Characterization of basic helix-loop-helix muscle regulatory factors in mammalian cells and in budding yeast
Abstract
MRF4 is a member of the basic helix-loop-helix (bHLH) muscle regulatory factor family (MRF). Over-expression of MRF4 converts permissive cells of mesodermal origin into muscle stem cells referred to as myoblasts. Upon induction by growth factor withdrawal, the committed myoblasts undergo terminal differentiation and fuse into multi-nucleated myofibers. A subset of co-transfected contractile protein genes can be differentially trans-activated in these experiments, indicating that MRF4 functions as a muscle-specific transcription factor. Using a GAL4-DNA binding (DB) system, the MRF4 transcription activation domain (TAD) was identified and found to be located within the amino terminus. Subsequent studies showed that amino acids 10 to 30 constituted the minimum MRF4 activation domain. By adopting a domain swapping strategy, the contribution of the MRF4 TAD to trans-activation specificity also was examined. Results showed that the MRF4 TAD imparts partial trans-activation specificity. Interestingly, the carboxyl end of MRF4 also was found to provide a very important supportive role in both the myogenic induction and the trans-activation functions of MRF4. Prior to using a yeast two-hybrid screening system to identify MRF4 co-regulators, various trans-activation properties of MRF4 were characterized in the budding yeast, Saccharomyces cerevisae. When tested in yeast as a GAL4-DB-MRF-TAD fusion protein, both the MyoD and MRF4 TADs were active towards reporters driven by GAL4 UAS. However, only MRF4 was capable of trans-activating an (E-box)$\sb4$-his3 reporter gene in this system. Further domain swapping experiments showed that the TADs of the MRFs solely dictate the trans-activation specificity of each MRF in yeast. Furthermore, the MRF4 TAD and the GAL4 TAD were shown to be functionally interchangeable, suggesting that both TADs activate transcription through similar mechanisms. The feasibility of using a yeast two-hybrid screening strategy to screen for MRF4 co-regulators was further confirmed by the successful demonstration of a bHLH interaction between MRF4 and E12 in yeast. After confirmation of the correct expression of various MRF4 fusion proteins in yeast, several preliminary screens were performed. Alternative screening strategies also are discussed with regard to isolating co-regulators.
Degree
Ph.D.
Subject Area
Molecular biology
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