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  • 1.
    Antonsson, Åsa
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hughes, Kate
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Edin, Sofia
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Regulation of c-Rel Nuclear Localization by Binding of Ca2+/Calmodulin2003In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 23, no 4, p. 1418-1427Article in journal (Refereed)
    Abstract [en]

    The NF-κB/Rel family of transcription factors participates in the control of a wide array of genes, including genes involved in embryonic development and regulation of immune, inflammation, and stress responses. In most cells, inhibitory IκB proteins sequester NF-κB/Rel in the cytoplasm. Cellular stimulation results in the degradation of IκB and modification of NF-κB/Rel proteins, allowing NF-κB/Rel to translocate to the nucleus and act on its target genes. Calmodulin (CaM) is a highly conserved, ubiquitously expressed Ca2+ binding protein that serves as a key mediator of intracellular Ca2+ signals. Here we report that two members of the NF-κB/Rel family, c-Rel and RelA, interact directly with Ca2+-loaded CaM. The interaction with CaM is greatly enhanced by cell stimulation, and this enhancement is blocked by addition of IκB. c-Rel and RelA interact with CaM through a similar sequence near the nuclear localization signal. Compared to the wild-type protein, CaM binding-deficient mutants of c-Rel exhibit increases in both nuclear accumulation and transcriptional activity on the interleukin 2 and granulocyte macrophage colony-stimulating factor promoters in the presence of a Ca2+ signal. Conversely, for RelA neither nuclear accumulation nor transcriptional activity on these promoters is increased by mutation of the sequence interacting with CaM. Our results suggest that CaM binds c-Rel and RelA after their release from IκB and can inhibit nuclear import of c-Rel while letting RelA translocate to the nucleus and act on its target genes. CaM can therefore differentially regulate the activation of NF-κB/Rel proteins following stimulation.

  • 2.
    Bergqvist, Ingela
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Eriksson, Maria
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Saarikettu, Juha
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Eriksson, Björn
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Corneliussen, Brit
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Holmberg, Dan
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    The basic helix-loop-helix transcription factor E2-2 is involved in T lymphocyte development2000In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 30, no 10, p. 2857-2863Article in journal (Refereed)
    Abstract [en]

    E2A, HEB and E2-2 genes encode a group of basic helix-loop-helix (bHLH) transcription factors that are structurally and functionally similar. Deletion of the genes encoding either of these proteins leads to early lethality and a block in B lymphocyte development. Evidence for a function in T lymphocyte development has, however, only been reported for E2A and HEB. To further elucidate the role of E2-2 at developmental stages that have proven difficult to study due to the early lethality phenotype of mice defective in E2-2, we generated and analyzed mice conditionally mutated in the E2-2 gene. These mice are mosaic with respect to E2-2 expression, consisting of cells with either one functional and one null mutated E2-2 allele or two null mutated alleles. Using this experimental model, we find that cells with a homozygous null mutated E2-2 gene are under-represented in B lymphocyte as well as T lymphocyte cell lineages as compared to other hematopoietic or non-hematopoietic cell lineages. Our data suggests that E2-2 deficiency leads to a partial block in both B and T lymphocyte development. The block in T cell development appears to occur at an early stage in differentiation, since skewing in the mosaicism is observed already in CD4+8+ double-positive thymocytes.

  • 3.
    Bäckström, Stefan
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Huang, Shenghua
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Wolf-Watz, Magnus
    Chemistry.
    Xie, X Q
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Härd, Torleif
    Grundström, Thomas
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Crystallization and preliminary studies of the DNA-binding runt domain of AML1.2001In: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 57, no Pt 2, p. 269-71Article in journal (Refereed)
    Abstract [en]

    The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.

  • 4.
    Bäckström, Stefan
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Wolf-Watz, Magnus
    Chemistry.
    Grundström, Christine
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Härd, Torleif
    Grundström, Thomas
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    The RUNX1 Runt domain at 1.25A resolution: a structural switch and specifically bound chloride ions modulate DNA binding.2002In: J Mol Biol, ISSN 0022-2836, Vol. 322, no 2, p. 259-72Article in journal (Refereed)
    Abstract [en]

    The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.

  • 5.
    Edin, Sofia
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Oruganti, Sreenivasa Rao
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Interaction of calmodulin with Bcl10 modulates NF-kappaB activation.2010In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 47, no 11-12, p. 2057-2064Article in journal (Refereed)
    Abstract [en]

    Calcium signals resulting from antigen receptor activation are important in determining the responses of a T or B lymphocyte to an antigen. Calmodulin (CaM), a multi-functional sensor of intracellular calcium (Ca(2+)) signals in cells, is required in the pathway from the T cell receptor (TCR) to activation of the key transcription factor NF-kappaB. Here we searched for a partner in direct interaction with CaM in the pathway, and found that CaM interacts specifically with the signaling adaptor Bcl10. The binding is Ca(2+) dependent and of high affinity, with a K(d) of approximately 160 nM. Proximity of CaM and Bcl10 in vivo is induced by increases in the intracellular Ca(2+) level. The interaction is localized to the CARD domain of Bcl10, which interacts with the CARD domain of the upstream signaling partner Carma1. Binding of CaM to Bcl10 is shown to inhibit the ability of Bcl10 to interact with Carma1, an interaction that is required for signaling from the TCR to NF-kappaB. Furthermore, a mutant of Bcl10 with reduced binding to CaM shows increased activation of an NF-kappaB reporter, which is further enhanced by activating stimuli. We propose a novel mechanism whereby the Ca(2+) sensor CaM regulates T cell responses to antigens by binding to Bcl10, thereby modulating its interaction with Carma1 and subsequent activation of NF-kappaB.

