"Formation and properties of hairpin and tetraplex structures of guanine-rich regulatory sequences of muscle-specific genes".

Anat Yafe, Shulamit Etzioni, Pnina Weisman-Shomer and Michael Fry*

Department of Biochemistry, Rappaport Faculty of Medicine, Technion–Israel Institute of Technology PO Box 9649 Bat Galim, Haifa 31096, Israel

*To whom correspondence should be addressed. Tel: +972 4 829 5328; Fax: +972 4 851 0735;
Email: mickey@tx.technion.ac.il

Abstract:

Clustered guanine residues in DNA readily generate hairpin or a variety of tetrahelical structures. The myogenic determination protein MyoD was reported to bind to a tetrahelical structure of guanine-rich enhancer sequence of muscle creatine kinase (MCK) more tightly than to its target E-box motif [K. Walsh and A. Gualberto (1992) J. Biol. Chem., 267, 13714–13718], suggesting that tetraplex structures of regulatory sequences of muscle-specific genes could contribute to transcriptional regulation. In the current study we show that promoter or enhancer sequences of various muscle-specific genes display a disproportionately high incidence of guanine clusters. The sequences derived from the guanine-rich promoter or enhancer regions of three muscle-specific genes, human sarcomeric mitochondrial creatine kinase (sMtCK), mouse MCK and a7 integrin formed diverse secondary structures. The sMtCK sequence folded into a hairpin structure; the a7 integrin oligonucleotide generated a unimolecular tetraplex; and sequences from all three genes associated to generate bimolecular tetraplexes. Furthermore, two neighboring non-contiguous guanine-rich tracts in the a7 integrin promoter region also paired to form a tetraplex structure. We also show that homodimeric MyoD bound bimolecular tetraplex structures of muscle-specific regulatory sequences more efficiently than its target E-box motif. These results are consistent with a role of tetrahelical structures of DNA in the regulation of muscle-specific gene expression.

Introduction:

Clusters of contiguous guanine residues in DNA can associate in vitro under physiological-like conditions to form four-stranded structures designated DNA tetraplexes or quadruplexes. At the core of these structures are Hoogsteen hydrogen-bonded, cation coordinated stacked guanine quartets [for reviews see (1,2)]. Tetrahelical DNA species are grouped into three major classes according to the stoichiometry of the DNA strands: monomolecular tetraplexes, bimolecular tetraplexes and G4 four-molecular tetraplexes. In addition, different types of tetraplex DNA are also distinguished by the orientation of their strands. The DNA strands of monomolecular and bimolecular tetraplexes may be positioned in antiparallel (1,2) or parallel (3) orientation, whereas the four strands of G4 DNA are oriented parallel to one another. The various types of quadruplex DNA are in addition set apart by parameters, such as the molecular geometry of the tetrahelix, their glycosidic torsion angles, participation of non-guanine nucleotides in tetrad formation, type of the coordinating cation and the base composition of spacer stretches that separate guanine clusters (1,2).

Although the formation of DNA tetraplexes in vitro is well established, direct indications for their existence in vivo are just beginning to appear (4–6). At the same time, several indirect lines of evidence point to the existence of tetrahelical structures in genomic DNA and to their potential participation in various physiological and pathological processes (2,7). Tetraplex structures that might be formed by biologically important guanine-rich DNA sequences were implicated in the execution of specific roles in vivo. For example, tetrahelices formed by the telomeric G-strand were proposed to contribute to the regulation of telomere extension and to the protection of chromosome ends (8,9). Furthermore, the pairing of homologous chromosomes was purported to be mediated by the transient formation of interchromosomal tetraplex structures (10,11). In another case, folding of the d(CGG) trinucleotide repeat sequence in the FMR-1 gene into tetraplex structures was suggested to cause polymerase pausing and slippage that result in the expansion of the repeat sequence and in the silencing of the FMR-1 gene, setting off the fragile X syndrome (12,13). Arguments for the existence of tetraplex DNA and RNA structures in vivo are supported by the identification of multiple cellular proteins that interact with tetrahelical nucleic acids. Proteins from diverse organisms bind tetraplex DNA preferentially and at high affinity (14–20). Other proteins process or modulate the structure of tetraplex DNA. Such are the nucleases in fission yeast (21,22), mouse (23) and human cells (24) that specifically hydrolyze DNA (19,20,22) and RNA (21) next to quadruplex structures. Other proteins modify the equilibrium between single-stranded DNA and tetraplex guanine-rich DNA. For instance, the ß-subunit of an Oxytricha telomere binding protein (25,26) and the yeast RAP1 protein (27,28) enhance the formation of tetraplex structures by the guanine-rich strand of telomeric DNA. Conversely, yeast and human helicases of the RecQ family (29–32) as well as members of the hnRNP family (33,34) and other proteins (35) were shown to unwind or destabilize tetraplex structures of guanine-rich sequences in DNA or RNA.

The myogenic determination protein MyoD was reported to bind a tetrahelical structure of a guanine-rich tract from muscle creatine kinase (MCK) enhancer sequence more tightly than its target E-box motif (36). Tetraplex structures of guanine-rich stretches in regions upstream to genes, such as c-MYC (37) and insulin (38), were implicated in the regulation of their transcription. In analogy, it is possible that the preferential binding of MyoD to tetraplex structures of regulatory DNA sequences modulates the expression of muscle-specific genes. We thus examined in this work, the formation in vitro of secondary structures of guanine-rich DNA sequences derived from enhancer or promoter regions of muscle-specific genes and studied their properties. We show that guanine clusters in regulatory sequences of several muscle-specific genes readily form hairpin and monomolecular or bimolecular tetraplex structures. We also report that MyoD homodimers bind to bimolecular tetraplex structures of muscle-specific gene regulatory sequences more efficiently than to their E-box target motif.

Materials and Methods:

... Please see:  http://nar.oxfordjournals.org/cgi/content/full/33/9/2887

Additional References:

1. Hovsepian JA, and Frenster JH, "Sense and antisense during RNA initiation of the DNA transcription bubble". (Sense vs. antisense during initiation of gene transcription).

2. Frenster JH, Allfrey VG, and Mirsky, AE, "Metabolism and Morphology of Ribonucleoprotein Particles from the Cell Nucleus of Lymphocytes". (Intranuclear Na ⁺ and K ⁺ effects).

3. Halder K,  and Chowdhury S, "Kinetic resolution of bimolecular hybridization versus intramolecular folding in nucleic acids by surface plasmon resonance: application to G-quadruplex/duplex competition in human c-myc promoter". (Human c-myc gene promoters).

4. Ling J, Baibakov B, Pi W, Emerson BM, and Tuan D, "The HS2 Enhancer of the b-globin Locus Control Region Initiates Synthesis of Non-coding, Polyadenylated RNAs Independent of a cis-linked Globin Promoter".

5. Simonssen T, "G-Quadruplex DNA Structures -Variations on a Theme", Biol. Chem. vol. 382, pp. 621-628 April, 2001). http://www.bcbp.gu.se/simonsson/BC_2001.pdf
 

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