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Calcium regulation and functions of basic Helix-Loop-Helix transcription factors
Umeå University, Faculty of Medicine, Molecular Biology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The members of the ubiquitously expressed E-protein subfamily of basic Helix-Loop-Helix (bHLH) transcription factors, E12/E47, SEF2-1 and HEB, have important roles as regulators of gene expression in various differentiation processes, including lymphocyte development and myogenesis. In myogenesis, E-proteins are proposed to function as obligate heterodimer partners for members of the MyoD family of muscle-specific bHLH transcription factors.

The calcium ion (Ca2+) is a universal cellular messenger involved in regulation of a variety of cellular functions, including transcription. The Ca2+-bound form of the Ca2+-binding protein calmodulin (Ca2+/CaM) has been shown to inhibit DNA binding of E-proteins, but not tissue specific bHLH transcription factors, through direct physical interaction with the DNA binding basic sequence. The main focus of this thesis is on the role of Ca2+-binding proteins in regulation of bHLH transcription factors.

Solution structure analysis of CaM in complex with the CaM-binding basic sequence of an E-protein revealed a novel type of protein-protein interaction with alternative binding modes in a complex of a CaM dimer surrounding the dimer of the E-protein sequence. This model for the interaction was further supported by mutational analysis, since every amino-acid substitution in the CaM binding basic sequence of E12 only partially affected the interaction with CaM.

The mechanism of Ca2+/CaM regulation of transcriptional activation by E-proteins was studied using a cell culture system. CaM overexpression inhibited transcriptional activation by E12, E47 and SEF2-1 but not by MyoD. Ca2+/CaM inhibition of DNA binding in vitro directly correlated with the inhibitory effects of Ca2+ stimulation and CaM overexpression on transcription in vivo in a series of E12 basic sequence mutants. Furthermore, in vivo DNA binding of E12, but not a CaM resistant mutant of E12, was inhibited by overexpression of CaM. The data indicate that Ca2+/CaM can inhibit transcriptional activation by E-proteins through formation of a CaM-E-protein complex that can not bind DNA.

An in vitro myogenesis system was used to investigate the potential role of the CaM-E-protein interaction in regulation of differentiation. CaM resistant mutants of E12 were inhibitory in MyoD initiated myogenic conversion of transfected fibroblasts, and inducers of intracellular Ca2+ activated, and Ca2+-channel blockers inhibited, transcriptional activation by E12, but not by a CaM resistant mutant of E12, with MyoD. The data support a model that Ca2+/CaM plays a role in initiation of myogenic differentiation through inhibition of E-protein dimers that can function as competitors to the CaM resistant MyoD/E-protein heterodimers required for myogenesis.

The potential involvement of the Ca2+-binding calretinin proteins in regulation of bHLH transcription factors was also studied. Calretinin and the alternative splice variant calretinin-22k have been proposed to function as Ca2+-buffer proteins. Calretinin expression is restricted primarily to neuronal tissues. Calretinin and calretinin-22k are also found expressed in colon cancers, but not in normal colon tissue, and a role for calretinins in tumorigenesis has been proposed. We show that calretinins can inhibit DNA binding and transcriptional activation by E12 through basic sequence interaction. Endogenous E12/E47 and calretinin co-localize in a subset of cells in a proliferating colon cancer cell line and can be co-immunoprecipitated from the cell extract. A model is proposed in which calretinin overexpression can contribute to tumorigenesis through inhibition of the anti-proliferative function of E-proteins.

The role of the E-protein E2-2 in lymphocyte development was studied using genetically altered mice with mosaic deletion of the E2-2 gene. The proportion of cells with a functional E2-2 allele was increased in the B- and T-lymphocyte populations, indicating a role for E2-2 not only in B-cell development, as reported before, but also in T-cell development.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi , 2005. , 62 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 968
Keyword [en]
Molecular biology, calcium, calmodulin, calretinin, transcription, bHLH, E-protein, E2-2
Keyword [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-537ISBN: 91-7305-885-8 (print)OAI: oai:DiVA.org:umu-537DiVA: diva2:143752
Public defence
2005-06-02, Major Groove, 6L, Institutionen för Molekylärbiologi, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2005-05-13 Created: 2005-05-13 Last updated: 2009-11-27Bibliographically approved
List of papers
1. A third principle of protein:protein recognition: the calmodulin dimer interaction with basic-helix-loop-helix transcription factors.
Open this publication in new window or tab >>A third principle of protein:protein recognition: the calmodulin dimer interaction with basic-helix-loop-helix transcription factors.
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(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4588 (URN)
Available from: 2005-05-13 Created: 2005-05-13 Last updated: 2011-03-30Bibliographically approved
2. Calcium/calmodulin inhibition of transcriptional activity of E-proteins by prevention of their binding to DNA.
Open this publication in new window or tab >>Calcium/calmodulin inhibition of transcriptional activity of E-proteins by prevention of their binding to DNA.
2004 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 39, 41004-41011 p.Article in journal (Refereed) Published
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.

Keyword
Amino Acid Sequence, Animals, Blotting; Western, Calcium/*metabolism, Calmodulin/chemistry/*metabolism, Cell Nucleus/metabolism, Cells; Cultured, Chromatin/metabolism, Cytoplasm/metabolism, DNA/*chemistry/metabolism, DNA; Complementary/metabolism, DNA-Binding Proteins/*metabolism, Humans, Mice, Microscopy; Fluorescence, Molecular Sequence Data, Mutagenesis; Site-Directed, Mutation, Nerve Tissue Proteins, Plasmids/metabolism, Precipitin Tests, Protein Binding, Protein Structure; Tertiary, Sepharose/chemistry, Sequence Homology; Amino Acid, Signal Transduction, TCF Transcription Factors, Thapsigargin/pharmacology, Trans-Activation (Genetics), Transcription Factors/*metabolism, Transcription; Genetic, Transfection
Identifiers
urn:nbn:se:umu:diva-16488 (URN)10.1074/jbc.M408120200 (DOI)15280352 (PubMedID)
Available from: 2007-10-03 Created: 2007-10-03 Last updated: 2017-12-14Bibliographically approved
3. Calcium/Calmodulin Regulation of Myogenesis Through Differential Inhibition of Basic Helix-Loop-Helix Transcription Factors.
Open this publication in new window or tab >>Calcium/Calmodulin Regulation of Myogenesis Through Differential Inhibition of Basic Helix-Loop-Helix Transcription Factors.
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4590 (URN)
Available from: 2005-05-13 Created: 2005-05-13 Last updated: 2010-01-13Bibliographically approved
4. Identification of Calretinins as Inhibitors of DNA Binding of E-proteins.
Open this publication in new window or tab >>Identification of Calretinins as Inhibitors of DNA Binding of E-proteins.
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4591 (URN)
Available from: 2005-05-13 Created: 2005-05-13 Last updated: 2010-01-13Bibliographically approved
5. The basic helix-loop-helix transcription factor E2-2 is involved in T lymphocyte development
Open this publication in new window or tab >>The basic helix-loop-helix transcription factor E2-2 is involved in T lymphocyte development
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2000 (English)In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 30, no 10, 2857-2863 p.Article in journal (Refereed) Published
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.

Identifiers
urn:nbn:se:umu:diva-4592 (URN)10.1002/1521-4141(200010)30:10<2857::AID-IMMU2857>3.0.CO;2-G (DOI)
Available from: 2005-05-13 Created: 2005-05-13 Last updated: 2017-12-14

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