Genomic distribution and functional analyses of potential G-quadruplex-forming sequences in Saccharomyces cerevisiae

Abstract

Although well studied in vitro, the in vivo functions of G-quadruplexes (G4-DNA and G4-RNA) are only beginning to be defined. Recent studies have demonstrated enrichment for sequences with intramolecular G-quadruplex forming potential (QFP) in transcriptional promoters of humans, chickens and bacteria. Here we survey the yeast genome for QFP sequences and similarly find strong enrichment for these sequences in upstream promoter regions, as well as weaker but significant enrichment in open reading frames (ORFs). Further, four findings are consistent with roles for QFP sequences in transcriptional regulation. First, QFP is correlated with upstream promoter regions with low histone occupancy. Second, treatment of cells with N-methyl mesoporphyrin IX (NMM), which binds G-quadruplexes selectively in vitro, causes significant upregulation of loci with QFP-possessing promoters or ORFs. NMM also causes downregulation of loci connected with the function of the ribosomal DNA (rDNA), which itself has high QFP. Third, ORFs with QFP are selectively downregulated in sgs1 mutants that lack the G4-DNA-unwinding helicase Sgs1p. Fourth, a screen for yeast mutants that enhance or suppress growth inhibition by NMM revealed enrichment for chromatin and transcriptional regulators, as well as telomere maintenance factors. These findings raise the possibility that QFP sequences form bona fide G-quadruplexes in vivo and thus regulate transcription.

In vivo veritas: Using yeast to probe the biological functions of G-quadruplexes

“…The yeast genome has a QFP density that is approximately an order of magnitude less than the human genome. For example, approximately 40% and 1.5% of human and yeast upstream promoter sequences have QFP, using the same definition of QFP in both organisms [6,9]. The smaller number of sequences with QFP in yeast might provide advantages for certain analyses.…”

“…Nonetheless, a good deal of information demonstrating or strongly suggesting their functions in vivo has emerged in recent years. For example, telomeric G4-DNA has been proven to exist in Stylonichia lemnae [4,5], and sequences with intramolecular quadruplex-forming potential (QFP) have been shown to be highly overrepresented in the promoter regions of diverse organisms and to be connected with control of gene expression [6][7][8][9][10][11][12]. In addition, a number of small molecule ligands have been identified that bind to and stabilize quadruplexes (Fig.…”

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress

“…Antibodies to G4DNA have been used to visualize G4DNA in cells (7)(8)(9)(10). Small molecules designed to interact specifically with G4DNA have been found to alter gene expression in a G4DNA-selective manner (11,12). The Pif1 helicase localizes to G4DNA sequence motifs and binds and unwinds G4DNA structures (13,14).…”

Targeting G-Quadruplex DNA with Small Molecules

“…[103] Recently, NMM was found to up-regulate genes having promoters with a high potential for G-quadruplex formation, and it also suppressed rDNA activity in yeast. [38] The binding of rDNA (known to have a high potential for G-quadruplex formation) and the subsequent suppression of ribosome biogenesis is the reported mode of action of CX-3543 (quarfloxin), a small molecule drug candidate currently in phase II clinical trials. [105] Telomestatin (SOT-095), a natural product isolated from Streptomyces anulatus 3533-SV4, [106] is one of the strongest and most specific inhibitors of telomerase reported to date (IC 50 ≅ 1 µM).…”

“…[33][34][35] No consensus sequence for G-quadruplex folding has been experimentally determined, but approximately 370'000 sequences with the putative G-quadruplex-forming motif (G 3+ N 1-7 ) 4+ (where G = guanosine and N = any base) are dispersed throughout the human genome. [36,37] These sequences are concentrated in promoter regions, [14,38,39] introns, [36] 5' and 3' UTRs, [40] and at the ends of eukaryotic chromosomes. [41,42] G-rich sequences having this motif can fold into highly stable intramolecular Gquadruplex structures (T m > 90 o C) [19] that usually fold and unfold very slowly at room temperature in vitro (k f ~0.001 s -1 ).…”