  • 6.
    Grundström, Christine
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kumar, Anjani
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Priya, Anshu
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Negi, Neema
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    ETS1 and PAX5 transcription factors recruit AID to Igh DNA2018In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 48, no 10, p. 1687-1697Article in journal (Refereed)
    Abstract [en]

    B lymphocytes optimize antibody responses by class switch recombination (CSR), which changes the expressed constant region exon of the immunoglobulin heavy chain (IgH), and by somatic hypermutation (SH) that introduces point mutations in the variable regions of the antibody genes. Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates both these antibody diversification processes by deaminating cytosine to uracil. Here we asked the question if transcription factors can mediate the specific targeting of the antibody diversification by recruiting AID. We have recently reported that AID is together with the transcription factors E2A, PAX5 and IRF4 in a complex on key sequences of the Igh locus. Here we report that also ETS1 is together with AID in this complex on key sequences of the Igh locus in splenic B cells of mice. Furthermore, we show that both ETS1 and PAX5 can directly recruit AID to DNA sequences from the Igh locus with the specific binding site for the transcription factor. Taken together, our findings support the notion of a targeting mechanism for the selective diversification of antibody genes with limited genome wide mutagenesis by recruitment of AID by PAX5 and ETS1 in a transcription factor complex.

  • 7.
    Grundström, Thomas
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hauser, Jannek
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kumar, Anjani
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Priya, Anshu
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kumar, Ramesh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Regulation of diversification and affinity maturation of antibodies2016In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 38, p. S43-S43Article in journal (Other academic)
  • 8.
    Grundström, Thomas
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hauser, Jannek
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kumar, Ramesh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ahmed, Tanzeel
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Mechanisms controlling diversification and affinity maturation of antibodies2015In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 36, p. S43-S43Article in journal (Other academic)
  • 9.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Allelic Exclusion of IgH through Inhibition of E2A in a VDJ Recombination Complex2014In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 192, no 5, p. 2460-2470Article in journal (Refereed)
    Abstract [en]

    A key feature of the immune system is the paradigm that one lymphocyte has only one Ag specificity that can be selected for or against. This requires that only one of the alleles of genes for AgR chains is made functional. However, the molecular mechanism of this allelic exclusion has been an enigma. In this study, we show that B lymphocytes with E2A that cannot be inhibited by calmodulin are dramatically defective in allelic exclusion of the IgH locus. Furthermore, we provide data supporting that E2A, PAX5, and the RAGs are in a VDJ recombination complex bound to key sequences on the Igh gene. We show that pre-BCR activation releases the VDJ recombination complex through calmodulin binding to E2A. We also show that pre-BCR signaling downregulates several components of the recombination machinery, including RAG1, RAG2, and PAX5, through calmodulin inhibition of E2A.

  • 10.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kumar, Ramesh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Regulated localization of an AID complex with E2A, PAX5 and IRF4 at the Igh locus2016In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 80, p. 78-90Article in journal (Refereed)
    Abstract [en]

    Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates somatic hypermutation (SH) and class switch recombination (CSR) by deaminating cytosine to uracil. The targeting of AID and therefore SH and CSR to Ig genes is a central process of the immune system, but the trans-acting factors mediating the specific targeting have remained elusive. Here we show that defective calmodulin inhibition of the transcription factor E2A after activation of the B cell receptor (BCR) leads to reduced BCR, IL4 plus CD40 ligand stimulated CSR to IgE and instead CSR to other Ig classes. AID that initiates CSR is shown to be in a complex with the transcription factors E2A, PAX5 and IRF4 on key sequences of the Igh locus. Calmodulin shows proximity with each of them after BCR stimulation. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks these reductions. AID binds directly to the bHLH domain of E2A and to the PD domain of PAX5. E2A, AID, PAX5 and IRF4 are components of a CSR complex that is redistributed on the Igh locus by BCR signaling through calmodulin binding.

  • 11.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kumar, Ramesh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Signal regulated localisation of a mutagenic protein complex at the Igh locus2015In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, p. 13-13Article in journal (Other academic)
    Abstract [en]

    Our system to produce antibodies is critical for our survival against numerous infections, but it causes also many tumors. B-lymphocytes can modify their immunoglobulin (Ig) genes to generate specific antibodies with a new isotype and enhanced affinity against an antigen. Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates these processes by deaminating cytosine to uracil. How somatic hypermutation (SH) and class switch recombination (CSR) are targeted is key to understanding the defect DNA integrity in lymphomas and also in other tumors where inflammatory signals aberrantly induces AID. The trans-acting factors mediating specific targeting of AID and thereby SH and CSR have remained elusive. Here we show that mutant E2A with defect inhibition by the Ca2+sensor protein calmodulin results in reduced B cell receptor- (BCR-), IL4-plus CD40 ligand-stimulated CSR to IgE and instead aberrant CSR. AID is shown to be together with the transcription factors E2A, PAX5 and IRF4 in a complex on key sequences of the Igh locus in activated mouse splenic B cells. Calmodulin shows proximity with each of them after BCR stimulation. Direct protein-protein interactions enable formation of the complexes. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks reduction of binding to these target sites and increases binding to other target sites. Thus, E2A, AID, PAX5 and IRF4 are components of a CSR and SH complex that is redistributed on the IgH gene by BCR signaling through calmodulin binding.

