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 ␣-actin, Desmin, and ␣-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.The mouse Muscle creatine kinase (MCK) gene serves as a useful model for understanding muscle-specific gene transcription since its expression is restricted to terminally differentiated striated muscle, where it is one of the most abundantly expressed genes. Transcription of MCK is regulated by an upstream enhancer (Ϫ1256 to Ϫ1050), a proximal promoter (Ϫ358 to ϩ7), and an intron 1 modulatory region (ϩ740 to ϩ1731). The upstream enhancer confers muscle-specific expression to reporter constructs in cell culture, transgenic mice, and virus-mediated gene transfer (1,13,24,30,31,56,61) and contains at least seven control elements, which were identified by footprinting, deletion/mutation analysis, and gel mobility shift assays (1,8,16,39,41). The sequences and relative positions of elements in the MCK enhancer (CArG/SRE, AP2, MEF3, A/T-rich, left and right E-boxes, and MEF2) are highly conserved between mammalian species, and many of the associated transcription factors have been identified (11,19,20,27,39,68).In contrast to the upstream enhancer, very little is known about the highly conserved (Ϫ358 to ϩ7) region comprising the MCK proximal promoter. Studies with cultured cells and transgenic mice have demonstrated that the MCK promoter alone is capable of driving muscle-specific expression (30,56) and that it is ϳ40-fold more active in skeletal than in cardiac myocytes (C. L. Himeda and S. D. Hauschka, unpublished data). However, the MCK promoter also synergizes with the upstream enhancer to drive much higher expression of reporter constructs in both types of striated muscle (13,56). The MCK promoter is known to contain a highly conserved E-box and CA...

Co-expression of FBN1 with mesenchyme-specific genes in mouse cell lines: implications for phenotypic variability in Marfan syndrome

“…MAZ (mycassociated zinc-finger protein) has been shown to regulate musclespecific gene expression. 30 RP58 has an essential role in skeletal myogenesis, 31 and BLIMP1 is involved in myocyte differentiation. 32 CIZ is implicated in regulation of bone mass biology.…”

Systematic measurement of transcription factor-DNA interactions by targeted mass spectrometry identifies candidate gene regulatory proteins

“…Another approach for studying TF-TRE interactions that does not require genetic engineering or protein detection reagents, and can readily provide information about the ensemble of TFs associated with a TRE, is DNA affinity purification followed by "shotgun" mass spectrometry (MS) analysis (8)(9)(10). This approach takes advantage of the ability of MS to identify large numbers of proteins in complex mixtures, and, when performed in a quantitative manner, can identify specific TF-DNA binding events even in the presence of a high background of nonspecifically copurifying proteins.…”

KLF3 Regulates Muscle-Specific Gene Expression and Synergizes with Serum Response Factor on KLF Binding Sites

“…The reporter plasmids Ϫ80MCKCAT, eϪ80MCKCAT, Ϫ358MCKCAT, eϪ358MCKCAT, (MPEX-mt)Ϫ80MCKCAT, and pUCSV2PAP have been described previously (1,25,54), as have constructs containing full-length mouse KLF3 cDNA in pMT2 (12), full-length and all truncated mouse KLF3 cDNAs in pMT3 (46), FLAG-tagged mouse KLF3 cDNA in pMT3 (46), and full-length KLF4 cDNA in pcDNA3 (a generous gift of Gary Owens [35]). Full-length human SRF cDNA in pCGN (11) was a generous gift of Robert Schwartz.…”

“…In a previous study, we used quantitative proteomics to identify factors binding the MCK promoter MPEX site and demonstrated that one of these candidates, MAZ, regulates muscle-specific genes through both established and divergent binding motifs (25). Here, we investigate the role of Kruppellike factor 3 (KLF3), which was also identified as a candidate MPEX-binding factor (MPEX-BF) by proteomic analysis.…”

“…The MCK promoter contains a conserved CArG site, an E box, and a GC-rich MPEX site, and the last two elements contribute to expression in both types of striated muscle (25,41). Interestingly, in contrast to the MCK enhancer, where high sequence conservation occurs only within control elements, the promoter is highly conserved throughout (ϳ70% identical between human and mouse), suggesting complex regulatory functions and control elements that have yet to be identified.…”