טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShklover Jeny
SubjectStructure-Function Relationship of the Binding of the
Transcription Factor MyoD to Tetraplex DNA
DepartmentDepartment of Medicine
Supervisor Professor Michael Fry
Full Thesis textFull thesis text - English Version


Abstract

Guanine clusters in DNA can form Hoogsteen bonded G-quartets that serve as core for alkali cation-coordinated quadruplex structures. Tetraplex structures were implicated in the in vivo regulation of specific functions such as telomere extension and gene transcription. This work investigated the interaction of myogenic transcription factors (MRFs) with quadruplex structures of promoter sequences of muscle-specific genes.


Heterodimers with E-proteins of four MRFs; MyoD, Myf-5, MRF4 and Myogenin activate muscle-specific gene expression by binding to E-box motifs in their promoters or enhancers. Our laboratory demonstrated in the past that whereas MyoD-E47
heterodimers associated most tightly with E-box elements in sequences of muscle-specific gene promoters, MyoD homodimers bound preferentially to quadruplex structures of promoter sequences. However, unlike homodimeric MyoD, Myogenin homodimers did not exhibit preference for quadruplex DNA.

In the first part of this work I identified the protein structure elements of homodimeric MyoD that participate in the binding of E-box and quadruplex DNA. Deletion of a single basic amino acids triad in the MyoD basic domain resulted in complete loss of the E-box-binding capacity, while Kd values of complexes of mutant proteins with two deleted triads revealed that each remaining cluster sufficed for a relatively tight binding of quadruplex DNA. Molecular modeling indicated that the relative contribution of each amino acids triad to the binding affinity was related to its distance from the bound DNA. These results suggested that the binding of double-stranded E-box DNA or tetraplex structures of muscle-specific genes promoter was mediated by overlapping but distinct elements in the basic region of MyoD.

In the second part of this work I investigated the protein structure basis for the different affinities of homodimeric MyoD and Myogenin for quadruplex DNA. I first showed that replacing different residues in the basic amino acid triads of Myogenin with those of MyoD did not increase its quadruplex affinity. Next by constructing chimerical MyoD and Myogenin proteins with reciprocally switched basic regions I demonstrated that both the basic region and peripheral domains of MyoD were required for its high avidity for tetraplex DNA. This conclusion was sustained by a comparison of the quadruplex DNA affinities of isolated bHLH regions of MyoD and Myogenin.

In summary, in this first systematic investigation of the interaction of transcription factors with potential quadruplex domains in gene promoters we defined protein elements that determine the differential affinities of two myogenic factors for quadruplex and E-box DNA.