  • 12.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Regulation of allelic exclusion of IgH by calmodulin inhibition of E2AManuscript (preprint) (Other academic)
  • 13.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kumar, Ramesh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wallenius, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ahmed, Tanzeel
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Regulation of diversification and affinity maturation of antibodies2014In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 34, p. S50-S50Article in journal (Other academic)
  • 14.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Saarikettu, Juha
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Calcium regulation of myogenesis by differential calmodulin inhibition of basic helix-loop-helix transcription factors2008In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 19, no 6, p. 2509-2519Article in journal (Refereed)
    Abstract [en]

    The members of the MyoD family of basic helix-loop-helix (bHLH) transcription factors are critical regulators of skeletal muscle differentiation that function as heterodimers with ubiquitously expressed E-protein bHLH transcription factors. These heterodimers must compete successfully with homodimers of E12 and other E-proteins to enable myogenesis. Here, we show that E12 mutants resistant to Ca(2+)-loaded calmodulin (CaM) inhibit MyoD-initiated myogenic conversion of transfected fibroblasts. Ca(2+) channel blockers reduce, and Ca(2+) stimulation increases, transcription by coexpressed MyoD and wild-type E12 but not CaM-resistant mutant E12. Furthermore, CaM-resistant E12 gives lower MyoD binding and higher E12 binding to a MyoD-responsive promoter in vivo and cannot rescue myogenic differentiation that has been inhibited by siRNA against E12 and E47. Our data support the concept that Ca(2+)-loaded CaM enables myogenesis by inhibiting DNA binding of E-protein homodimers, thereby promoting occupancy of myogenic bHLH protein/E-protein heterodimers on promoters of myogenic target genes.

  • 15.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sveshnikova, Natalia
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wallenius, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Baradaran, Sanna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Saarikettu, Juha
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    B-cell receptor activation inhibits AID expression through calmodulin inhibition of E-proteins2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 4, p. 1267-1272Article in journal (Refereed)
    Abstract [en]

    Upon encountering antigens, B-lymphocytes can adapt to produce a highly specific and potent antibody response. Somatic hypermutation, which introduces point mutations in the variable regions of antibody genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both somatic hypermutation and CSR. The mutagenic AID enzyme has to be tightly controlled. Here, we show that engagement of the membrane-bound antibodies of the B-cell receptor (BCR), which signals that good antibody affinity has been reached, inhibits AID gene expression and that calcium (Ca(2+)) signaling is essential for this inhibition. Moreover, we show that overexpression of the Ca(2+) sensor protein calmodulin inhibits AID gene expression, and that the transcription factor E2A is required for regulation of the AID gene by the BCR. E2A mutated in the binding site for calmodulin, and thus showing calmodulin-resistant DNA binding, makes AID expression resistant to the inhibition through BCR activation. Thus, BCR activation inhibits AID gene expression through Ca(2+)/calmodulin inhibition of E2A.

  • 16.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Verma-Gaur, Jiyoti
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Broad feedback inhibition of pre-B-cell receptor signaling components2013In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 54, no 3-4, p. 247-253Article in journal (Refereed)
    Abstract [en]

    During B lymphocyte development, first immunoglobulin heavy chain gene segments and then immunoglobulin light chain gene segments are rearranged to create antibody diversity. Early in the development, expression of a pre-B-cell receptor (pre-BCR) that has membrane-bound Ig heavy chain protein associated with surrogate light chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces survival and clonal expansion to select cells with good heavy chains, but it also down-regulates transcription of the genes for the SLC proteins and CD19 and limits its own proliferative signaling. Here we have analyzed whether the down-regulation is limited to the SLC proteins and CD19, and we show that the pre-BCR of primary mouse pre-B-cells instead is subject to a broad feedback inhibition of pre-BCR signaling components. Activation of signaling leads to down-regulation of the receptor proteins, many co-receptors and proteins participating in signal pathways from the receptor. Thus the down-regulation of the pre-BCR is much broader than previously assumed. We also show that Ca2+/calmodulin inhibition of the transcription factor E2A is required for the feedback inhibition of the pre-BCR signaling proteins. (C) 2012 Elsevier Ltd. All rights reserved.

  • 17.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Verma-Gaur, Jiyoti
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wallenius, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Mechanisms controlling diversification and affinity maturation of antibodies2013In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 32, p. S45-S45Article in journal (Other academic)
  • 18.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Verma-Gaur, Jiyoti
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wallenius, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Initiation of antigen receptor-dependent differentiation into plasma cells by calmodulin inhibition of E2A2009In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 183, no 2, p. 1179-1187Article in journal (Refereed)
    Abstract [en]

    Differentiation of B lymphocytes into Ab-secreting plasmablasts and plasma cells is Ag driven. The interaction of Ag with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. However, not much is known about the coupling between BCR activation and the shift in transcription factor network from that of a B cell to that of ASC differentiation. Our genome-wide analysis shows that Ab-secreting cell differentiation of mouse B cells is induced by BCR activation through very fast regulatory events from the BCR. We identify activation of IFN regulatory factor-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Spi-B gene expression as immediate early events. Furthermore, the transcription factor E2A is required for the rapid key down-regulations after BCR activation, and the Ca(2+) sensor protein calmodulin has the corresponding regulatory effect as BCR activation. Moreover, mutants in the calmodulin binding site of E2A show that Ca(2+) signaling through calmodulin inhibition of E2A is essential for the rapid down-regulation of immediate early genes after BCR activation in initiation of plasma cell differentiation.

