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

Abstract

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.

We thank C. Bjornson, D. Kimelman, Q.-G. Nguyen, R. Palmiter, and E. Wang for many constructive discussions and for critical comments on the manuscript. We thank J. Eng, C. Fabre-Suver, Q.-G. Nguyen, K. Vaughn, and E. Yi for technical assistance. We are grateful to A. Weiner for providing a starter culture of HeLa S cells, C. Thornton for providing Six5 antisera, E. Olson for the MEF2C expression plasmid, A. Lassar for the MyoD expression construct, L. Kedes for pHcTnC4500CAT/I1-500-S, K. Hidaka and T. Mukai for pM202CAT and pM202CAT(mut C1), E. Hardeman for hTnIsUSE/-2000hSkeletalActin/CAT, J. D. Engel for providing GATA-2 cDNA, and S. Orkin for GATA-2 antisera.

This work was supported by National Institutes of Health grants AR18860 and R24 64387 to S.D.H. and predoctoral training grant GM07270 to C.L.H., as well as by the Muscular Dystrophy Association and federal funds from the National Heart, Lung, and Blood Institute, National Institutes of Health, under contract no. NO1-HV-28179.