Change search
ReferencesLink to record
Permanent link

Direct link
Calcium/calmodulin inhibition of transcriptional activity of E-proteins by prevention of their binding to DNA.
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). (Grundström)
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). (Grundström)
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). (Grundström)
2004 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, 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.

Place, publisher, year, edition, pages
2004. Vol. 279, no 39, 41004-41011 p.
Keyword [en]
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
URN: urn:nbn:se:umu:diva-16488DOI: 10.1074/jbc.M408120200PubMedID: 15280352OAI: diva2:156161
Available from: 2007-10-03 Created: 2007-10-03 Last updated: 2009-11-27Bibliographically approved
In thesis
1. Calcium regulation and functions of basic Helix-Loop-Helix transcription factors
Open this publication in new window or tab >>Calcium regulation and functions of basic Helix-Loop-Helix transcription factors
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.
Umeå University medical dissertations, ISSN 0346-6612 ; 968
Molecular biology, calcium, calmodulin, calretinin, transcription, bHLH, E-protein, E2-2, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:umu:diva-537 (URN)91-7305-885-8 (ISBN)
Public defence
2005-06-02, Major Groove, 6L, Institutionen för Molekylärbiologi, Umeå, 13:00 (English)
Available from: 2005-05-13 Created: 2005-05-13 Last updated: 2009-11-27Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Grundström, Thomas
By organisation
Molecular Biology (Faculty of Medicine)
In the same journal
Journal of Biological Chemistry

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 71 hits
ReferencesLink to record
Permanent link

Direct link