Quantitative Proteomic Identification of MAZ as a Transcriptional Regulator of Muscle-Specific Genes in Skeletal and Cardiac Myocytes

Abstract

We identified a conserved sequence within the Muscle creatine kinase (MCK) promoter that is critical for high-level activity in skeletal and cardiac myocytes (MCK Promoter Element X [MPEX]). After selectively enriching for MPEX-binding factor(s) (MPEX-BFs), ICAT-based quantitative proteomics was used to identify MPEX-BF candidates, one of which was MAZ (Myc-associated zinc finger protein). MAZ transactivates the MCK promoter and binds the MPEX site in vitro, and chromatin immunoprecipitation analysis demonstrates enrichment of MAZ at the endogenous MCK promoter and other muscle gene promoters (Skeletal α-actin, Desmin, and α-Myosin heavy chain) in skeletal and cardiac myocytes. Consistent with its role in muscle gene transcription, MAZ transcripts and DNA-binding activity are upregulated during skeletal myocyte differentiation. Furthermore, MAZ was shown to bind numerous sequences (e.g., CTCCTCCC and CTCCACCC) that diverge from the GA box binding motif. Alternate motifs were identified in many muscle promoters, including Myogenin and MEF2C, and one motif was shown to be critical for Six4 promoter activity in both skeletal and cardiac myocytes. Interestingly, MAZ occupies and is able to transactivate the Six4 promoter in skeletal but not cardiac myocytes. Taken together, these findings are consistent with a previously unrecognized role for MAZ in muscle gene regulation.

ACKNOWLEDGMENTS

We thank J. Angello, J. Buskin, D. Helterline, Q. Nguyen, P. Tai, and R. Welikson for technical assistance and/or critical discussions. We are grateful to J. Klimek and D. Martin at the Institute for Systems Biology proteomics facility for help with mass spectrometry.

This work was supported by NIH-RO1-AR18860 (to S.D.H.), NIH-T32-HL007312, Experimental Pathology of Cardiovascular Disease (to C.L.H.), contract no. N01-HV-28179 from the National Heart, Lung, and Blood Institute (to S.D.H. and C.L.H.), and grant no. P50GMO76547 from the National Institute of General Medical Sciences (to J.A.R.).