Murine Twist is a bHLH Regulator that Inhibits Myogenesis by Multiple Molecular Mechanisms

Citation

Yun, Kyuson (1997) Murine Twist is a bHLH Regulator that Inhibits Myogenesis by Multiple Molecular Mechanisms. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/kt6j-sg39. https://resolver.caltech.edu/CaltechTHESIS:07182025-151320548

Abstract

Twist is a member of the basic helix-loop-helix (bHLH) class of transcc1iption factors. From its expression patterns during fly and mouse embryogenesis, it was chosen to be studied as a potential upstream- or cross-regulator of myogenic regulatory factors (MRFs). Additional motivation for studying twist in the context of myogenesis comes from the shared structural motif among MRFs, E-proteins ("universal" partners for tissue-specific factors), and twist that would allow direct physical association. Helix-loop- helix domain allows combinatorial dimerization that results in distinct DNA-binding complexes. Depending on the partner choice and availability, twist and other bHLHs can form complexes with different activities.

In E8.5d mouse embryo, twist and myf5 overlap in their expression domains in the developing somites, indicating the physiological relevance of the twist and MRF interaction. This observation suggests that twist and myf5 (MRF) may interact with each other, directly or indirectly through E-proteins. Supporting data for this idea are presented in chapter 2. In addition, twist is demonstrated to form active DNA binding complexes with different E-proteins. Since twist titrates available E-proteins from MyoD in vitro and in vivo, twist acts as a dominant negative regulator of myogenesis. However, the fact that twist:E complex is an active DNA binding factor suggests that twist may regulate the expression of downstream target genes.

Studies using a tethered dimer between MyoD and E47 (MyoD-E47) support the likelihood of transcriptional regulaton by twist, presented in chapter 3. Results from chapters 2 and 3 suggest that although bHLH competition and MEF2 titration are viable mechanisms of myogenic inhibition by twist, the most potent inhibitory activity of twist is likely to involve another mechanism.

Since twist inhibits myogenesis initiated by all combinations of MRFs and MEF2 tested in transfected cells, the cell cycle status of these cells were tested since forced proliferation would account for failure to differentiate. MyoD expressing cells are found to arrest normally in the presence of twist, suggesting twist inhibition is specific to differentiation and not an overall inactivation of MyoD function. Furthermore, twist inhibits the onset of myogenin suggesting this early myogenic event is blocked. However, this is not the only restriction point targeted by twist since late twist expression (driven by the myogenin promoter) can still inhibit muscle differentiation. These observations together show that twist is a potent inhibitor of myogenesis and suggest that twist may be involved in regulating proper muscle differentiation in developing embryos.

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