  • 19.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Verma-Gaur, Jiyoti
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wallenius, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Grundström, Thomas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mechanisms regulating diversification and affinity maturation of antibodies2012In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 30, no Suppl 1, p. S42-S42Article in journal (Other academic)
  • 20.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Verma-Gaur, Jiyoti
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wallenius, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Regulatory mechanisms controlling diversification and affinity maturation of antibodies2011Conference paper (Refereed)
  • 21.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wallenius, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sveshnikova, Natalia
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Saarikettu, Juha
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Calmodulin inhibition of E2A stops expression of surrogate light chains of the pre-B-cell receptor and CD192010In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 47, no 5, p. 1031-1038Article in journal (Refereed)
    Abstract [en]

    To create antibody diversity, B lymphocyte development is characterized by the ordered rearrangement of first immunoglobulin (Ig) heavy chain gene segments and then Ig light-chain gene segments. Early in B-cell development, expression of a pre-B-cell receptor (pre-BCR) composed of membrane-bound Ig heavy chain protein associated with surrogate light-chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces clonal expansion, but it also turns off transcription of the genes for the SLC proteins lambda5 and VpreB, which limits this proliferation. Here we show that signaling from the pre-BCR rapidly down-regulates lambda5 and VpreB and also the co-receptor CD19 in primary pre-B-cells. We show that calcium (Ca(2+)) signaling is essential for this silencing of the SLC and CD19 genes. The SLC genes are activated by the E2A transcription factor, and we show that E2A is required for pre-BCR-mediated regulation of the genes. E2A mutated in its binding site for the Ca(2+) sensor protein calmodulin, and thus with calmodulin-resistant DNA binding, makes lambda5, VpreB and CD19 expression resistant to the inhibition following pre-BCR activation. Thus, Ca(2+) down-regulates SLC and CD19 gene expression upon pre-BCR activation through inhibition of E2A by Ca(2+)/calmodulin.

  • 22.
    Hermann, Stefan
    et al.
    Department of Biosciences, Karolinska institute, Novum, Huddinge, Sweden.
    Saarikettu, Juha
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Onions, Jacqueline
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hughes, Kate
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Calcium regulation of basic helix-loop-helix transcription factors1998In: Cell Calcium, ISSN 0143-4160, E-ISSN 1532-1991, Vol. 23, no 2-3, p. 135-142Article in journal (Refereed)
    Abstract [en]

    The basic helix-loop-helix (bHLH) family of transcription factors is essential for numerous developmental and growth control processes. The regulation of bHLH proteins occurs at many levels, including tissue specific expression, differential oligomerization and DNA binding specificities, interaction with negatively acting HLH proteins and post-translational modifications. This review focuses on what is emerging as another level of bHLH protein regulation, calcium regulation through interaction with Ca2+ loaded calmodulin and S-100 proteins. The mechanism and implications of these Ca2+ regulated interactions are discussed.

  • 23.
    Hughes, Kate
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Antonsson, Åsa
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Calmodulin dependence of NFκB activation1998In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 441, no 1, p. 132-136Article in journal (Refereed)
    Abstract [en]

    The NF kappa B family of transcription factors is regulated by inhibitory I kappa B proteins. A diversity of stimuli leads to the phosphorylation and subsequent degradation of I kappa B, releasing NF kappa B to act on its target genes. Calmodulin (CaM) is a key regulator of numerous cellular processes and is the predominant intracellular receptor for Ca2+ signals. Here me report that several CaM antagonists inhibit the activation of NF kappa B, and that this is due to the prevention of inducible I kappa B phosphorylation. Our results suggest that CaM is involved in the phosphorylation of I kappa B, a finding that may help in elucidating the mechanism of this critical step of NF kappa B activation.

  • 24.
    Hughes, Kate
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Edin, Sofia
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Antonsson, Åsa
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Calmodulin-dependent Kinase II Mediates T Cell Receptor/CD3- and Phorbol Ester-induced Activation of IκB Kinase2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 38, p. 36008-36013Article in journal (Refereed)
  • 25.
    Hughes, Kate
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Saarikettu, Juha
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gene expression in transfected cells2002In: Calcium-Binding Protein Protocols: Volume 2: Methods and Techniques / [ed] Hans J. Vogel, Totowa, NJ: Humana Press, 2002, Vol. 173, p. 355-363Chapter in book (Refereed)
    Abstract [en]

    A general approach to address the biological function of a calcium-binding protein, or another protein, in living cells is to increase or decrease the activity of the protein in the cell and analyze the effects on cell functions. In many cases, it is desirable to determine the effects of overexpressing the protein or a constitutively active or dominantly negative derivative, or to express the protein in a cell that normally lacks it. This is achieved by introducing its gene exogenously. The cDNA for the protein is cloned downstream of an active promoter in a plasmid designed for expression in mammalian cells. This expression plasmid is then transfected into the cell.

