow of transgenic mice that constitutively express Gfi1b under the control of the pan-hematopoietic vav promoter. Sequence analysis of the Gfi1b promoter showed the presence of potential Gfi1/Gfi1b-binding sites close to the mRNA start site. The expression of reporter gene constructs containing the Gfi1b core promoter appended to the luciferase gene were strongly repressed in the presence of exogenous Gfi1b. Moreover, analysis of combinatorial mutant mice that carry the vav-Gfi1b transgene and a green fluorescent protein-tagged Gfi1 gene locus demonstrated that the Gfi1 gene can be repressed by Gfi1b. Direct binding of Gfi1b and Gfi1 to the potential binding sites in the Gfi1b promoter could be demonstrated by gel-shift analyses in vitro. Chromatin-immunoprecipitation experiments showed that both the Gfi1b and the Gfi1 promoter are indeed occupied by Gfi1b in vivo. Hence, we conclude from our data that Gfi1b can auto-repress its own expression, but, in addition, is also able to cross-repress expression of the Gfi1 gene most likely in a cell type specific manner.

Animals↗
PubMed2005

The zinc finger transcription factor Growth factor independence 1 (Gfi1).

Growth Factor Independence 1 (Gfi1) is a transcriptional repressor with a molecular weight between 47 and 55 kDa The protein has six C-terminal C2H2-type zinc-finger domains and a characteristic stretch of 20 amino acids, called the SNAG-domain, at its N-terminus. Expression of Gfi1 ranges from the hematopoietic and lymphoid system, to sensory epithelia, lung and parts of the CNS. Gene knock-out studies revealed that Gfi1 is essential for the development of granulocytes and plays a role in T-cell differentiation and macrophage-dependent cytokine production, indicating that this protein shares responsibility for different lines of defense against pathogens. Also, Gfi1 is required for the proper development of inner ear hair cells illustrated by ataxia and deafness in knock-out mice. While hereditary hearing loss has so far not been associated with Gfi1 malfunction, crippling mutations in the Gfi1 gene have been reported in patients suffering from neutropenia suggesting an important role of Gfi1 in this human disease.

Amino Acid Sequence↗
PubMed2005

The serine/threonine kinase Pim-1.

The human pim-1 gene encodes a serine/threonine kinase, which belongs to the group of calcium/calmodulin-regulated kinases (CAMK). It contains a characteristic kinase domain, a so-called ATP anchor and an active site. In mouse and human, two Pim-1 proteins are produced from the same gene by using an alternative upstream CUG initiation codon, a 44 kD and another, shorter 34 kD form that both contain the kinase domain. Expression of Pim-1 is widespread and ranges from the hematopoietic and lymphoid system to prostate, testis and oral epithelial cells. Two other proteins with significant sequence similarities exist, Pim-2 and Pim-3; both are also serine/threonine kinases and have largely overlapping functions. Pim-1 is able to phosphorylate different targets, most of which are involved in cell cycle progression or apoptosis. Pim-1 expression can be induced by several external stimuli in particular by a number of cytokines relevant in the immune system, which led to the labeling of Pim-1 as a "booster" for the immune response.

Animals↗
PubMed2005

The transcriptional repressor Gfi1 controls STAT3-dependent dendritic cell development and function.

The mechanisms controlling the differentiation of dendritic cells (DCs) remain largely unknown. Using a transcriptional profiling approach, we identified Gfi1 as a novel critical transcription factor in DC differentiation. Gfi1-/- mice showed a global reduction of myeloid and lymphoid DCs in all lymphoid organs whereas epidermal Langerhans cells were enhanced in number. In vivo, Gfi1-/- DCs showed striking phenotypic and functional alterations such as defective maturation and increased cytokine production. In vitro, Gfi1-/- hematopoietic progenitor cells were unable to develop into DCs. Instead, they differentiated into macrophages, suggesting that Gfi1 is a critical modulator of DC versus macrophage development. Analysis of hematopoietic chimeras and retrovirus-reconstituted hematopoietic progenitor cells established a cell autonomous and nonredundant role for Gfi1 in DC development. The developmental defect of Gfi1-/- progenitor cells was associated with decreased STAT3 activation. In conclusion, we have identified Gfi1 as a critical transcription factor that controls DC versus macrophage development and dissociates DC maturation and activation.

Animals↗
PubMed2005

Novel target genes of the Wnt pathway and statistical insights into Wnt target promoter regulation.

