Abstract
G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III1 of the c-MYC promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII1 located upstream of the P1 promoter regulates up-to 80% of c-MYC transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII1 sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII1 resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the c-MYC and SOX2 promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common regulatory mechanism for genes whose promoters contain these sequences.
Highlights
The presence of secondary structure in guanine-rich oligonucleotides was first documented in the late 1980’s [1]
Most of the Pu27 homologous sequences are located within gene transcription regions including the noncoding region of SOX2 /Pu3+, GRM6 /Pu5, and NAV2 /Pu11+ or on the noncoding strand of the genes (Pu27/ c-MYC, Pu20-/CDH4) (Table 1)
We have previously shown that Pu27 inhibits cell growth and downregulates c-MYC transcription in leukemic cell lines [42]
Summary
The presence of secondary structure in guanine-rich oligonucleotides was first documented in the late 1980’s [1]. Four adjacent guanines (on one strand or on different strands of DNA) can spontaneously arrange in a square planar structure which is stabilized by Hoogsteen hydrogen bonds called G-tetrads. This structure is further stabilized by monovalent cations at physiological concentrations [1, 2]. G-quadruplex forming sequences are preferentially located near the promoter regions of eukaryotic genes, especially of oncogenes including c-MYC [8, 9], KRAS [10], c-KIT [11] and BCL2 [12]. The past 20 years have seen an evolving interest in G-quadruplex structures as targets for cancer therapy primarily due to the putative regulatory role of these structures [14, 15]
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