  • 26.
    Kumar, Anjani
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Priya, Anshu
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ahmed, Tanzeel
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Negi, Neema
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Regulation of the DNA Repair Complex during Somatic Hypermutation and Class-Switch Recombination2018In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 200, no 12, p. 4146-4156Article in journal (Refereed)
    Abstract [en]

    B lymphocytes optimize Ab responses by somatic hypermutation (SH), which introduces pointmutations in the variable regions of the Ab genes and by class-switch recombination (CSR), which changes the expressed C region exon of the IgH. These Ab diversification processes are initiated by the deaminating enzyme activation-induced cytidine deaminase followed by many DNA repair enzymes, ultimately leading to deletions and a high mutation rate in the Ab genes, whereas DNA lesions made by activation-induced cytidine deaminase are repaired with low error rate on most other genes. This indicates an advanced regulation of DNA repair. In this study, we show that initiation of Ab diversification in B lymphocytes of mouse spleen leads to formation of a complex between many proteins in DNA repair. We show also thatBCR activation, which signals the end of successful SH, reduces interactions between some proteins in the complex and increases other interactions in the complex with varying kinetics. Furthermore, we show increased localization of SH-and CSR-coupled proteins on switch regions of the Igh locus upon initiation of SH/CSR and differential changes in the localization upon BCR signaling, which terminates SH. These findings provide early evidence for a DNA repair complex or complexes that may be of functional significance for carrying out essential roles in SH and/or CSR in B cells.

  • 27. Lai, Dazhi
    et al.
    Wan, Mimi
    Wu, Jie
    Preston-Hurlburt, Paula
    Kushwaha, Ritu
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Imbalzano, Anthony N
    Chi, Tian
    Induction of TLR4-target genes entails calcium/calmodulin-dependent regulation of chromatin remodeling.2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 4, p. 1169-1174Article in journal (Refereed)
    Abstract [en]

    Upon toll-like receptor 4 (TLR4) signaling in macrophages, the mammalian Swi/Snf-like BAF chromatin remodeling complex is recruited to many TLR4 target genes where it remodels their chromatin to promote transcription. Here, we show that, surprisingly, recruitment is not sufficient for chromatin remodeling; a second event, dependent on calcium/calmodulin (CaM), is additionally required. Calcium/CaM directly binds the HMG domain of the BAF57 subunit within the BAF complex. Calcium/CaM antagonists, including a CaM-binding peptide derived from BAF57, abolish BAF-dependent remodeling and gene expression without compromising BAF recruitment. BAF57 RNAi and BAF57 dominant negative mutants defective in CaM binding similarly impair the induction of BAF target genes. Our data implicate calcium/CaM in TLR4 signaling, and reveal a previously undescribed, recruitment-independent mode of regulation of the BAF complex that is probably achieved through a direct CaM-BAF interaction.

  • 28.
    Larsson, Göran
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Onions, Jacqueline
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hermann, Stefan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wijmenga, Sybren S
    Backbone dynamics of a symmetric calmodulin dimer in complex with the calmodulin-binding domain of the basic-helix-loop-helix transcription factor SEF2-1/E2-2: a highly dynamic complex.2005In: Biophys J, ISSN 0006-3495, Vol. 89, no 2, p. 1214-26Article in journal (Refereed)
    Abstract [en]

    Calmodulin (CaM) interacts specifically as a dimer with some dimeric basic-Helix-Loop-Helix (bHLH) transcription factors via a novel high affinity binding mode. Here we report a study of the backbone dynamics by (15)N-spin relaxation on the CaM dimer in complex with a dimeric peptide that mimics the CaM binding region of the bHLH transcription factor SEF2-1. The relaxation data were measured at multiple magnetic fields, and analyzed in a model-free manner using in-house written software designed to detect nanosecond internal motion. Besides picosecond motions, all residues also experience internal motion with an effective correlation time of approximately 2.5 ns with squared order parameter (S(2)) of approximately 0.75. Hydrodynamic calculations suggest that this can be attributed to motions of the N- and C-terminal domains of the CaM dimer in the complex. Moreover, residues with significant exchange broadening are found. They are clustered in the CaM:SEF2-1mp binding interface, the CaM:CaM dimer interface, and in the flexible helix connecting the CaM N- and C-terminal domains, and have similar exchange times (approximately 50 micros), suggesting a cooperative mechanism probably caused by protein:protein interactions. The dynamic features presented here support the conclusion that the conformationally heterogeneous bHLH mimicking peptide trapped inside the CaM dimer exchanges between different binding sites on both nanosecond and microsecond timescales. Nature has thus found a way to specifically recognize a relatively ill-fitting target. This novel mode of target-specific binding, which neither belongs to lock-and-key nor induced-fit binding, is characterized by dimerization and continuous exchange between multiple flexible binding alternatives.

