A CpG island promoter drives the CXXC5 gene expression.

CXXC5 is a member of the zinc-finger CXXC family that binds to unmethylated CpG dinucleotides. CXXC5 modulates gene expressions resulting in diverse cellular events mediated by distinct signaling pathways. However, the mechanism responsible for CXXC5 expression remains largely unknown. We found here that of the 14 annotated CXXC5 transcripts with distinct 5' untranslated regions encoding the same protein, transcript variant 2 with the highest expression level among variants represents the main transcript in cell models. The DNA segment in and at the immediate 5'-sequences of the first exon of variant 2 contains a core promoter within which multiple transcription start sites are present. Residing in a region with high G-C nucleotide content and CpG repeats, the core promoter is unmethylated, deficient in nucleosomes, and associated with active RNA polymerase-II. These findings suggest that a CpG island promoter drives CXXC5 expression. Promoter pull-down revealed the association of various transcription factors (TFs) and transcription co-regulatory proteins, as well as proteins involved in histone/chromatin, DNA, and RNA processing with the core promoter. Of the TFs, we verified that ELF1 and MAZ contribute to CXXC5 expression. Moreover, the first exon of variant 2 may contain a G-quadruplex forming region that could modulate CXXC5 expression.

Doxorubicin exhibits strong and selective association with VEGF Pu22 G-quadruplex.

BACKGROUND: Vascular endothelial growth factor (VEGF), is upregulated in tumor cells and thus became a potential therapeutic target for anti-cancer drugs. Recent reports suggested the use of Doxorubicin (Dox) with VEGF-targeting siRNAs for an enhanced decrease in VEGF expression. Besides, VEGF-B gene therapy was found to suppress the cardiotoxicity effects of Dox. On the other hand, even though Dox is a commonly used anti-cancer agent, its mechanism of actions isn't completely mapped out. Herein, the interactions between a G4 structure formed by the VEGF promoter region Pu22 and Dox were investigated. METHODS: The Dox-G4 interactions were examined via competition dialysis, UV-vis Absorption, Circular Dichroism (CD) and Fluorescence spectroscopy. RESULTS: The results demonstrated that Dox was stabilizing the VEGF Pu22 G4 structure and the calculated association constant for VEGF Pu22-G4 complex (Ka = 7.50 × 106) was very close to the reported Ka values for Dox-dsDNA complexes. Additionally, the competition dialysis experiments revealed the selectivity of Dox to Pu22 compared to other G4 structures formed in telomeric repeats and promoter regions such as BCL-2 and C-myc. CONCLUSIONS: Dox exhibits strong and selective association with VEGF Pu22 G4 structure that was comparable to its well-known association with dsDNA. GENERAL SIGNIFICANCE: The results presented here might be useful in the general area of antitumor drug-DNA interactions. Doxorubicin's significant affinity to VEGF Pu22 G4 might be one of the plausible mechanisms behind its anti-tumor activity.

A conjugated gold nanoparticle-azacyanine off-on-off fluorescence probe for sensitive and selective detection of G-quadruplexes.

G-quadruplex secondary structures have gained significant recognition due to the discovery of their involvement in regulation of gene expression and their association with many diseases such as cancer and neurological disorders. Consequently, the need for the recognition and characterization of G-quadruplex structures has increased considerably. Here, we present a rapid, facile and sensitive off-on-off in vitro platform for G-quadruplex detection, based on the gold nanoparticle-azacyanine5 (AuNP-Aza5) conjugated fluorescence probe. The conjugated probe is governed by Fluorescence Resonance Energy Transfer (FRET) mechanism between the fluorophore molecule, Aza5, and AuNPs. The fluorescence of Aza5 that was hindered by AuNPs (off), was restored in the presence of L-cysteine (on) until the addition of a G-quadruplex structure (off). The developed sensing platform selectively responds to G-quadruplex structures formed within the promoter regions of VEGF-Pu22, K-RAS, C-myc and BCL-2. It doesn't exhibit a similar response to the other secondary structures such as single, double or triple stranded nucleic acid structures. The detailed investigation of the probe with VEGF-Pu22 as a model G-quadruplex structure revealed a linear response between the concentration range of 0.032-0.347 µM with a detection limit of 12.66 nM.