טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentEtzioni Shulamit
SubjectInteraction of the MyoD Protein with Tetrahelical DNA
DepartmentDepartment of Medicine
Supervisors Professor Michael Fry
Professor Eyal Bengal
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Clusters of guanine in DNA readily associate to form hairpin or tetrahelical structures. Tetraplex forms of guanine-rich sequences in the promoters of several genes were implicated in the repression or activation of their transcription.

Myogenic transcription is activated by the binding of heterodimers of the bHLH proteins MyoD with E proteins to a consensus E-box sequence d(CANNTG) in promoter or enhancer regions of muscle-specific genes. Homodimers of MyoD bind E-box less tightly and are less efficient activators of transcription. Regions from promoters or enhancers of the muscle-specific genes; human sacrcomric Mitochondrial Creatine Kinase (sMtCK) and mouse α7 integrin display a disproportionate high incidence of guanine clusters. Sequences derived from these regions readily formed diverse secondary structures; hairpin and mono- and bi-molecular tetraplex structures. Further, MyoD was reported to bind a tetrahelical structure of guanine-rich enhancer sequence of Muscle Creatine Kinase more tightly than E-box.

In this work we show that homodimers of MyoD form complexes with bimolecular tetraplex structures of muscle-specific regulatory sequences but not with their double-stranded, hairpin or unimolecular tetraplex forms. Preferential binding of homodimeric MyoD to bimolecular tetraplex DNA structures over E-box DNA is reflected by the 18.7 to 39.9-fold lower dissociation constants, Kd, of the MyoD-tetraplex DNA complexes. Conversely, MyoD-E47 heterodimers form tighter complexes with E-box as indicated by their 6.8 to 19.0-fold lower Kd relative to complexes with bimolecular tetraplex DNA structures. We speculate that the favored binding of MyoD homodimers to tetraplex DNA structures lowers their ability to activate muscle-specific gene transcription whereas the preferential binding of MyoD-E47 heterodimers to E-box DNA enhances their transcriptional potency.

In a second part of the work we report that the basic region which is responsible for E-box DNA binding is also necessary for the binding of tetraplex DNA. However, point mutations and deletions of selected amino acids that diminish E-box binding, do not affect tetraplex DNA binding. We conclude that the capacity of MyoD to bind tetraplex DNA structures is less stringently dependent on the integrity of the basic region than the binding of E-box, apparently because the tetraplex binding requires a different conformation of the basic region.

Last, we report that deletion of each of the MyoD helix regions (helix1/2), essential for dimerization of bHLH proteins, decreased its capacity to bind tetraplex DNA without completely abrogating it, while completely obliterating the binding to E-box DNA. Again, showing the different binding to tetraplex DNA