  • 29.
    Liu, Hebin
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Carlsson, Leif
    Umeå Centre for Molecular Medicine (UCMM).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Identification of an N-terminal transactivation domain of Runx1 that separates molecular function from global differentiation function.2006In: J Biol Chem, ISSN 0021-9258, Vol. 281, no 35, p. 25659-69Article in journal (Refereed)
    Abstract [en]

    RUNX1, or AML1, is a transcription factor that is the most frequent target for chromosomal gene translocations in acute leukemias. RUNX1 is essential for definitive hematopoiesis in embryos and profoundly influences adult steady-state hematopoiesis both positively and negatively. To investigate this wide range of normal activities and the pathological role of RUNX1, it is important to define the functions of different domains of the protein. RUNX1, RUNX2, and RUNX3 are highly conserved in their DNA binding runt homology domain and contain divergent sequences of unknown function N-terminal to this domain. Here we analyzed the role of the N-terminal sequence and the alpha-helix of the runt homology domain of Runx1 in DNA binding, transactivation, and megakaryocytopoiesis. Both the N terminus and the alpha-helix were found to reduce DNA binding of Runx1 and be essential for transactivation of the granulocyte-macrophage colony-stimulating factor and Ialpha1 promoters by Runx1. The N terminus of Runx1, including the alpha-helix, was also required for transactivation of a Gal4 reporter when expressed as fusion proteins with a Gal4 DNA binding domain, and the N terminus alone was capable of stimulating transcription when fused to the Gal4 DNA binding domain. The N terminus and the alpha-helix, however, were not required for megakaryocyte development from embryonic stem cells differentiated in vitro. Thus, our findings define a second transactivation domain of Runx1 that is differentially required for activation of transcription of some Runx1-dependent promoters and megakaryocytopoiesis.

  • 30.
    Liu, Hebin
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Calcium regulation of GM-CSF by calmodulin-dependent kinase II phosphorylation of Ets12002In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 13, no 12, p. 4497-4507Article in journal (Refereed)
    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.

  • 31.
    Liu, Hebin
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Holm, Magnus
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Xie, Xiao-Qi
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Chemistry.
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    AML1/Runx1 recruits calcineurin to regulate granulocyte macrophage colony-stimulating factor by Ets1 activation.2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 28, p. 29398-29408Article in journal (Refereed)
    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.

  • 32. Mårtensson, Annica
    et al.
    Xie, Xiao-Qi
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Persson, Christine
    Holm, Magnus
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Mårtensson, Inga-Lill
    PEBP2 and c-myb sites crucial for lambda5 core enhancer activity in pre-B cells.2001In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 31, no 11, p. 3165-3174Article in journal (Refereed)
    Abstract [en]

    The lambda5 gene is expressed exclusively in precursor (pre-) B cells where its gene product, as part of the pre-B cell receptor, is crucial for the proliferation of these cells. Several DNA regions regulate the activity and expression pattern of the lambda5 gene. Amongst these is an enhancer, B(lambda5), located 5' of the gene. Here we analyze the lambda5 enhancer core, b(lambda5), which in earlier experiments was demonstrated to retain 50% of the enhancer activity, and show that this activity is restricted to pre-B cells. We identify a DNA element within b(lambda5), PEBP2(lambda5), which is essential for enhancer activity: mutation within this site dramatically reduces core enhancer activity in pre-B cells. The PEBP2(lambda5) site binds bacterially produced polyoma enhancer binding proteins (PEBP) (Runx/AML/CBFA). Furthermore, PEBP2 proteins present in nuclear extracts from murine pre-B cells bind to the PEBP2(lambda5) element. PEBP2 proteins in mature B cells also bind to the PEBP2(lambda5 )element, implying that if PEBP2 proteins are responsible for the stage-specific expression, they have to be non-activating or inhibiting in mature B cells. We also demonstrate that a described partner of PEBP2, c-myb, binds to a sequence termed myb(lambda5) located just upstream of the PEBP2(lambda5) site in the core enhancer. The myb(lambda5) element is also crucial for enhancer activity, since mutating the myb site reduces core enhancer activity to the same extent as mutating the PEBP2 site. Earlier reports have shown that c-myb is expressed at high levels in pre-B cell lines whereas its expression is down-regulated in more mature B cell lines. Thus, c-myb may be involved in determining the stage-specific expression of the lambda5 gene.

  • 33.
    Onions, Jacqueline
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Hermann, Stefan
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    A novel type of calmodulin interaction in the inhibition of basic helix-loop-helix transcription factors.2000In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, no 15, p. 4366-4374Article in journal (Refereed)
    Abstract [en]

    Calmodulin is the predominant intracellular receptor for Ca(2+) signals, mediating the regulation of numerous cellular processes. Previous studies have shown that calcium-loaded calmodulin can bind to and inhibit the activity of certain basic helix-loop-helix (bHLH) transcription factors. The basic sequence within the bHLH domain is the primary target for calmodulin binding, and sequences modulating the calmodulin interaction reside directly N-terminal to the basic sequence. Here we show that the interaction of calmodulin with bHLH proteins is of a novel type, displaying characteristics very different from those of previously characterized calmodulin-target complexes. We show that calmodulin interacts much stronger with a dimeric basic sequence than with the monomeric form. The calmodulin-bHLH protein complex contains equimolar amounts of calmodulin and bHLH chains. The interaction is unusual in being to a large extent polar in nature, and it is highly resistant to tested calmodulin inhibitors. Both the N-terminal and C-terminal domains of calmodulin can independently bind to and inhibit the DNA binding of bHLH proteins. The C-terminal domain preferentially binds to the basic sequence, whereas the N-terminal domain is essential for the effect of the modulatory sequence. We propose a model for the calmodulin-bHLH complex where two calmodulin molecules interact with one bHLH dimer, with one domain of calmodulin preferentially binding to the basic sequence of bHLH proteins and the other domain interacting with the modulatory sequence.

