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RUNX1/AML1 functions and mechanisms regulating granulocyte-macrophage colony-stimulating factor transcription
Umeå University, Faculty of Medicine, Molecular Biology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine involved in the production and function of hematopoietic cells, and GM-CSF plays in particular a major role in responses to infection and physiological and pathological inflammatory processes. GM-CSF is produced in many cell types, and increases in the intracellular Ca2+ concentration are, like in many other systems, of major importance in the intracellular signaling that determines GM-CSF expression after receptor stimulation of the cells. Previous studies have shown that the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) mediates stimulation of GM-CSF transcription in response to Ca2+. This thesis shows that Ca2+ signaling also regulates GM-CSF transcription negatively through Ca2+/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Mutation of the CaMK II target serines increased transactivation of the GM-CSF promoter/enhancer and decreased the sensitivity to inhibition by increased Ca2+ or constitutively active CaMK II. The Ca2+-dependent phosphorylation of Ets1 was also shown to reduce the binding of Ets1 to the GM-CSF promoter in vivo.

RUNX1, also known as acute myeloid leukemia 1 (AML1), is one of three mammalian RUNX transcription factors and has many essential functions in hematopoiesis. RUNX1 has also many important roles in the immune system, and RUNX1 is the most frequent target for chromosomal translocation of genes in acute human leukemias. This thesis shows that RUNX1 directly interacts with both subunits of CN and that the strongest interaction is localised to the regulatory CN subunit and the DNA binding domain of the RUNX protein. Constitutively active CN was shown to activate the promoter/enhancer of GM-CSF synergistically with RUNX1, RUNX2 or RUNX3, and the Ets1 binding site of the promoter was shown to be essential for the synergy between RUNX1 and CN in Jurkat T cells. The analysis suggests that Ets1 phosphorylated by the protein kinase glycogen synthase kinase-3β is the target of RUNX1-recruited CN phosphatase at the GM-CSF promoter.

Transforming growth factor-β (TGF-β) is another multipotent cytokine that often has a role opposite to that of GM-CSF in inflammatory responses since it is a potent suppressor of immune cells and therefore is anti-inflammatory. This thesis shows that TGF-β can decrease transcription from a GM-CSF promoter/enhancer. Certain constitutively active TGF-β receptors and the TGF-β activated transcription factor Smad3 could also repress GM-CSF transcription, whereas several other Smad proteins did not have this inhibitory effect. The inhibition required intact DNA binding ability of Smad3, and the 125 bp upstream of the transcription initiation site, which was sufficient for the inhibition, contains several weak Smad binding sites near the TATA box next to an Ets1 site of the promoter. Smad3 was able to bind to the promoter DNA together with Ets1 and could also be in complex with Ets1 in the absence of DNA. Surface plasmon resonance analysis revealed that Ets1 interacted with the DNA binding domain of Smad3, and the binding constant of this interaction was about 1 µM. The results identify a negative regulation of the GM-CSF promoter by TGF-β signaling through direct Smad3 binding and indicate that the mechanism is by Smad3 interaction with Ets1 and perhaps other proteins around the TATA box of the promoter.

This thesis also identifies a novel transactivation domain in the N-terminal of RUNX1 including the N-terminal α-helix in the DNA binding domain. The domain was also required for RUNX2 and RUNX3 transactivation. Despite this, the N-terminal domain of RUNX1 was not essential for RUNX1 function in megakaryocytopoiesis in vitro from mouse embryonic stem cells.

Place, publisher, year, edition, pages
2005. , 74 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 957
Keyword [en]
RUNX1/AML1, Granulocyte-macrophage colony-stimulating factor (GM-CSF), Calcineurin, CaMKII, Ets1, Transforming growth factor-b (TGF-b), Smad3
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-486ISBN: 91-7305-856-4 (print)OAI: oai:DiVA.org:umu-486DiVA: diva2:143565
Public defence
2005-04-28, Föreläsningssalen Major Groove, Institutionen för Molekyärbiologi, Umeå Universitet, Umeå, 13:00 (English)
Opponent
Available from: 2005-03-30 Created: 2005-03-30 Last updated: 2009-11-25Bibliographically approved
List of papers
1. Calcium regulation of GM-CSF by calmodulin-dependent kinase II phosphorylation of Ets1.
Open this publication in new window or tab >>Calcium regulation of GM-CSF by calmodulin-dependent kinase II phosphorylation of Ets1.
2002 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 13, no 12, 4497-4507 p.Article in journal (Refereed) Published
Abstract [en]

