doi: 10.1128/MCB.19.4.2853.
Activated notch inhibits myogenic activity of the MADS-Box transcription factor myocyte enhancer factor 2C
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- PMID:10082551
- PMCID: PMC84078
- DOI: 10.1128/MCB.19.4.2853
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Activated notch inhibits myogenic activity of the MADS-Box transcription factor myocyte enhancer factor 2C
J Wilson-Rawls et al. Mol Cell Biol.1999 Apr.
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Abstract
Skeletal muscle gene expression is dependent on combinatorial associations between members of the MyoD family of basic helix-loop-helix (bHLH) transcription factors and the myocyte enhancer factor 2 (MEF2) family of MADS-box transcription factors. The transmembrane receptor Notch interferes with the muscle-inducing activity of myogenic bHLH proteins, and it has been suggested that this inhibitory activity of Notch is directed at an essential cofactor that recognizes the DNA binding domains of the myogenic bHLH proteins. Given that MEF2 proteins interact with the DNA binding domains of myogenic bHLH factors to cooperatively regulate myogenesis, we investigated whether members of the MEF2 family might serve as targets for the inhibitory effects of Notch on myogenesis. We show that a constitutively activated form of Notch specifically blocks DNA binding by MEF2C, as well as its ability to cooperate with MyoD and myogenin to activate myogenesis. Responsiveness to Notch requires a 12-amino-acid region of MEF2C immediately adjacent to the DNA binding domain that is unique to this MEF2 isoform. Two-hybrid assays and coimmunoprecipitations show that this region of MEF2C interacts directly with the ankyrin repeat region of Notch. These findings reveal a novel mechanism for Notch-mediated inhibition of myogenesis and demonstrate that the Notch signaling pathway can discriminate between different members of the MEF2 family.
Figures
Schematic diagrams of Notch proteins. Structures of full-length Notch and the various deletion mutants used in this study are shown. The extracellular region of Notch consists of 34 to 36 EGF repeats followed by three novel Notch/lin12 domains. The transmembrane domain (TM) is shown with the orientation relative to the plasma membrane. The intracellular region of Notch contains six ankyrin repeats flanked by putative nuclear localization signals (nls), and a PEST sequence is located at the C terminus. NotchΔ lacks the ankyrin repeats and NotchIC contains only the intracellular region. AnkR consists of the ankyrin repeats and the N-terminal intracellular residues, along with an HA epitope tag at the C terminus. Unless otherwise specified, most experiments were performed with wild-type or mutant forms of human Notch2.
NotchIC inhibits cooperative activation of E-box- and MEF2-dependent reporters by myogenic bHLH proteins and MEF2C. (A) 10T1/2 cells were transfected with 3 μg of each reporter, 1 μg of each activator, and 3 μg of NotchIC, and CAT assays were performed as described in Materials and Methods. The data is presented as the fold activity versus that observed with the reporter gene alone and represents the mean ± the standard error of the mean for three experiments performed with at least two different preparations of the plasmids. (A) MEF2x2-CAT was used as the reporter. A schematic of the putative protein-protein interactions required for reporter gene activation is shown to the right. (B) 4R-tkCAT was used as the reporter. A schematic of the putative protein-protein interactions required for reporter gene activation is shown on the right.
NotchIC inhibits the ability of MEF2C to transactivate a MEF2-dependent reporter gene. 10T1/2 cells were transfected with 2 μg of each reporter, 2 μg of each MEF2 plasmid, and 2 μg of NotchIC, and CAT assays were performed as described in Materials and Methods. (A) MEF2x2CAT was used as the reporter. The ratio of plasmids used was based on data from titration experiments (data not shown). The data is presented as the fold activity versus that observed with the reporter gene alone and represents the mean ± the standard error of the mean for three experiments performed with at least two different preparations of the plasmids. (B) RSV-CAT, tk-CAT, and SV2CAT were used as reporters. Values represent the results of a representative experiment and are expressed as CAT activity relative to that with each reporter gene alone, which was assigned a value of 100.
NotchIC inhibits the ability of MEF2C to bind DNA. MEF2 and NotchIC proteins were translated from plasmid templates by using TNT reticulocyte lysates and T7 polymerase. The efficiency of translation was determined with duplicate Trans-[35S]-labeled reaction mixtures (data not shown). A32P-end-labeled double-stranded oligonucleotide representing the MCK MEF2 site was used as the probe. DNA binding reactions were carried out as described in Materials and Methods, and protein-DNA complexes were analyzed on 5% 0.5× TBE polyacrylamide gels. (A) The ability of MEF2C to bind DNA was specifically inhibited when cotranslated with NotchIC. (B) Wild-type MEF2C or C-terminal truncation mutants 1–105 or 1–117 were translated in the presence or absence of NotchIC and tested for DNA binding activity. MEF2C/1–117 was inhibited by NotchIC, whereas MEF2C/1–105 was not. Lysate alone is shown at the right. (C) Schematic diagrams of the MEF2C proteins used in panel B.
Detection of MEF2-Notch interaction by using a two-hybrid assay. (A) Schematic diagrams of Notch and the GAL4 fusion proteins used in two-hybrid assays. The GAL4 DNA binding domain was fused to the entire intracellular portion of mouse Notch1 (GAL4/Notch1751–2294) or the residues N terminal (GAL4/Notch1751–1869) or C terminal (GAL4/Notch2042–2294) to the ankyrin repeats. TM, transmembrane domain; nls, putative nuclear localization signal. (B) 10T1/2 cells were transfected with 2 μg of the pG5E1bCAT reporter, 2 μg of MEF2C, and 2 μg of each GAL4/Notch plasmid, and the CAT activity was determined as described in Materials and Methods. The data are presented as the fold activity versus that observed with the reporter gene alone and represents the average ± the standard error of four experiments performed with at least two different preparations of the plasmids. Significant reporter gene activation was seen only with MEF2C and Gal4/NotchIC1751–2294.
Coimmunoprecipitation of Notch and MEF2C from cell extracts. COS cells were transfected with expression vectors encoding the ankyrin repeat region of Notch fused to an HA epitope tag (AnkR-HA) and MEF2C fused to a FLAG tag. Cell lysates were immunoprecipitated with anti-FLAG antibody, immunoprecipitates were separated by SDS–12% PAGE, followed by Western blot with anti-HA antibody and anti-HRP. (A) Schematic of the experiment. (B) Western blot of extracts from cells transfected with the indicated expression vectors and35S-labeled in vitro translation products in adjacent lanes as markers. AnkR-HA, which migrates as a doublet of approximately 45 and 64 kDa, coimmunoprecipitates with wild-type MEF2C-FLAG (lane 5), but not with the alternate isoform of MEF2C lacking the Notch-binding domain (lane 3).
Schematic of the influence of Notch on MEF2C and myogenic bHLH factor interactions. Notch signaling interferes with the myogenic activity of MEF2C. In addition, the activity of MyoD and other myogenic bHLH factors is blocked by Notch through a MEF2C-independent pathway.
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