טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentYafe Anat
SubjectTetrahelical DNA Structures in Regulatory Regions of
Myogenic Genes and their Interaction with Myogenic
Transcription Factors
DepartmentDepartment of Medicine
Supervisors Professor Michael Fry
Professor Eyal Bengal
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

DNA tracts enriched in clusters of contiguous guanine residues have been identified in several biologically important regions of the genomes of bacteria, yeast, protozoa and mammals. These sequences can associate in vitro under physiological-like conditions to form four-stranded structures designated DNA tetraplexes or quadruplexes. An extensively researched proposed role of tetraplex DNA structures is their function in the regulation of gene transcription.

Formation of tetraplex structures by regulatory sequences of muscle-specific genes and their interaction with myogenic regulatory factors (MRFs) is the subject of this work.

Four MRFs that comprise a subgroup within the superfamily of basic helix-loop-helix (bHLH) proteins; MyoD, Myf5, myogenin and MRF4, regulate the coordinated transcription of multiple muscle-specific genes during myogenesis. Studies of differentiation in vitro revealed that heterodimers of MRFs with E12 or E47 proteins activated transcription by their binding to a conserved E-box motifs, d(CANNTG), in the regulatory regions of muscle-specific genes.

We report that guanine clusters are represented at a disproportional high incidence in regulatory regions of muscle-specific genes. Examined sequences of the muscle-specific genes; human sarcomeric mitochondrial creatine kinase (sMtCK), mouse MCK and α7 integrin are shown to readily fold in vitro into secondary structures. Specifically, sMtCK forms hairpin structure and α7 integrin folds into a parallel monomolecular tetraplex structure. The sequences of all three genes also form parallel bimolecular tetraplex DNA structures.

In earlier work, we found that MyoD homodimers bound G'2 bimolecular tetraplexes much more tightly than E-box DNA. Conversely, MyoD/E47 heterodimers were similarly found to bind E-box more tightly than G'2 bimolecular tetraplex DNA structures.

We show here that homodimeric MRF4 displays a higher affinity for G’2 Integrin DNA relative to G’2 sMtCK DNA, and that Myogenin exhibits similarly low affinities for both G’2 Integrin DNA and G’2 sMtCK DNA. The two proteins in their homodimeric forms have low affinity for E-box DNA. In contrast to the respective homodimers, the affinities of MRF4/E47 and Myogenin/E47 heterodimers for E-box DNA were found to be ~50-fold higher than their affinity for the G'2 bimolecular tetraplex DNA structures.

In the last part of this work we examined the in vivo effect of the tetraplex structures on MyoD-mediated gene activation. We show that introduction of G’2 Integrin 26 DNA into HEK293 cells synergistically amplifies the MyoD-dependent expression of a firefly luciferase reporter gene.