The multipotent cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is involved in particular in the physiological response to infection and in inflammatory responses. GM-CSF is produced by many cell types, including T lymphocytes responding to T-cell receptor activation and mantle zone B lymphocytes. B-cell receptor and T-cell receptor activation generates two major signals: an increase in intracellular Ca(2+) concentration and a protein kinase cascade. Previous studies have shown that the Ca(2+)/calmodulin-dependent phosphatase calcineurin mediates stimulation of GM-CSF transcription in response to Ca(2+). In this study, we show that Ca(2+) signaling also regulates GM-CSF transcription negatively through Ca(2+)/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Wild-type Ets1 negatively affects GM-CSF transcription on Ca(2+) stimulation in the presence of cyclosporin A, which inhibits calcineurin. Conversely, Ets1 with mutated CaMK II target serines showed an increase in transactivation of the GM-CSF promoter/enhancer. Moreover, constitutively active CaMK II inhibited transactivation of GM-CSF by wild-type Ets1 but not by Ets1 with mutated CaMK II sites. Mutation of CaMK II target serines in Ets1 also relieves inhibition of cooperative transactivation of GM-CSF with the Runx1/AML1 transcription factor. In addition, the Ca(2+)-dependent phosphorylation of Ets1 reduces the binding of Ets1 to the GM-CSF promoter in vivo.

Identifiers
urn:nbn:se:umu:diva-29860 (URN)10.1091/mbc.E02-03-0149 (DOI)12475968 (PubMedID)
Available from: 2009-11-25 Created: 2009-11-25 Last updated: 2017-12-12
2. AML1/Runx1 recruits calcineurin to regulate granulocyte macrophage colony-stimulating factor by Ets1 activation.
Open this publication in new window or tab >>AML1/Runx1 recruits calcineurin to regulate granulocyte macrophage colony-stimulating factor by Ets1 activation.
Show others...
2004 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 279, no 28, 29398-29408 p.Article in journal (Refereed) Published
Abstract [en]

Acute myeloid leukemia 1 (AML1), also denoted Runx1, is a transcription factor essential for hematopoiesis, and the AML1 gene is the most common target of chromosomal translocations in human leukemias. AML1 binds to sequences present in the regulatory regions of a number of hematopoiesis-specific genes, including certain cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) up-regulated after T cell receptor stimulation. Here we show that both subunits of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN), which is activated upon T cell receptor stimulation, interact directly with the N-terminal runt homology domain-containing part of AML1. The regulatory CN subunit binds AML1 with a higher affinity and in addition also interacts with the isolated runt homology domain. The related Runx2 transcription factor, which is essential for bone formation, also interacts with CN. A constitutively active derivative of CN is shown to activate synergistically the GM-CSF promoter/enhancer together with AML1 or Runx2. We also provide evidence that relief of the negative effect of the AML1 sites is important for Ca(2+) activation of the GM-CSF promoter/enhancer and that AML1 overexpression increases this Ca(2+) activation. Both subunits of CN interact with AML1 in coimmunoprecipitation analyses, and confocal microscopy analysis of cells expressing fluorescence-tagged protein derivatives shows that CN can be recruited to the nucleus by AML1 in vivo. Mutant analysis of the GM-CSF promoter shows that the Ets1 binding site of the promoter is essential for the synergy between AML1 and CN in Jurkat T cells. Analysis of the effects of inhibitors of the protein kinase glycogen synthase kinase-3beta and in vitro phosphorylation/dephosphorylation analysis of Ets1 suggest that glycogen synthase kinase-3beta-phosphorylated Ets1 is a target of AML1-recruited CN phosphatase at the GM-CSF promoter.

Keyword
Animals, Calcineurin/genetics/*metabolism, Calcium/metabolism, Core Binding Factor Alpha 1 Subunit, Core Binding Factor Alpha 2 Subunit, Cyclosporine/pharmacology, DNA-Binding Proteins/genetics/*metabolism, Enhancer Elements (Genetics), Enzyme Inhibitors/pharmacology, Gene Expression Regulation/drug effects, Genes; Reporter, Glycogen Synthase Kinase 3/antagonists & inhibitors/metabolism, Granulocyte-Macrophage Colony-Stimulating Factor/genetics/*metabolism, Humans, Ionomycin/pharmacology, Ionophores/pharmacology, Jurkat Cells, Mice, Neoplasm Proteins/genetics/metabolism, Phosphorylation, Promoter Regions (Genetics), Protein Structure; Tertiary, Protein Subunits/genetics/*metabolism, Protein Transport/physiology, Proto-Oncogene Protein c-ets-1, Proto-Oncogene Proteins/genetics/*metabolism, Proto-Oncogene Proteins c-ets, Transcription Factors/genetics/*metabolism
Identifiers
urn:nbn:se:umu:diva-16489 (URN)10.1074/jbc.M403173200 (DOI)15123671 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2009-11-25Bibliographically approved
3. TGF-β inhibition of the GM-CSF promoter by Smad3 interaction with Ets1
Open this publication in new window or tab >>TGF-β inhibition of the GM-CSF promoter by Smad3 interaction with Ets1
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4449 (URN)
Available from: 2005-03-30 Created: 2005-03-30 Last updated: 2010-01-13Bibliographically approved
4. The N-terminus of RUNX1 contains a transactivation domain
Open this publication in new window or tab >>The N-terminus of RUNX1 contains a transactivation domain
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4450 (URN)
Available from: 2005-03-30 Created: 2005-03-30 Last updated: 2010-01-13Bibliographically approved

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