  • 34.
    Oruganti, Sreenivasa Rao
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Edin, Sofia
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    CaMKII targets Bc110 in T-cell receptor induced activation of NF-κB2011In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 48, no 12-13, p. 1448-1460Article in journal (Refereed)
    Abstract [en]

    Recognition of antigen by T- or B-cell receptors leads to formation of an immunological synapse and initiation of signalling events that collaborate to determine the nature of the adaptive immune response. Activation of NF-κB transcription factors has a key role in regulation of numerous genes with important functions in immune responses and inflammation and is of great importance for lymphocyte activation and differentiation. The activation of NF-κB depends on changes in intracellular Ca2+ levels, and both calmodulin (CaM) and a CaM-dependent kinase, CaMKII, help regulate NF-κB activation after T-cell receptor (TCR) stimulation, but the mechanisms are not well characterized. Here we have analyzed the functional role of CaMKII in the signalling pathway from the TCR to activation of IKK, the kinase that phosphorylates the NF-κB inhibitor IκB. We show that CaMKII is recruited to the immunological synapse where it interacts with and phosphorylates the signalling adaptor protein Bcl10. Furthermore, phosphorylation of the CARD domain of Bcl10 by CaMKII regulates the interactions within the important Carma1, Bcl10, Malt1 signalling complex and the essential signal induced ubiquitinations of Bcl10 and IKKγ. We propose a novel mechanism whereby Ca2+ signals can be integrated at the immunological synapse through CaMKII-dependent phosphorylation of Bcl10.

  • 35.
    Oruganti, Sreenivasa Rao
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Edin, Sofia
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    CaMKII targets Bcl10 in T-cell receptor induced activation of NF-κB2011In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 48, no 12-13, p. 1448-1460Article in journal (Refereed)
    Abstract [en]

    Recognition of antigen by T- or B-cell receptors leads to formation of an immunological synapse and initiation of signalling events that collaborate to determine the nature of the adaptive immune response. Activation of NF-κB transcription factors has a key role in regulation of numerous genes with important functions in immune responses and inflammation and is of great importance for lymphocyte activation and differentiation. The activation of NF-κB depends on changes in intracellular Ca(2+) levels, and both calmodulin (CaM) and a CaM-dependent kinase, CaMKII, help regulate NF-κB activation after T-cell receptor (TCR) stimulation, but the mechanisms are not well characterized. Here we have analyzed the functional role of CaMKII in the signalling pathway from the TCR to activation of IKK, the kinase that phosphorylates the NF-κB inhibitor IκB. We show that CaMKII is recruited to the immunological synapse where it interacts with and phosphorylates the signalling adaptor protein Bcl10. Furthermore, phosphorylation of the CARD domain of Bcl10 by CaMKII regulates the interactions within the important Carma1, Bcl10, Malt1 signalling complex and the essential signal induced ubiquitinations of Bcl10 and IKKγ. We propose a novel mechanism whereby Ca(2+) signals can be integrated at the immunological synapse through CaMKII-dependent phosphorylation of Bcl10.

  • 36.
    Saarikettu, Juha
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sveshnikova, Natalia
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Calcium/calmodulin inhibition of transcriptional activity of E-proteins by prevention of their binding to DNA.2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 39, p. 41004-41011Article in journal (Refereed)
    Abstract [en]

    The Ca2+ sensor protein calmodulin can interact with the DNA binding basic helix-loop-helix (bHLH) domain of E12, E47, and SEF2-1 (E2-2), which belong to the E-protein subclass of bHLH transcription factors. This interaction inhibits the DNA binding of these bHLH proteins in vitro, and an ionophore that increases intracellular Ca2+ can inhibit transcriptional activation by the E-proteins. Here we have attempted to determine if these phenomena reflect a direct calmodulin-dependent inhibition of DNA binding by E-proteins in vivo. We show that calmodulin overexpression inhibits the transcriptional activity of E12, E47, and SEF2-1. We have compared calmodulin effects on DNA binding in vitro and on activation of transcription in vivo using a series of E12 mutants harboring defined alterations within the basic sequence of the bHLH domain that reduce their ability to bind calmodulin to varying degrees. We find a striking direct correlation between the ability of calmodulin to inhibit their DNA binding in vitro and the ability of overexpressed calmodulin or cellular Ca2+ mobilization to inhibit their transcriptional activity in vivo. Furthermore, E12 and overexpressed calmodulin were co-localized in the nucleus, and calmodulin pull-down experiments with cell extracts showed a Ca2+-dependent interaction between calmodulin and E12 but not with a calmodulin inhibition-deficient E12 mutant. Chromatin immunoprecipitation showed that calmodulin overexpression leads to decreased binding of E12 and E47, but not a calmodulin inhibition-deficient E12 mutant, to the DNA recognition sequence in vivo. The data suggest that Ca2+ signaling can inhibit the transcriptional activities of E-proteins through direct binding of Ca2+/calmodulin to the basic sequence of E-proteins, resulting in inhibition of their DNA binding. Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.

  • 37.
    Verma-Gaur, Jiyoti
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hauser, Jannek
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Negative feedback regulation of antigen receptors through calmodulin inhibition of E2A2012In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 188, no 12, p. 6175-6183Article in journal (Refereed)
    Abstract [en]

    Signaling from the BCR is used to judge Ag-binding strengths of the Abs of B cells. BCR signaling enables the selection for successive improvements in the Ag affinity over an extremely broad range of affinities during somatic hypermutation. We show that the mouse BCR is subject to general negative feedback regulation of the receptor proteins, as well as many coreceptors and proteins in signal pathways from the receptor. Thus, the BCR can downregulate itself, which can enable sensitive detection of successive improvements in the Ag affinity over a very large span of affinities. Furthermore, the feedback inhibition of the BCR signalosome and most of its proteins, as well as most other regulations of genes by BCR stimulation, is to a large extent through inhibition of the transcription factor E2A by Ca(2+)/calmodulin.

