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Calcium/Calmodulin Regulation of Myogenesis Through Differential Inhibition of Basic Helix-Loop-Helix Transcription Factors.
Umeå University, Faculty of Medicine, Molecular Biology.
Manuscript (Other academic)
Identifiers
URN: urn:nbn:se:umu:diva-4590OAI: oai:DiVA.org:umu-4590DiVA: diva2:143749
Available from: 2005-05-13 Created: 2005-05-13 Last updated: 2010-01-13Bibliographically 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.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 968
Keyword
Molecular biology, calcium, calmodulin, calretinin, transcription, bHLH, E-protein, E2-2, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
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)
Opponent
Supervisors
Available from: 2005-05-13 Created: 2005-05-13 Last updated: 2009-11-27Bibliographically approved

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