The Wnt pathway controls biological processes via the regulation of target gene expression. The expression of direct Wnt target genes, e.g. cyclin D1 and MYC, is activated by the transcription factor TCF, which binds to specific sequence motifs in the promoter. Indirect target genes are regulated via transcription regulators, which are targets of the Wnt pathway. As an example, MYC regulates the MYC interacting zinc finger protein-1 (MIZ-1), which is able to inhibit the expression of the indirect target p21WAF1. We intended to identify new Wnt target genes and to get a deeper insight into the regulatory mechanisms of Wnt target gene expression. For this we analyzed the differential expression pattern of Wnt-1 activated cells by microarray analysis. We identified 43 sequences including eight expressed sequence tags (ESTs), which showed increased transcript levels, and 104 sequences including 19 ESTs with decreased RNA levels. Northern blot and real-time quantitative PCR analysis of the differential expression levels of 15 genes confirmed the differential expression trends of eight candidate genes. When the Wnt pathway was regulated at the lower level of glycogen synthase kinase-3 beta (GSK-3 beta) or adenomatous polyposis coli (APC), we detected discrepant expression trends. We compared the number of binding sites of transcription factors in the genomic regions of all candidate target genes with the number of sites in control genes. We found that the genomic regions of the down-regulated genes include an increased number of putative MIZ-1 binding sites. Our study introduces several new Wnt target genes and provides indications that the specific gene expression pattern depends on the type of the activation trigger or the level of interference with the Wnt pathway. Furthermore, our data indicate that a high proportion of Wnt target genes are regulated by indirect mechanisms.

Animals↗
PubMed2004

Transcription factor Gfi1 regulates self-renewal and engraftment of hematopoietic stem cells.

The generation of all blood cells depends on the ability of hematopoietic stem cells (HSCs) for self-renewal and multilineage differentiation. We show here that the transcription factor Gfi1 is expressed in HSCs and in more mature cells such as common lymphoid progenitors (CLPs) and granulo/monocytic progenitors, but is absent in common myeloid progenitors and megakaryocyte/erythroid progenitors. When Gfi1 is deleted in mice, HSC frequencies are significantly reduced and CLPs all but disappear from the bone marrow. This specific requirement of Gfi1 for the maintenance of HSC numbers is cell autonomous. Transplantation of Gfi1-deficient bone marrow results in a compromised radioprotection and lower numbers of colony forming units in the spleen of wild-type recipients. Strikingly, Gfi1-/- bone marrow cells are severely impaired in competitive long-term reconstituting abilities after transplantation and show a surprisingly high proportion of actively cycling HSCs, suggesting that Gfi1 restrains proliferation of HSCs and thereby regulates their self-renewal and long-term engraftment abilities.

Alleles↗
PubMed2004

The oncogenic serine/threonine kinase Pim-1 phosphorylates and inhibits the activity of Cdc25C-associated kinase 1 (C-TAK1): a novel role for Pim-1 at the G2/M cell cycle checkpoint.

The Pim-1 oncogene encodes a serine-threonine kinase that relays signals from cytokine receptors and contributes to the formation of lymphoid tumors when expressed at high levels. Here we show that the protein kinase Cdc25 C-associated kinase 1 (C-TAK1) is a binding partner and a substrate of Pim-1. A physical interaction of Pim-1 and C-TAK1 could be shown biochemically and in yeast two-hybrid assays. Immunofluorescence experiments suggested that Pim-1.C-TAK1 complexes are predominantly cytoplasmic. When transiently transfected, Pim-1 was also found in the nucleus and could recruit C-TAK1 to this compartment. Both Pim-1 and C-TAK1 underwent autophosphorylation, but only Pim-1 was able to phosphorylate C-TAK1 but not vice versa. Mass spectrometry analysis of C-TAK1 suggested that the sites of autophosphorylation and Pim-1-mediated phosphorylation are distinct and not overlapping. Phosphorylation by Pim-1 decreased C-TAK1 kinase activity significantly, in particular its ability to phosphorylate and inactivate Cdc25C, a protein that actively promotes cell cycle progression at the G(2)/M phase. Hence our findings directly suggest a novel role for Pim-1 as a positive regulator at the G(2)/M transition of the cell cycle.

Animals↗
PubMed2004

Transcription profiling of inner ears from Pou4f3(ddl/ddl) identifies Gfi1 as a target of the Pou4f3 deafness gene.

Pou4f3 (Brn3.1, Brn3c) is a class IV POU domain transcription factor that has a central function in the development of all hair cells in the human and mouse inner ear sensory epithelia. A mutation of POU4F3 underlies human autosomal dominant non-syndromic progressive hearing loss DFNA15. Through a comparison of inner ear gene expression profiles of E16.5 wild-type and Pou4f3 mutant deaf mice using a high density oligonucleotide microarray, we identified the gene encoding growth factor independence 1 (Gfi1) as a likely in vivo target gene regulated by Pou4f3. To validate this result, we performed semi-quantitative RT-PCR and in situ hybridizations for Gfi1 on wild-type and Pou4f3 mutant mice. Our results demonstrate that a deficiency of Pou4f3 leads to a statistically significant reduction in Gfi1 expression levels and that the dynamics of Gfi1 mRNA abundance closely follow the pattern of expression for Pou4f3. To examine the role of Gfi1 in the pathogenesis of Pou4f3-related deafness, we performed comparative analyses of the embryonic inner ears of Pou4f3 and Gfi1 mouse mutants using immunohistochemistry and scanning electron microscopy. The loss of Gfi1 results in outer hair cell degeneration, which appears comparable to that observed in Pou4f3 mutants. These results identify Gfi1 as the first downstream target of a hair cell specific transcription factor and suggest that outer hair cell degeneration in Pou4f3 mutants is largely or entirely a result of the loss of expression of Gfi1.

Animals↗