Calcium regulation and functions of basic Helix-Loop-Helix transcription factors

• Main Author: Saarikettu, Juha
• Format: Doctoral Thesis
• Language: English
• Published: Umeå universitet, Molekylärbiologi (Teknat- och Medfak) 2005
• Subjects: Molecular biology; calcium; calmodulin; calretinin; transcription; bHLH; E-protein; E2-2; Molekylärbiologi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-537; http://nbn-resolving.de/urn:isbn:91-7305-885-8

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.