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The basic helix-loop-helix transcription factor E2-2 is involved in T lymphocyte development
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
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2000 (English)In: European Journal of Immunology, ISSN 0014-2980, 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.

Place, publisher, year, edition, pages
2000. Vol. 30, no 10, 2857-2863 p.
URN: urn:nbn:se:umu:diva-4592DOI: 10.1002/1521-4141(200010)30:10<2857::AID-IMMU2857>3.0.CO;2-GOAI: diva2:143751
Available from: 2005-05-13 Created: 2005-05-13 Last updated: 2011-03-14
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

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