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Molecular and Cellular Biology, March 2004, p. 2132-2143, Vol. 24, No. 5
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.5.2132-2143.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Quantitative Proteomic Identification of Six4 as the Trex-Binding Factor in the Muscle Creatine Kinase Enhancer

Charis L. Himeda,1 Jeffrey A. Ranish,2 John C. Angello,1 Pascal Maire,3 Ruedi Aebersold,2 and Stephen D. Hauschka1*

Department of Biochemistry, University of Washington, Seattle, Washington 98195,1 Institute for Systems Biology, Seattle, Washington 98103-8904;,2 Departement Genetique, Developpement et Pathologie Moleculaire, Institut Cochin, INSERM 567, CNRS UMR 8104, Universite Paris V, 75014 Paris, France3

Received 18 July 2003/ Returned for modification 9 September 2003/ Accepted 5 December 2003

Transcriptional regulatory element X (Trex) is a positive control site within the Muscle creatine kinase (MCK) enhancer. Cell culture and transgenic studies indicate that the Trex site is important for MCK expression in skeletal and cardiac muscle. After selectively enriching for the Trex-binding factor (TrexBF) using magnetic beads coupled to oligonucleotides containing either wild-type or mutant Trex sites, quantitative proteomics was used to identify TrexBF as Six4, a homeodomain transcription factor of the Six/sine oculis family, from a background of ~900 copurifying proteins. Using gel shift assays and Six-specific antisera, we demonstrated that Six4 is TrexBF in mouse skeletal myocytes and embryonic day 10 chick skeletal and cardiac muscle, while Six5 is the major TrexBF in adult mouse heart. In cotransfection studies, Six4 transactivates the MCK enhancer as well as muscle-specific regulatory regions of Aldolase A and Cardiac troponin C via Trex/MEF3 sites. Our results are consistent with Six4 being a key regulator of muscle gene expression in adult skeletal muscle and in developing striated muscle. The Trex/MEF3 composite sequence ([C/A]ACC[C/T]GA) allowed us to identify novel putative Six-binding sites in six other muscle genes. Our proteomics strategy will be useful for identifying transcription factors from complex mixtures using only defined DNA fragments for purification.

* Corresponding author. Mailing address: Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195. Phone: (206) 543-1797. Fax: (206) 685-1792. E-mail: haus{at}u.washington.edu.

Molecular and Cellular Biology, March 2004, p. 2132-2143, Vol. 24, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.5.2132-2143.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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