  • 38. Verma-Gaur, Jiyoti
    et al.
    Hauser, Jannek
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Negative feedback regulation of antigen receptors through calmodulin inhibition of E2AManuscript (preprint) (Other academic)
  • 39.
    Wallenius, Anders
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hauser, Jannek
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Aas, Per Arne
    Sarno, Antonio
    Kavli, Bodil
    Krokan, Hans E.
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Expression and recruitment of uracil-DNA glycosylase are regulated by E2A during antibody diversification2014In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 60, no 1, p. 23-31Article in journal (Refereed)
    Abstract [en]

    B-lymphocytes can modify their immunoglobulin (Ig) genes to generate specific antibodies with a new isotype and enhanced affinity against an antigen. Activation-induced cytidine deaminase (AID), which is positively regulated by the transcription factor E2A, is the key enzyme that initiates these processes by deaminating cytosine to uracil in Ig genes. Nuclear uracil-DNA glycosylase (UNG2) is subsequently required for uracil processing in the generation of high affinity antibodies of different isotypes. Here we show that the transcription factor E2A binds to the UNG2 promoter and represses UNG2 expression. Inhibition of E2A by binding of Ca2+-activated calmodulin alleviates this repression. Furthermore, we demonstrate that UNG2 preferentially accumulates in regions of the Ig heavy chain (IgH) gene containing AID hotspots. Calmodulin inhibition of E2A strongly enhances this UNG2 accumulation, indicating that it is negatively regulated by E2A as well. We show also that over-expression of E2A can suppress class switch recombination. The results suggest that E2A is a key factor in regulating the balance between AID and UNG2, both at expression and Ig targeting levels, to stimulate Ig diversification and suppress normal DNA repair processes. (c) 2014 Elsevier Ltd. All rights reserved.

  • 40. Wolf-Watz, M
    et al.
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Härd, T
    Structure and backbone dynamics of Apo-CBFbeta in solution.2001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 38, p. 11423-11432Article in journal (Refereed)
    Abstract [en]

    Runx proteins constitute a family of mammalian transcription factors that interact with DNA through their evolutionarily conserved Runt domain. CBFbeta, alternatively denoted PEBP2beta, is the non-DNA-binding heterodimer partner and acts to enhance the DNA binding affinity of Runx proteins. Runx proteins and CBFbeta are associated with a variety of biological functions and human diseases; they are, for example, together the most frequent targets for chromosomal rearrangements in acute human leukemias. We have determined the solution structure and characterized the backbone dynamics of C-terminally truncated fragments containing residues 1-141 of CBFbeta. The present apo-CBFbeta structure is very similar to that seen in a Runt-CBFbeta complex. An evaluation of backbone (15)N NMR relaxation parameters shows that CBFbeta is a rigid molecule with high order parameters throughout the backbone; the only regions displaying significant dynamics are a long loop and the C-terminal alpha-helix. A few residues display relaxation behavior indicating conformational exchange on microsecond to millisecond time scales, but only one of these is located at the Runt binding surface. Our structure and dynamics analysis of CBFbeta therefore suggests that the protein binds to Runt without large conformational changes or induced folding ("lock-and-key" interaction). The apo-CBFbeta structure presented here exhibits several significant differences with two other published NMR ensembles of very similar protein fragments. The differences are located in four regions outside of the central beta-barrel, whereas the beta-barrel itself is almost identical in the three NMR structures. The comparison illustrates that independently determined NMR structures may display rather large differences in backbone conformation in regions that appear to be well-defined in each of the calculated NMR ensembles.

  • 41.
    Wolf-Watz, Magnus
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Bäckström, Stefan
    Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Grundström, Thomas
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sauer, Uwe
    Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Härd, Torleif
    Chloride binding by the AML1/Runx1 transcription factor studied by NMR2001In: FEBS Letters, ISSN 0014-5793, Vol. 488, no 1-2, p. 81-4Article in journal (Refereed)
    Abstract [en]

    It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.

  • 42.
    Xie, Xiao-Qi
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Pardali, Evangelia
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Holm, Magnus
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sideras, Paschalis
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    AML and Ets proteins regulate the I alpha1 germ-line promoter.1999In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 29, no 2, p. 488-498Article in journal (Refereed)
    Abstract [en]

    The immunoglobulin heavy chain (IgH) class switch recombination of B lymphocytes preferentially targets unrearranged IgH genes that have already been rendered transcriptionally active. Transcription of the germ-line IgH genes is controlled by intervening (I) regions upstream of their switch regions. The I alpha1 promoter activates transcription of the human germ-line C alpha1 gene for IgA1 and mediates the transforming growth factor (TGF)-beta1 responsiveness of this locus. Here we show that the I alpha1 promoter contains several binding sites for the AML/PEBP2/CBF family of transcription factors and that AML and Ets proteins are major regulators of the basal and TGF-beta-inducible promoter activity. Our data constitute a starting point for studies to elucidate the molecular mechanism by which TGF-beta regulates IgA production.

1 - 42 of 42
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