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J. Biol. Chem., Vol. 279, Issue 39, 41004-41011, September 24, 2004

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Calcium/Calmodulin Inhibition of Transcriptional Activity of E-proteins by Prevention of Their Binding to DNA^*

Juha Saarikettu, Natalia Sveshnikova, and Thomas Grundström

From the Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden

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.

Received for publication, July 19, 2004 , and in revised form, July 26, 2004.

^* This work was supported by grants from the Swedish Research Council for Engineering Sciences and the Swedish Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 46-90-7852531; Fax: 46-90-771420; E-mail: Thomas.Grundstrom{at}molbiol.umu.se.

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